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Recent Advances in
iPSCs for Therapy,
Volume 3
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Advances in Stem Cell Biology
Recent Advances in
iPSCs for Therapy,
Volume 3
Edited by
Alexander Birbrair
Federal University of Minas Gerais
Department of Pathology
Belo Horizonte, Minas Gerais, Brazil
Columbia University Medical Center
Department of Radiology
New York, NY, United States
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Notices
Knowledge and best practice in this field are constantly changing. As new research and
experience broaden our understanding, changes in research methods, professional
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This book is dedicated to my mother, Marina Sobolevsky, of blessed memory, who passed
away during the creation of this volume. Professor of Mathematics at the State University of
Ceara
´(UECE), she was loved by her colleagues and students, whom she inspired by her
unique manner of teaching. All success in my career and personal life I owe to her.
My beloved father, Lev Birbrair, and my beloved mom, Marina Sobolevsky, of blessed memory (July 28, 1959eJune 3, 2020)
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Contents
Dedication...............................................................................................v
Contributors........................................................................................... xv
About the editor .................................................................................... xxi
Preface ...............................................................................................xxiii
CHAPTER 1 Strategies to utilize iPS cells for hair follicle
regeneration and the treatment of hair loss
disorders ................................................................ 1
Manabu Ohyama
1. Introduction ....................................................................... 2
2. HF morphology and physiology............................................. 4
2.1 Morphogenesis and morphology of HF..............................4
2.2 Physiology of HF focusing on the hair cycle ......................6
3. Experimental approaches to HF regeneration ........................... 6
3.1 Principles of experimental HF regeneration ........................6
3.2 Use of hiPSCs for human HF regeneration .........................8
4. Relationship between HF and iPSCs: the HF as a favorable
iPSC material ..................................................................... 8
5. Regeneration of human HFs using hiPSCs............................... 8
5.1 Basic principles.............................................................8
5.2 Induction of folliculogenic KCs from hiPSCs .....................9
5.3 Generation of dermal cells with trichogenic activity from
hiPSCs....................................................................... 11
5.4 Assembly of HF structures using hiPSC-derived HF
components ................................................................ 13
5.5 Use of hiPSCs for HF formation mimicking normal
organogenesis.............................................................. 15
6. Strategies for treating hair loss disorders using hiPSCs .............15
6.1 Development of pathophysiologically specific therapeutic
approaches.................................................................. 15
6.2 Cell-based therapies to reverse HF miniaturization . ........... 15
6.3 Suppression of immune responses involving HFs by
hiPSC-derived cells...................................................... 16
7. Conclusion........................................................................17
List of abbreviations................................................................17
Acknowledgment....................................................................18
References.............................................................................18
vii
CHAPTER 2 iPSCs and cell therapy for Parkinson’s disease.......23
Jeffrey S. Schweitzer, Bin Song and Kwang-Soo Kim
1. Introduction ......................................................................25
2. PD as an established target for cell therapy.............................26
3. Developing a cell therapy approach for PD.............................27
3.1 Disease models for tests of efficacy............................... 27
3.2 Sources of transplantable tissue .. .................................. 27
3.3 Fetal cell transplantation as early proof-of-concept
studiesdlessons and challenges.................................... 27
3.4 hESC cell sources for PD therapy ................................. 28
3.5 Non-iPSC autologous cell sources................................. 29
3.6 iPSC-derived DA cells ................................................ 29
3.7 Identification and generation of clinical grade hiPSCs ...... 30
3.8 Genomic stability of hiPSCs and epigenetics . ................. 31
3.9 Generation of transplantable mDA progenitors from
patient-derived hiPSCs................................................ 31
3.10 Safety of hiPSC-derived cell grafts................................ 33
4. Clinical factors ..................................................................33
4.1 Variable reprogramming potential and differentiation
specificity................................................................... 34
4.2 Variable response of different forms of the disease ............ 34
4.3 Expression of the disease within the graft ........................ 34
4.4 Implantation target site and cell number .......................... 35
4.5 Graft survival and outgrowth ......................................... 35
4.6 Surgical technique ....................................................... 35
5. Toward clinical implementation of iPSC-based therapy
for PD..............................................................................37
5.1 Production of clinical-grade iPSCs.................................. 37
5.2 Optimization and standardization of in vitro
differentiation protocols ................................................ 37
5.3 Patient selection and follow-up monitoring criteria ............ 38
5.4 Regulatory and socioeconomic acceptance ....................... 38
6. Perspective........................................................................38
Abbreviations.........................................................................39
Acknowledgments...................................................................39
References.............................................................................39
CHAPTER 3 Induced pluripotent stem cells as a potential
treatment for Huntington’s disease..........................49
B. Srinageshwar, G.L. Dunbar and J. Rossignol
1. Huntington’s disease...........................................................50
2. HTT gene and protein function.............................................51
viii Contents
3. Epigenetics and HD............................................................51
4. Signs and symptoms of HD..................................................52
5. Stem cell therapy for HD.....................................................53
5.1 Mesenchymal stem cells (MSCs) and HD ........................ 54
5.2 Neural stem cells (NSCs) and HD .................................. 54
5.3 Embryonic stem cells (ESCs) and HD .. ........................... 55
6. Induced pluripotent stem cells (iPSCs). ..................................55
6.1 Induced pluripotent stem cells (iPSCs) and HD................. 55
6.2 iPSC-based therapy for HD ........................................... 56
7. Stem cellsebased HD clinical trials.......................................59
8. Summary and conclusions ...................................................61
Acknowledgments...................................................................62
References.............................................................................62
CHAPTER 4 Present and future of adult stem cells and
induced pluripotent stem cells therapy for
ischemic stroke .....................................................67
Ana Bugallo-Casal, Marı
´
aPe
´rez-Mato and Francisco Campos
1. Introduction ......................................................................68
2. Protective and recovery approaches for ischemic stroke ............69
3. Adult stem cells: NSCs and mesenchymal stem cells................74
3.1 Protective effects of NSCs in the treatment of stroke ......... 74
3.2 Therapeutic potential of MSCs in stroke: immune
modulation, neuroprotection, and neurorepair .. ................. 75
4. Induced pluripotent stem cells: a multifaceted role in the
study of neurological diseases ..............................................81
4.1 iPSCs in stroke............................................................ 83
4.2 iPSCs and CADASIL ................................................... 84
5. Conclusion and future perspectives........................................87
Glossary................................................................................89
References.............................................................................90
CHAPTER 5 Stem cell therapy in Alzheimer’s disease................97
Milena Pinto, PhD, Christian Camargo, MD,
Michelle Marrero, MD and Bernard Baumel, MD
1. Introduction ......................................................................98
2. Preclinical studies on the use of stem cells in dementias ......... 100
3. Endogenous approach .......................................................100
4. Exogenous approach.........................................................101
4.1 Classification of stem cells for exogenous therapeutic
approaches.................................................................101
4.2 Embryonic stem cells ..................................................102
Contents ix
4.3 Induced pluripotent stem cells.......................................102
4.4 Neuronal stem cells.....................................................103
4.5 Mesenchymal stem cells ..............................................104
4.6 MSCs and Alzheimer’s disease: preclinical experience......113
5. Previous human experience with MSCs................................114
5.1 Choice of stem cell for clinical studies ...........................114
5.2 Use of human MSCs in neurodegenerative diseases ..........115
5.3 Route of administration considerations ...........................117
5.4 Cell dosage................................................................119
5.5 Concluding remarks ....................................................121
References...........................................................................122
CHAPTER 6 Stem cell therapies for glaucoma and optic
neuropathy .............................................. ............ 133
Ziming Luo, Michael Nahmou and Kun-Che Chang
1. Introduction ....................................................................134
2. Retinal cell fate specification..............................................134
2.1 Regulatory mechanisms of retinal ganglion cell
differentiation.............................................................134
2.2 Stem cell to retinal ganglion cell differentiation ...............136
2.3 Retinal organoid differentiation.....................................137
3. Retinal cell transplantation.................................................139
3.1 Primary retinal ganglion cell transplantation....................139
3.2 Stem cellederived retinal ganglion cell transplantation .....140
3.3 Retinal organoid transplantation ....................................140
3.4 iPSCs for cell replacement therapy ................................143
4. iPSCs for modeling of familial glaucoma .. ...........................143
5. Summary and future directions ...........................................146
References...........................................................................146
CHAPTER 7 Induced Pluripotent Stem Cells (iPSC) in
Age-related Macular Degeneration (AMD) ............. 155
Graham Anderson, PhD, Pierre Bagnaninchi, PhD and
Baljean Dhillon, BMedSci(Hons), BMBS FRCS(Glasg),
FRCOphth, FRCS(Ed), FRCPE
1. Background.....................................................................156
1.1 AMD........................................................................156
1.2 The RPE’s Role in the Subretinal Niche . ........................156
2. Cellular Therapies for AMD ..............................................159
2.1 Induced Pluripotent Stem Cell (iPSC)-derived RPE ..........161
2.2 Clinical Trials Utilizing iPSC-RPE in AMD....................162
xContents
2.3 Genomic Instability in iPSC-RPE, is Direct
Reprogramming the Answer?........................................164
3. Future Directions .............................................................166
References...........................................................................167
CHAPTER 8 Considerations in using human pluripotent stem
cellederived pancreatic beta cells to treat type 1
diabetes .......................................... .................... 173
Wei Xuan Tan, Hwee Hui Lau, Nguan Soon Tan,
Chin Meng Khoo and Adrian Kee Keong Teo
1. Introduction ....................................................................174
2. hPSC-based clinical trials ..................................................175
3. Considerations for hPSCs for clinical trials...........................176
3.1 hESCs versus hiPSCs ..................................................181
3.2 Genotype of donors.....................................................182
3.3 Allogeneic versus autologous transplantation...................183
4. hiPSC biobanking efforts...................................................183
5. Generation of hPSCs.........................................................185
5.1 Regulations on the generation of hPSCs for clinical use ....185
5.2 Tissue acquisition and somatic cell isolation....................186
5.3 Reprogramming into hiPSCs.........................................187
5.4 hiPSC culture and expansion ........................................189
6. Manufacturing hPSC-derived pancreatic beta cells for cell
therapy...........................................................................189
6.1 Challenges in generating cGMP-grade hPSC-derived
pancreatic beta cells for therapy ....................................190
6.2 Strategies to prevent tumor formation.............................192
7. hPSC-based therapy for type 1 diabetes ...............................193
7.1 Manufacturing of PEC-01 cells .....................................194
7.2 VC-01 clinical trial .. ...................................................194
7.3 VC-02 clinical trials....................................................195
7.4 Preclinical study by Semma therapeutics.........................196
8. Future outlook and concluding thoughts...............................196
Acknowledgments.................................................................197
References...........................................................................197
CHAPTER 9 Induced pluripotent stem cells for treatment
of heart failure........................................... .......... 205
Shigeru Miyagawa and Yoshiki Sawa
1. Introduction ....................................................................206
2. Trend of cell therapy ........................................................207
Contents xi
3. Production of iPSCs and establishment of a cardiogenic
differentiation method.......................................................208
3.1 Cytokine-based CM differentiation of iPSCs....................208
3.2 Small moleculeebased myocardial differentiation
of iPSCs....................................................................209
3.3 Direct reprogramming for cardiomyocyte generation.........209
4. Large-scale cell culture for cell transplantation therapy...........209
4.1 Bioreactor system for generating CMs for cell
transplantation therapy.................................................210
5. Proof of concept in iPSC-CM sheet for heart failure and
immunologic study of iPSCs .. ............................................211
6. Removal of undifferentiated iPSCs to confirm safety for
clinical applications..........................................................213
7. Development of new drug-based heart failure therapy using
disease-specific iPSCs.......................................................214
7.1 Disease-specific iPSCs and drug discovery screening
in cardiovascular disease..............................................214
7.2 Drug discovery screening using iPSC-CM tissue .. ............215
7.3 Future plans and perspectives in drug screening using
iPSCs........................................................................217
References...........................................................................218
CHAPTER 10 Induced pluripotent stem cells in liver disease ... 225
M. Teresa Donato, Marı
´
a Pelecha
´and Laia Tolosa
1. Introduction ....................................................................226
2. Generation of iPSCs and differentiation into hepatic
phenotype .. .....................................................................227
2.1 Differentiation of iPSCs into hepatocyte-like cells ............228
2.2 Correction of iPSCs ....................................................228
2.3 Differentiation of iPSCs into other hepatic cells ...............229
2.4 Cocultures and 3D cultures of iPSC-derived cells.............230
3. Use of iPSCs in liver disease..............................................231
3.1 Indications of cell therapy for liver diseases ....................231
3.2 Bioartificial liver systems to bridge patients to liver
transplantation............................................................233
3.3 iPSCs in animal models of liver disease.. .. ......................233
3.4 Limitations and challenges of the clinical use of iPSCs .....235
4. iPSC for modeling liver disease ..........................................236
5. Hepatotoxicity studies.......................................................238
6. Conclusions.....................................................................241
Abbreviations.......................................................................241
Acknowledgments.................................................................241
References...........................................................................242
xii Contents
CHAPTER 11 Induced pluripotent stem cells: potential therapeutic
application for improving fertility in humans and
animals ............................................. ................ 251
Oscar A. Peralta, Vı
´
ctor H. Parraguez and Cristian G. Torres
1. Biology of iPSC in mammals .............................................252
2. Stem cells as candidates for in vitro germ cell derivation........ 253
3. Potential strategies for the use of iPSC-derived germ cells
in human, livestock, and wild animal reproductive
biotechnology..................................................................255
4. Paracrine control and gene signaling pathways involved in
in vivo male germ differentiation: potential approaches for
in vitro derivation of germ cells from iPSC........................... 257
5. In vitro approaches for derivation of germ cells from iPSCs .... 260
6. Conclusion......................................................................262
Funding ..............................................................................262
References...........................................................................262
CHAPTER 12 Induced pluripotent stem cells in wound
healing........................................................... ... 269
Xixiang Gao, Jolanta Gorecka, Umber Cheema,
Yongquan Gu, Yingfeng Wu and Alan Dardik
1. Introduction ....................................................................270
2. Background.. ...................................................................271
3. Stem cell therapy in wound healing.....................................272
3.1 Embryonic stem cells ..................................................274
3.2 Mesenchymal stem cells ..............................................274
4. Induced pluripotent stem cells .. ..........................................275
4.1 iPSC advantages.........................................................276
4.2 iPSC-derived endothelial cells.......................................276
4.3 iPSC-derived fibroblasts...............................................280
4.4 iPSC-derived mesenchymal stem cells............................281
4.5 iPSC-derived extracellular vesicles ................................281
5. Challenges and solutions of iPSC in wound healing ............... 282
6. Conclusion......................................................................283
7. Future directions ..............................................................284
References...........................................................................284
CHAPTER 13 Induced pluripotent stem cells for periodontal
regeneration......................... ............................. 291
Ryan Bloomquist and Mahmood S. Mozaffari
1. Prevalence of periodontal diseases.......................................292
Contents xiii
2. Overview of pathogenesis of periodontitis ............................293
3. Current therapies for periodontitis and their limitations........... 295
4. Development of the dental complex.....................................296
5. Stem cells and tissue regeneration.......................................298
6. iPSCs in regenerative dentistry ...........................................301
7. Clinical application of iPSCs.............................................. 305
8. Conclusions and perspective...............................................306
Acknowledgments.................................................................308
References...........................................................................308
CHAPTER 14 Current development in iPSC-based therapy for
autoimmune diseases................................ ......... 315
Anil Kumar, Jugal Kishore Das, Hao-Yun Peng, Liqing Wang,
Yijie Ren, Xiaofang Xiong and Jianxun Song
1. Introduction ....................................................................316
2. Cellular components in autoimmunity..................................318
3. Treg cells in autoimmune disease........................................319
4. Dendritic cells (DCs) in autoimmune disease .. ......................320
5. Autoimmune disease.........................................................323
6. Diabetes mellitus..............................................................323
7. Rheumatoid arthritis (RA) .................................................325
8. Multiple sclerosis (MS).....................................................328
9. Conclusion and future challenges........................................330
Acknowledgments.................................................................331
References...........................................................................331
Index...................................................................................................339
xiv Contents
Contributors
Graham Anderson, PhD
Centre for Regenerative Medicine, Institute for Regeneration and Repair, The
University of Edinburgh, Edinburgh BioQuarter, Edinburgh, United Kingdom
Pierre Bagnaninchi, PhD
Centre for Regenerative Medicine, Institute for Regeneration and Repair, The
University of Edinburgh, Edinburgh BioQuarter, Edinburgh, United Kingdom
Bernard Baumel, MD
Department of Neurology, University of Miami Miller School of Medicine, Miami,
FL, United States
Alexander Birbrair
Department of Pathology, Federal University of Minas Gerais, Belo Horizonte,
MG, Brazil; Department of Radiology, Columbia University Medical Center,
New York, NY, United States
Ryan Bloomquist
Department of Restorative Sciences, The Dental College of Georgia, Augusta
University, Augusta, GA, United States
Ana Bugallo-Casal
Clinical Neurosciences Research Laboratory (LINC), Health Research Institute of
Santiago de Compostela (IDIS), Santiago de Compostela, A Corun
˜a, Spain
Christian Camargo, MD
Department of Neurology, University of Miami Miller School of Medicine, Miami,
FL, United States
Francisco Campos
Clinical Neurosciences Research Laboratory (LINC), Health Research Institute of
Santiago de Compostela (IDIS), Santiago de Compostela, A Corun
˜a, Spain
Kun-Che Chang
Spencer Center for Vision Research, School of Medicine, Stanford University,
Palo Alto, CA, United States; Department of Ophthalmology, Louis J. Fox Center
for Vision Restoration, University of Pittsburgh School of Medicine, Pittsburgh,
PA, United States
Umber Cheema
UCL Institute of Orthopaedics & Musculoskeletal Sciences, UCL Division of
Surgery & Interventional Sciences, University College London, London,
United Kingdom
Alan Dardik
Vascular Biology and Therapeutics Program and the Department of Surgery, Yale
University School of Medicine, New Haven, CT, United States
xv
Jugal Kishore Das
Department of Microbial Pathogenesis and Immunology, Texas A&M University
Health Science Center, Bryan, TX, United States
Baljean Dhillon, BMedSci(Hons), BMBS FRCS(Glasg), FRCOphth, FRCS(Ed), FRCPE
Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh
Bioquarter, Edinburgh, United Kingdom; NHS Lothian, Clinical Ophthalmology,
Princess Alexandria Eye Pavilion, Edinburgh, United Kingdom
M. Teresa Donato
Unidad de Hepatologı
´a Experimental, Instituto de Investigacio
´n Sanitaria La Fe,
Valencia, Spain; Departamento de Bioquı
´mica y Biologı
´a Molecular, Facultad de
Medicina, Universidad de Valencia, Valencia, Spain
G.L. Dunbar
Field Neurosciences Institute Laboratory for Restorative Neurology, Central
Michigan University, Mt. Pleasant, MI, United States; Program in Neuroscience,
Central Michigan University, Mt. Pleasant, MI, United States; Department of
Psychology, Central Michigan University, Mt. Pleasant, MI, United States; Field
Neurosciences Institute, St. Mary’s of Michigan, Saginaw, MI, United States
Xixiang Gao
Department of Vascular Surgery, Xuanwu Hospital, Capital Medical University
and Institute of Vascular Surgery, Capital Medical University, Beijing, China;
Vascular Biology and Therapeutics Program and the Department of Surgery, Yale
University School of Medicine, New Haven, CT, United States
Jolanta Gorecka
Vascular Biology and Therapeutics Program and the Department of Surgery, Yale
University School of Medicine, New Haven, CT, United States
Yongquan Gu
Department of Vascular Surgery, Xuanwu Hospital, Capital Medical University
and Institute of Vascular Surgery, Capital Medical University, Beijing, China
Chin Meng Khoo
Yong Loo Lin School of Medicine, National University of Singapore, Singapore,
Singapore; Division of Endocrinology, National University Hospital, Singapore,
Singapore
Kwang-Soo Kim
Department of Psychiatry; Molecular Neurobiology Laboratory, McLean Hospital,
Harvard Medical School, Belmont, MA, United States
Anil Kumar
Department of Microbial Pathogenesis and Immunology, Texas A&M University
Health Science Center, Bryan, TX, United States
xvi Contributors
Hwee Hui Lau
Stem Cells and Diabetes Laboratory, Institute of Molecular and Cell Biology,
A*STAR, Singapore, Singapore; School of Biological Sciences, Nanyang
Technological University, Singapore, Singapore
Ziming Luo
Spencer Center for Vision Research, School of Medicine, Stanford University,
Palo Alto, CA, United States
Michelle Marrero, MD
Department of Neurology, University of Miami Miller School of Medicine, Miami,
FL, United States
Shigeru Miyagawa
Department of Cardiovascular Surgery, Osaka University Graduate School of
Medicine, Suita, Osaka, Japan
Mahmood S. Mozaffari
Department of Oral Biology and Diagnostic Sciences, The Dental College of
Georgia, Augusta University, Augusta, GA, United States
Michael Nahmou
Spencer Center for Vision Research, School of Medicine, Stanford University,
Palo Alto, CA, United States
Manabu Ohyama
Department of Dermatology, Kyorin University Faculty of Medicine, Mitaka-shi,
Tokyo, Japan
Vı
´ctor H. Parraguez
Department of Biological Sciences, Faculty of Veterinary and Animal Sciences,
University of Chile, Santiago, Chile; Department of Animal Production, Faculty of
Agrarian Sciences, University of Chile, Santiago, Chile
Marı
´a Pelecha
´
Unidad de Hepatologı
´a Experimental, Instituto de Investigacio
´n Sanitaria La Fe,
Valencia, Spain
Hao-Yun Peng
Department of Microbial Pathogenesis and Immunology, Texas A&M University
Health Science Center, Bryan, TX, United States
Oscar A. Peralta
Department of Animal Production Sciences, Faculty of Veterinary and Animal
Sciences, University of Chile, Santiago, Chile; Department of Biomedical
Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary
Medicine, Virginia Tech, Blacksburg, VA, United States
Contributors xvii
Milena Pinto, PhD
Department of Neurology, University of Miami Miller School of Medicine, Miami,
FL, United States
Marı
´aPe
´rez-Mato
Neuroscience and Cerebrovascular Research Laboratory, Department of
Neurology and Stroke Center, La Paz University Hospital, Neuroscience Area of
IdiPAZ Health Research Institute, Universidad Auto
´noma de Madrid, Madrid,
Spain
Yijie Ren
Department of Microbial Pathogenesis and Immunology, Texas A&M University
Health Science Center, Bryan, TX, United States
J. Rossignol
College of Medicine, Central Michigan University, Mt. Pleasant, MI, United States;
Field Neurosciences Institute Laboratory for Restorative Neurology, Central
Michigan University, Mt. Pleasant, MI, United States; Program in Neuroscience,
Central Michigan University, Mt. Pleasant, MI, United States
Yoshiki Sawa
Department of Cardiovascular Surgery, Osaka University Graduate School of
Medicine, Suita, Osaka, Japan
Jeffrey S. Schweitzer
Department of Neurosurgery, Massachusetts General Hospital, Harvard Medical
School, Boston, MA, United States
Bin Song
Department of Psychiatry; Molecular Neurobiology Laboratory, McLean Hospital,
Harvard Medical School, Belmont, MA, United States
Jianxun Song
Department of Microbial Pathogenesis and Immunology, Texas A&M University
Health Science Center, Bryan, TX, United States
B. Srinageshwar
College of Medicine, Central Michigan University, Mt. Pleasant, MI, United States;
Field Neurosciences Institute Laboratory for Restorative Neurology, Central
Michigan University, Mt. Pleasant, MI, United States; Program in Neuroscience,
Central Michigan University, Mt. Pleasant, MI, United States
Wei Xuan Tan
Stem Cells and Diabetes Laboratory, Institute of Molecular and Cell Biology,
A*STAR, Singapore, Singapore; Yong Loo Lin School of Medicine, National
University of Singapore, Singapore, Singapore
xviii Contributors
Nguan Soon Tan
School of Biological Sciences, Nanyang Technological University, Singapore,
Singapore; Lee Kong Chian School of Medicine, Nanyang Technological
University, Singapore, Singapore
Adrian Kee Keong Teo
Stem Cells and Diabetes Laboratory, Institute of Molecular and Cell Biology,
A*STAR, Singapore, Singapore; Yong Loo Lin School of Medicine, National
University of Singapore, Singapore, Singapore
Laia Tolosa
Unidad de Hepatologı
´a Experimental, Instituto de Investigacio
´n Sanitaria La Fe,
Valencia, Spain
Cristian G. Torres
Department of Clinical Sciences, Faculty of Veterinary and Animal Sciences,
University of Chile, Santiago, Chile
Liqing Wang
Department of Microbial Pathogenesis and Immunology, Texas A&M University
Health Science Center, Bryan, TX, United States
Yingfeng Wu
Department of Vascular Surgery, Xuanwu Hospital, Capital Medical University
and Institute of Vascular Surgery, Capital Medical University, Beijing, China
Xiaofang Xiong
Department of Microbial Pathogenesis and Immunology, Texas A&M University
Health Science Center, Bryan, TX, United States
Contributors xix
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About the editor
ALEXANDER BIRBRAIR
Dr. Alexander Birbrair received his bachelor’s biomedical degree from Santa Cruz
State University in Brazil. He completed his PhD in Neuroscience, in the field of
stem cell biology, at the Wake Forest School of Medicine under the mentorship of
Osvaldo Delbono. Then, he joined as a postdoc in stem cell biology at Paul Fren-
ette’s laboratory at Albert Einstein School of Medicine in New York. In 2016, he
was appointed faculty at Federal University of Minas Gerais in Brazil, where he
started his own lab. His laboratory is interested in understanding how the cellular
components of different tissues function and control disease progression. His group
explores the roles of specific cell populations in the tissue microenvironment by us-
ing state-of-the-art techniques. His research is funded by the Serrapilheira Institute,
CNPq, CAPES, and FAPEMIG. In 2018, Alexander was elected affiliate member of
the Brazilian Academy of Sciences (ABC), and in 2019, he was elected member of
the Global Young Academy (GYA). He is the Founding Editor and Editor-in-Chief
of Current Tissue Microenvironment Reports and Associate Editor of Molecular
Biotechnology. Alexander also serves in the editorial board of several other interna-
tional journals: Stem Cell Reviews and Reports, Stem Cell Research, Stem Cells and
Development, and Histology and Histopathology.
xxi
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Preface
Alexander Birbrair
1,2
1
Department of Pathology, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil;
2
Department of Radiology, Columbia University Medical Center, New York, NY, United States
This book’s initial title was “iPSCs: Recent Advances.” Nevertheless, because of the
ongoing strong interest in this theme, we were capable of collecting more chapters
than would fit in one single volume, covering induced pluripotent stem cells (iPSCs)
biology from different perspectives. Therefore, the book was subdivided into several
volumes.
This volume “Recent Advances in iPSCs for Therapy” offers contributions by
known scientists and clinicians in the multidisciplinary areas of biological and med-
ical research. The chapters bring up-to-date comprehensive overviews of current ad-
vances in the field. This book describes the use of iPSCs for therapy of several
disorders. Further insights into the biology of these cells will have important impli-
cations for the possible use of iPSCs as cell therapy. The authors focus on the mod-
ern state-of-the-art methodologies and the leading-edge concepts in the field of stem
cell biology. In recent years, remarkable progress has been made in the obtention of
iPSCs and their differentiation into several cell types, tissues, and organs using state-
of-the-art techniques. These advantages facilitated identification of key targets and
definition of the molecular basis of several disorders. Thus, the present book is an
attempt to describe the most recent developments in the area of iPSCs biology,
which is one of the rising hot topics in the field of molecular and cellular biology
today. Here, we present a selected collection of detailed chapters on what we
know so far about the use of iPSCs for therapy of multiple diseases. Fourteen chap-
ters written by experts in the field summarize the present knowledge about iPSCs
therapeutic potential.
Manabu Ohyama from Kyorin University Faculty of Medicine discusses iPSCs
for hair follicle regeneration and the treatment of hair loss disorders. Jeffrey S.
Schweitzer and colleagues from Harvard Medical School describe iPSCs for Parkin-
son’s disease. Julien Rossignol and colleagues from Central Michigan University
compile our understanding of iPSCs as a potential treatment for Huntington’s dis-
ease. Francisco Campos and colleagues from Health Research Institute of Santiago
de Compostela update us with what we know about iPSCs for therapy of ischemic
stroke. Barry Baumel and colleagues from University of Miami Miller School of
Medicine summarize current knowledge on stem cell therapies for Alzheimer’s dis-
ease. Kun-Che Chang and colleagues from Stanford University address the impor-
tance of iPSCs for glaucoma and optic neuropathy. Graham Anderson and
colleagues from University of Edinburgh talk about iPSCs in age-related macular
degeneration. Adrian Kee Keong Teo and colleagues from National University of
Singapore, Singapore, focus on the use of iPSCs to treat type 1 diabetes. Shigeru
Miyagawa and Yoshiki Sawa from Osaka University Graduate School of Medicine
xxiii
give an overview of the use of iPSCs for the treatment of heart failure. Laia Tolosa
and colleagues from Universidad de Valencia present iPSCs for liver diseases. Oscar
A. Peralta and colleagues from University of Chile introduce what we know so far
about potential therapeutic applications of iPSCs for improving fertility in humans
and animals. Alan Dardik and colleagues from Yale University School of Medicine
discuss iPSCs for wound healing. Mahmood S. Mozaffari and colleagues from
Augusta University update us with information on the use of iPSCs for regeneration
of dental complex. Finally, Anil Kumar and colleagues from Texas A&M University
Health Science Center summarize our current status on the use of iPSCs for autoim-
mune diseases.
It is hoped that the articles published in this book will become a source of refer-
ence and inspiration for future research ideas. I would like to express my deep grat-
itude to my wife, Veranika Ushakova, and Ms. Billie Jean Fernandez and Ms.
Elisabeth Brown from Elsevier, who helped at every step of the execution of this
project.
Alexander Birbrair
Editor
xxiv Preface
Strategies to utilize iPS
cells for hair follicle
regeneration and the
treatment of hair loss
disorders
1
Manabu Ohyama
Department of Dermatology, Kyorin University Faculty of Medicine, Mitaka-shi, Tokyo, Japan
Chapter outline
1. Introduction ................................................................................................ ........... 2
2. HF morphology and physiology................................................................................ 4
2.1 Morphogenesis and morphology of HF ....................................................... 4
2.2 Physiology of HF focusing on the hair cycle ............................................... 6
3. Experimental approaches to HF regeneration ........................................................... 6
3.1 Principles of experimental HF regeneration................................................ 6
3.2 Use of hiPSCs for human HF regeneration ................................................. 8
4. Relationship between HF and iPSCs: the HF as a favorable iPSC material .................. 8
5. Regeneration of human HFs using hiPSCs ................................................................8
5.1 Basic principles ...................................................................................... 8
5.2 Induction of folliculogenic KCs from hiPSCs .............................................. 9
5.3 Generation of dermal cells with trichogenic activity from hiPSCs ............... 11
5.4 Assembly of HF structures using hiPSC-derived HF components................ 13
5.5 Use of hiPSCs for HF formation mimicking normal organogenesis.............. 15
6. Strategies for treating hair loss disorders using hiPSCs..........................................15
6.1 Development of pathophysiologically specific therapeutic approaches ........ 15
6.2 Cell-based therapies to reverse HF miniaturization ................................... 15
6.3 Suppression of immune responses involving HFs by hiPSC-derived cells .... 16
7. Conclusion ..........................................................................................................17
List of abbreviations.................................................................................................. 17
Acknowledgment....................................................................................................... 18
References ............................................................................................................... 18
Abstract
The hair follicle (HF) is a mammalian skin structure that provides physical pro-
tection, detects sensation, and enables thermoregulation. In humans, loss of hairs
CHAPTER
1
Recent Advances in iPSCs for Therapy, Volume 3. https://doi.org/10.1016/B978-0-12-822229-4.00013-9
Copyright ©2021 Elsevier Inc. All rights reserved.
on the head greatly affects the physical appearance, leading to altered quality of
life. Thus, vast demand exists for treatments for hair loss disorders, represented by
male and female pattern hair loss or alopecia areata. Regenerative medicine ap-
proaches, including HF bioengineering, can provide remedies for intractable hair
loss diseases caused by irreversible HF destruction. The basis for experimental HF
regeneration has been established in murine. However, human HF reconstitution
adopting that principle has been hampered by the paucity of starting materials,
including HF epithelial stem cells and hair-inductive dermal cells, and the loss of
their HF-prone properties during in vitro expansion. With their high-proliferative
capacity and multipotency, human induced pluripotent stem cells (hiPSCs) should
be useful for HF regeneration. Indeed, hiPSC-derived epithelial and mesenchymal
cells can contribute to in vivo HF-like structure regeneration. hiPSCs can also give
rise to 3D integumentary organ systems comprising HFs which can be isolated and
grafted onto areas of hair loss. Previous studies have focused mainly on the
reproduction of HF structures; however, considering that the regeneration of
complete HFs is not required to correct many common hair loss conditions, hiPSCs
may be better differentiated into trichogenic dermal cells for cell-based therapy. For
immune-mediated hair loss disorders, immunoregulatory cells can be induced from
hiPSCs and inoculated into the affected lesion. In summary, hiPSCs are a promising
cell source not only for HF bioengineering and but also for preparing cell pop-
ulations that may be able to mitigate hair loss.
Keywords:
Alopecia areata; Bulge; Dermal papilla; Dermal sheath; Differentiation; Female pattern
hair loss; Hair cycle; Hair follicle; Hair loss; Induced pluripotent stem cells; Keratinocyte; Male
pattern hair loss; Mesencymal stem/stromal cells; Primary scarring alopecia; Regeneration;
Treatment.
1.Introduction
The hair follicle (HF) is a skin appendage and a characteristic of mammals (Mon-
tagna and Parakkal, 1974). The HF is a multifunctional miniorgan that provides a
physical barrier, acts as sensory machinery, and enables thermoregulation (Paus
and Cotsarelis, 1999;Schneider et al., 2009). HFs can influence social interactions
(Paus and Cotsarelis, 1999). HFs greatly impact physical appearance in humans
(Rose, 2018;Ahluwalia and Fabi, 2019). Accordingly, there is much demand for
treatment of hair loss disorders (Rose, 2018;Endo et al., 2018). The so-called
“androgenetic alopecia” is a common form of hair loss and is subdivided into the
androgen-dependent male pattern hair loss (MPHL) and less androgen-dependent
and more heterogeneous female pattern hair loss (FPHL) (Olsen et al., 2005;
Carmina et al., 2019)(Fig. 1.1). Effective oral medications (for MPHL) and autol-
ogous HF transplantation (for MPHL and FPHL) are readily available; however,
their efficacy is limited for advanced cases (Jung et al., 2014;Adil and Godwin,
2017;Manabe et al., 2018). Alopecia areata (AA) is a commonly encountered
autoimmune-mediated hair loss disease (Strazzulla et al., 2018a)(Fig. 1.2). Despite
the development of potentially groundbreaking drugs for severe cases, no treatment
2CHAPTER 1 Strategies to utilize iPS cells for hair
FIGURE 1.1 Clinical and trichoscopic findings of MPHL and FPHL.
MPHL and FPHL are characterized by the distribution of hair loss areas (the
frontotemporal angle and the vertex in MPHL and the crown and the frontal area with
retention of the frontal hairline in FPHL); however, both conditions share the same
pathology of hair miniaturization as detected by trichoscopic examination.
FIGURE 1.2 Clinicopathological findings of AA.
(a) Typically, AA is characterized by round to oval patches of hair loss, which can merge to
affect larger areas. Total scalp or body hair loss can be observed. (b) AA is an autoimmune
disease. Peribulbar lymphocytic infiltration (arrows) is a histopathological hallmark of AA.
1. Introduction 3
has been approved by the Food and Drug Administration (Strazzulla et al., 2018b;
Jabbari et al., 2018). Furthermore, the treatment options for other forms of intrac-
table alopecia, such as congenital hypotrichosis, primary scarring alopecia leaving
permanent hair loss, and advanced-stage FPHL, are limited (Manabe et al., 2018;
Kinoshita-Ise et al., 2017;Harries et al., 2008).
Methods for experimental HF regeneration have been developed (Ohyama et al.,
2010;Yang and Cotsarelis, 2010;Ohyama and Veraitch, 2013;Ohyama, 2019).
Stem cell/progenitor populations of HF components, which maintain continuous
HF self-renewal cycles, have been identified and isolated as living cells (Ohyama
and Veraitch, 2013;Ohyama, 2019). Use of such plastic and highly proliferative
cells should facilitate full establishment of human HF reproduction technology,
which is hampered by major technical hurdles. Among them, the shortage of starting
human HF-derived materials and the loss of their “HF-prone” intrinsic properties
during in vitro expansion are important (Ohyama and Veraitch, 2013;Ohyama,
2019).
Human induced pluripotent stem cells (human iPSCs or hiPSCs), with their theo-
retically unlimited proliferative nature and the capacity to differentiate into multiple
cell lineages, may provide strategies to overcome the obstacles to human HF regen-
eration (Ohyama and Veraitch, 2013;Ohyama, 2019). In this chapter, HF
morphology and physiology and previously established experimental HF regenera-
tion techniques are first introduced. Next, past and current investigations using
hiPSCs for HF regeneration are described, with a focus on the advantage of hiPSCs
over conventionally adopted HF-derived cell subsets. Finally, the pathophysiology
of hair loss disorders and possible hiPSC-based remedies for individual conditions,
including alternatives to HF regeneration, are explained.
2.HF morphology and physiology
2.1 Morphogenesis and morphology of HF
HF morphogenesis depends on well-orchestrated epithelialemesenchymal interac-
tions (EMIs), which begin with the formation of the placode, a focal thickening
of the fetal epithelium, which is driven by WNT signals arising from the underlying
dermis (Sennett and Rendl, 2012;Saxena et al., 2019)(Fig. 1.3). In response to the
dermal signals from a cell condensate (the dermal condensate) formed beneath the
placode, the fetal epithelium and the basement membrane invaginate into the dermis
to form a hollow cylindrical structure consisting of multiple layers of keratinocytes
(KCs). This structure is surrounded by a collagenous connective tissue sheath (the
dermal sheath; DS), to form the main body of the HF (Figs. 1.3 and 1.4)(Paus
and Cotsarelis, 1999;Sennett and Rendl, 2012;Stenn and Paus, 2001;
Muller-Rover et al., 2001). The epithelial main body is divided into two major
layers; the outer root sheath (ORS) and the inner root sheath, which are separated
by the biochemically distinct companion layer (Fig. 1.4)(Stenn and Paus, 2001;
4CHAPTER 1 Strategies to utilize iPS cells for hair
FIGURE 1.3 HF morphogenesis and the hair cycle.
HF morphogenesis are enabled by well-orchestrated epithelialemesenchymal
interactions (EMI; arrows). HFs self-renew via the hair cycle consisting of anagen (growing
phase), catagen (regressing phase), telogen (resting phase), and exogen (hair-shedding
phase) stages, regulated by DP.
FIGURE 1.4 Morphological and histological characteristics of the HF.
Microdissected human anagen HF (left panel) and histopathological images (middle
panel; low magnification, right panel; close-up image of the bulb portion).
2. HF morphology and physiology 5
Muller-Rover et al., 2001;Sperling, 1991). A highly specialized mesenchymal cell
aggregate, the dermal papilla (DP), is located at the root of the HF bilaterally sur-
rounded by ORS and capped by hair matrix cells, a highly proliferative KC subset,
which divide in response to DP signals to extend the hair shaft (Paus and Cotsarelis,
1999;Stenn and Paus, 2001)(Fig. 1.4). Melanocytes reside around the DPehair ma-
trix border (Sperling, 1991). The HF has several subsidiary structures, such as the
sebaceous gland and the arrector pili muscle (Stenn and Paus, 2001;Muller-Rover
et al., 2001;Sperling, 1991)(Fig. 1.4). The insertion point of the arrector pili muscle
of ORS is termed the bulge and harbors HF epithelial stem cells (Cotsarelis et al.,
1990;Lyle et al., 1998;Ohyama et al., 2006).
2.2 Physiology of HF focusing on the hair cycle
Each adult HF periodically and randomly regenerates itself via the hair cycle. This
consists of the anagen (growing phase), catagen (regression phase), telogen (resting
phase), and exogen (hair-shedding phase) stages (Fig. 1.3)(Paus and Cotsarelis,
1999;Stenn and Paus, 2001;Muller-Rover et al., 2001). The aforementioned struc-
tural characteristics (Fig. 1.4) are those of anagen HFs, which predominate in the
scalp and therefore are clinically important. In the case of human HFs, after several
years of anagen phase, the proximal portion of the HF below the bulge regresses by
apoptosis during the 2e3 weeks of the catagen stage, leaving the distal HF portion
above the bulge (Fig. 1.3)(Paus and Cotsarelis, 1999;Stenn and Paus, 2001;Klig-
man, 1959). In the following telogen stage, which lasts for around 2 months, the
proximal end of the hair shaft is fully keratinized, allowing hair shedding in the sub-
sequent exogen stage (Stenn and Paus, 2001;Higgins et al., 2009). During this pro-
cess, DP relocates near the bulge, presumably enabling DP cells (DPCs) to
communicate with bulge epithelial stem cells to initiate the next round of the hair
cycle by inducing the anagen stage (Panteleyev et al., 2001).
The hair cycle continues throughout life and is maintained by stem/progenitor
cell populations of key HF components, such as bulge epithelial stem cells, subbulge
melanocyte stem cells, and DPC precursors residing in the cup-shaped bottom
portion of the DS (DS cup cells; DSCCs) (Fig. 1.4)(Ohyama and Veraitch, 2013;
Ohyama, 2019;Rahmani et al., 2014;Nishimura et al., 2002).
Dysregulation of the hair cycle, e.g., anagen HFs’ sudden and simultaneous entry
into telogen, results in a pathological increase in hair shedding (telogen effluvium)
(Harrison and Sinclair, 2002). In addition, acceleration of the hair cycle accompa-
nied by shortening of the anagen stage leads to HF miniaturization, which is typical
of MPHL and FPHL (Fig. 1.1)(Manabe et al., 2018;Whiting, 2001;Messenger and
Sinclair, 2006).
3.Experimental approaches to HF regeneration
3.1 Principles of experimental HF regeneration
Various approaches to experimental HF regeneration have been reported (Ohyama
et al., 2010;Yang and Cotsarelis, 2010;Ohyama and Veraitch, 2013;Ohyama,
6CHAPTER 1 Strategies to utilize iPS cells for hair
2019). Most methodologies involve modifications of the same principledcografting
of epithelial and trichogenic dermal (mesenchymal) cells into in vivo permissive
environments, e.g., the subcutaneous spaces or under the kidney capsule, of immu-
nodeficient mice (Fig. 1.5).
The chamber (epithelial and dermal cells are mixed and injected into silicone
chambers transplanted onto the back of a mouse) (Lichti et al., 1993;Weinberg
et al., 1993) and patch (a small amount of cell mixture is grafted into the subcutane-
ous space) (Zheng et al., 2005) techniques are the standard methods (Fig. 1.5).
Because HF formation requires EMIs (Fig. 1.3)(Sennett and Rendl, 2012;Saxena
et al., 2019), the absence of the epithelial or dermal component would prevent HF
regeneration (Ohyama and Veraitch, 2013;Ohyama, 2019). These assays usually
achieve a satisfactory HF yield using murine cells (Weinberg et al., 1993;Zheng
et al., 2005). Of note, use of bulge KCs or freshly isolated trichogenic dermal cells
improves the regeneration rate (Morris et al., 2004;Blanpain et al., 2004;Ito et al.,
2007), suggesting that both the receptivity of epithelial cells to hair-inductive dermal
signals and the intensity of trichogenic activity in dermal cells are pivotal determi-
nants of the magnitude of folliculogenic EMIs (Ohyama and Veraitch, 2013;
Ohyama, 2019).
FIGURE 1.5 Major approaches for HF regeneration.
The chamber assay and the path assay are the gold standard methods for HF
regeneration. In both assays, epithelial and mesenchymal cells are mixed and cografted
in vivo, typically into immunodeficient mice. HFs are formed by spontaneous cellecell
reassembly (arrowheads).
3. Experimental approaches to HF regeneration 7
3.2 Use of hiPSCs for human HF regeneration
Unlike murine HFs, experimental regeneration of human HFs is technically difficult.
Indeed, adaptation of the aforementioned experimental approaches to human HF
regeneration is often limited by the paucity of starting materials and the loss of
cell properties optimal for HF induction during in vitro expansion (Ohyama and
Veraitch, 2013;Ohyama, 2019). hiPSCs, with their theoretically unlimited prolifer-
ative properties and ability to differentiate into multiple cell lineages, show promise
for overcoming these technical hurdles (Ohyama, 2019;Ohyama and Okano, 2013).
4.Relationship between HF and iPSCs: the HF as a
favorable iPSC material
HFs are easily accessible and can be collected less invasively. Multiple cellular com-
ponents of HFs, e.g., ORS KCs, melanocytes, and DPCs, are reported to be reprog-
rammable as hiPSCs (Ohyama, 2019). Because these components can be collected
from a successfully plucked HF (Petit et al., 2012) and the differentiation potential
of hiPSC lines varies according to their origins, the HF may be a favorable material
for hiPSC generation, especially for HF regeneration purposes (Ohyama, 2019).
Intrinsic upregulation of several Yamanaka factors (Oct3/4, Sox2, Klf4, c-Myc)
was observed in melanocytes (Sox2) and in murine DPCs (Sox2 and Klf4)(Utikal
et al., 2009;Tsai et al., 2011). Taking advantage of this preferential gene expression
profile, murine DPCs were reprogrammed into iPSCs by introduction of Oct4 alone
(Tsai et al., 2011). Although human DPCs require transduction of all four Yamanaka
factors to generate hiPSCs, the efficiency of DPC reprogramming was up to 0.03%,
while that of dermal fibroblasts under the same condition was up to 0.01%, support-
ing the suitability of DPCs for hiPSC generation (Higgins et al., 2012;Muchkaeva
et al., 2014). Also, an improved protocol for reprogramming plucked HF-derived
KCs (HFKCs) into hiPSC without feeder cells and serum-containing medium using
integration-free Sendai virus has been established (Re et al., 2018), highlighting the
advantage of the HF as a starting material for hiPSC generation.
5.Regeneration of human HFs using hiPSCs
5.1 Basic principles
A method of regenerating HFs from hiPSCs would theoretically allow infinite HF
self-replication by reprogramming HF cells into hiPSCs (Ohyama, 2019). Expansion
of HF-derived hiPSCs and redifferentiation into HF cell subsets may enable highly
efficient regenerative treatments for hair loss disorders. To that end, two approaches
can be conceived (Fig. 1.6): (1) preparation of individual HF cell components
followed by reconstruction of the HF structure using prepared materials (Ohyama,
2019) and (2) direct HF regeneration from hiPSCs mimicking normal organogenesis
(Takagi et al., 2016). The former would facilitate both optimization of each
8CHAPTER 1 Strategies to utilize iPS cells for hair
component to enhance the HF regeneration efficiency and adjustment of the size of
the regenerated structures. Additional applications, e.g., hiPSC-derived HF-KC and
DPC coculture for drug discovery, would also be possible using this approach. How-
ever, this approach has multiple steps and is technically challenging (Ohyama,
2019). The latter approach would be more physiological, straightforward, and stable.
However, modification of the hiPSC-derived product during the regeneration pro-
cess, e.g., size or shape adjustment, can be more technically difficult than using
the former approach (Ohyama, 2019). We favor the first strategy and have attempted
to generate each HF component by a stepwise approach (Ohyama, 2019;Veraitch
et al., 2013,2017)(Fig. 1.6).
5.2 Induction of folliculogenic KCs from hiPSCs
Based on the protocol for KC generation from human embryonic stem cells using
retinoic acid (RA) and BMP4 (Metallo et al., 2008), well-differentiated KCs have
FIGURE 1.6 Approaches for experimental regeneration of human HFs using hiPSCs.
HF epithelial and hair-inductive dermal cell components are induced from hiPSCs to
construct the main body of HF and DPC aggregates, respectively, which assemble to
regenerate HF structures. Alternatively, hiPSCs-derived embryoid bodies are implanted
in vivo (e.g., into immunodeficient mice) to recapitulate organogenesis to give rise to
organoids containing HFs, which are isolated for downstream applications. 3D-IOS, 3D
integumentary organ system; HF, hair follicles. Three-dimensional images were obtained
in a pilot study conducted under JSPS KAKENHI: JP 16H05370.
5. Regeneration of human HFs using hiPSCs 9
been induced from hiPSCs (Fig. 1.6a, 7a)(Itoh et al., 2011,2013). Neonatal or fetal
human KCs were more receptive to hair-inductive dermal signals than adult KCs,
which formed HF structures in in vivo reconstitution assays (Ehama et al., 2007;
Thangapazham et al., 2014). These observations imply that use of juvenile and pu-
tatively plastic KCs enhances the stability of human HF regeneration.
Because most hair loss patients are adults, providing “aged” KCs, attempts have
been made to endow KCs with HF characteristics by exposure to activators of the
signaling pathways essential to HF development (e.g., WNT, SHH, ectodysplasin-
A, and BMP (Saxena et al., 2019)) or epidermal and basic fibroblast growth factors.
These efforts have met with some success (Sun et al., 2011).
Because hiPSCs recapitulate fetal KC differentiation during their differentiation
into KCs, progenitors with biological properties analogous to those of fetal KCs with
high receptivity to trichogenic dermal signals can be generated (Ohyama and
Veraitch, 2013;Veraitch et al., 2013).
In line with this hypothesis, ectodermal precursor cells (EPCs), which were
generated by exposing hiPSCs to RA-BMP4 and collected without passaging in
KC culture medium, expressed both keratin 14 and 18 (Fig. 1.7b), suggesting
them as being innate (Veraitch et al., 2013). When cocultured with human DPCs,
one EPC line more strongly upregulated HF-related keratin genes than normal hu-
man adult interfollicular KCs (Veraitch et al., 2013). That EPC line also showed
enhanced DPC biomarker gene expression in coexisting DPCs (Veraitch et al.,
2013). These observations supported their ability to elicit trichogenic EMIs. Impor-
tantly, when cotransplanted into immunodeficient mice with hair-inductive mouse
neonatal dermal cells by the patch method, these EPCs, but not normal human adult
FIGURE 1.7 Induction of keratinocytes from hiPSCs for HF regeneration.
(a) Keratinocyte (KC) induction from hiPSCs. Arrow indicates sequential differentiation
into KCs. (b) KCs with some characteristics of precursors expressing keratin (KRT14) 14
and KRT18 were obtained during differentiation from hiPSCs to fully differentiated KCs.
10 CHAPTER 1 Strategies to utilize iPS cells for hair
interfollicular KCs, gave rise to HF structures (Veraitch et al., 2013)(Fig. 1.8a
and b). Immunohistochemical examination detected human-specific signals in the
reconstructed HF structures, indicating that the hiPSC-derived EPCs contributed,
at least in part, to HF regeneration.
Yang and colleagues further improved the aforementioned KC induction proto-
col by integrating EGF supplementation and postinduction selective sorting of cells
highly expressing CD200 and ITGA6 (putative HF epithelial stem cell markers).
The generated cells differentiated into all HF epithelial lineages to regenerate HFs
and the interfollicular epidermis. Importantly, the cells exhibited the ability to differ-
entiate into sebocytes in vitro (Yang et al., 2014).
These findings suggested that folliculogenic KCs with biological properties anal-
ogous to those of multipotent bulge epithelial stem cells and with greater HF regen-
eration capacity could be generated from hiPSCs.
5.3 Generation of dermal cells with trichogenic activity from
hiPSCs
DPCs and adjacent DS cells, especially DSCCs, are the dermal components of HFs
with trichogenic activity (Ohyama et al., 2010;Yang and Cotsarelis, 2010). Unlike
the mouse, collection of these dermal components from human HFs is dependent on
laborious microdissection techniques mainly because the composition of the extra-
cellular matrix hampers enzymatic dissociation (Topouzi et al., 2017). In addition,
DPCs and most likely DSCCs lose their biological properties during in vitro expan-
sion (Ohyama et al., 2012;Higgins et al., 2013). These limitations hamper
FIGURE 1.8 Regeneration of HF structures using hiPSC-derived epithelial cells.
(a) Microdissected cysts regenerated by subcutaneous injection of hiPSC-derived KC
(ectodermal) precursors and hair-inductive mouse neonatal dermal cells contained HF
structures. (b) Histology showing that regenerated structures reproduced HF
components such as the outer root sheath, the inner root sheath, and the hair shaft.
5. Regeneration of human HFs using hiPSCs 11
preparation of sufficiently trichogenic human dermal cells for HF regeneration,
particularly in the context of regenerative medicine. iPSC technology may provide
a strategy to overcome such limitations (Ohyama, 2019).
Because multipotent mesenchymal stem/stromal cells (MSCs) have been suc-
cessfully differentiated from hiPSCs (Fukuta et al., 2014;Hynes et al., 2016), cells
with trichogenicity equivalent to DPCs or DPCCs may be induced from hiPSCs. The
culture conditions necessary to maintain or restore DPC properties, including their
hair inductive capacity, in long-term cultured human DPCs were determined using a
mixture of WNT, BMP, and FGF signaling activators (dermal papilla-activating cul-
ture; DPAC) (Ohyama et al., 2012). hiPSC-derived MSCs can be endowed with DPC
or DPCC-like trichogenicity taking advantage of DPAC. Cultivation of hiPSCs on a
humanized substrate and in MSC medium containing PDGF, TGF-b, and FGF
results in an MSC phenotype, spindle-like morphology (Fig. 1.9a), and the expres-
sion of CD29, CD44, CD90, and CD166. Induced hiPSC-derived cells were able to
differentiate into osteoblasts, adipocytes, and chondrocytes, supporting their MSC-
like differentiation capacity (Veraitch et al., 2017). After treatment with RA and
cultivation in DPAC, cells with some DPC properties (hiPSC-derived DPCs
FIGURE 1.9 Induction of trichogenic dermal cells from hiPSCs.
(a) hiPSC-derived mesenchymal stem/stromal cells (iMSCs) can be induced by seeding
hiPSCs on a humanized substrate and in mesenchymal stem cell medium containing
PDGF, TGF-b, and FGF. iMSCs were treated with retinoic acid and incubated under
dermal papilla activation culture (DPAC) conditions to generate induced dermal papilla-
like cells (iDPCs). (b) Microscopic HF-like structures (arrows) were regenerated by
cotransplantation of LNGFR(þ)THY-1(þ) iMSC-derived iDPCs (labeled cells;
arrowheads) and human keratinocytes.
12 CHAPTER 1 Strategies to utilize iPS cells for hair
[iDPCs]) were generated from hiPSC-derived MSCs (iMSCs) (Fig. 1.9A). The
LNGFR(þ)THY-1(þ) subset of MSCs has been reported to have a high proliferative
capacity and multipotency (Mabuchi et al., 2013). When this population was
selectively isolated from hiPSC-derived MSCs and propagated for DPC induction
(Veraitch et al., 2017), the resulting iDPCs expressed several DPC biomarkers,
such as ALPL,WIF1,HEY1,WNT5A,LRP4, and BAMBI (Veraitch et al., 2017).
Intriguingly, LNGFR(þ)THY-1(þ) iMSC-derived iDPCs reproduced some aspects
of HF-related EMIs when cocultured with human KCs. Furthermore, cotransplanta-
tion of iDPCs with human KCs into immunodeficient mice yielded microscopic
HF-like structures expressing HF-specific keratins, supporting their DPC-like hair
inductive capacity in vivo (Fig. 1.9b)(Veraitch et al., 2017).
These observations imply that fully competent DPC equivalent cells can be
induced from hiPSCs. However, they also indicate the necessity of further improve-
ment of the current induction approach. A global gene expression analysis showed
that the molecular signature of hiPSC-derived DPC equivalent cells was more
similar to that of hiPSC-derived MSCs than that of human DPCs (Veraitch et al.,
2017). The frequency of successful HF-like structure formation was far lower
than that obtained with human DPCs (Veraitch et al., 2017).
Several strategies can be conceived to achieve induction of fully functional DPCs
from hiPSCs. In utero, craniofacial DPCs originate from the neural crest (Wong
et al., 2006;Nagoshi et al., 2008); hiPSC-derived neural crest cells may be more
suitable for differentiation into DPCs (Gnedeva et al., 2015). DPAC can be refined
by incorporating activators of additional signaling pathways essential for HF
morphogenesis, e.g., the EDA, SHH, PDGFA, and TGFbpathways (Sennett and
Rendl, 2012;Saxena et al., 2019). Because cell aggregation potentiates the tricho-
genic activity of DPCs (Higgins et al., 2013;Osada et al., 2007;Ohyama et al.,
2012), hiPSC-derived DPCs can be further aggregated. In our pilot study (conducted
under JSPS KAKENHI: JP 16H05370), the efficiency of cell aggregation seemed to
be different among hiPSC lines, suggesting functional differences. Further upregu-
lation of some DPC marker genes occurs in iDPC aggregates compared with iDPCs
and iMSCs. However, the trend was not consistent for all genes examined, and so the
feasibility and advantages of this approach should be examined in further studies.
5.4 Assembly of HF structures using hiPSC-derived HF components
To date, most in vivo HF reconstitution assays require mice and cell-autonomous
reassembly of transplanted cells (Ohyama, 2019). As a consequence, the HF struc-
tures partially regenerated from hiPSC-derived cells tended to be smaller than bona
fide human HFs (Veraitch et al., 2013,2017). For clinical application, regenerated
HFs are required to be xeno-free and full size.
Abaci et al. developed an in vitro biomimetic developmental approach, which
may solve the aforementioned issues. The investigators used a three-dimensional
(3D)-printing technique to prepare plastic molds, allowing creation of arrays of
microwells mimicking the shape of human HFs in a type I collagen gel containing
5. Regeneration of human HFs using hiPSCs 13
fibroblasts (Abaci et al., 2018). Following seeding of DPCs into the precast micro-
wells, DPC aggregates were spontaneously formed in the bottom and were subse-
quently overlaid with KC columns to form HF-like structures with KCs covering
the gel surface (Abaci et al., 2018). After 1e3 weeks of culture, human skine
equivalent containing HF-like structures expressing HF-specific keratins and
producing hair fibers was obtained. Furthermore, forced expression of Lef-1 in
DPCs and vascularization of the skin equivalent further enhanced the efficiency of
formation (Abaci et al., 2018). As mentioned earlier, methods for the generation
of epithelial and dermal HF components from hiPSCs have been developed
(Veraitch et al., 2013,2017)(Fig. 1.5). A protocol for differentiating melanocytes
from hiPSCs has also been established (Ohta et al., 2011). By assembling individu-
ally prepared hiPSC-derived HF components by the biomimetic developmental
approach, HF structures morphologically and physiologically similar to human
HFs can be bioengineered (Ohyama, 2019). Indeed, the development of a simplified
in vitro 3D HF structure reconstitution method using hiPSC-derived DP (iDPCs) is
underway (Fig. 1.10).
FIGURE 1.10 Approaches for treating hair loss disorders using hiPSCs.
Possible therapeutic approaches using hiPSCs for hair loss disorders based on their
pathophysiology. Noninflammatory scarring hair loss can be treated by grafting
bioengineered HFs (left). For those with remaining miniaturized hair follicles,
supplementation of hiPSC-derived trichogenic dermal cells may be effective (middle).
Immune-mediated hair loss diseases can be ameliorated by hiPSC-derived
immunoregulatory cell subsets (right). The HF-like structure in the upper panel was
bioengineered utilizing hiPSCs in a pilot study under JSPS KAKENHI: JP 16H05370.
14 CHAPTER 1 Strategies to utilize iPS cells for hair
5.5 Use of hiPSCs for HF formation mimicking normal
organogenesis
In theory, hiPSCs can mimic the organogenesis of miniorgans of all lineages
(Ohyama, 2019). Cultured mouse gingivaederived iPSCs formed embryoid bodies
and incubation with Wnt10b predisposed them to a follicular fate (Takagi et al.,
2016). Subsequently, EB clusters were embedded in collagen gel and transplanted
into severe combined immunodeficient mice (clustering-dependent embryoid body
[CDB] transplantation method), resulting in the formation of a 3D integumentary or-
gan system (3D-IOS) containing HFs and sebaceous glands (Takagi et al., 2016)
(Fig. 1.6). The newly formed miniorgans were isolated and transplanted onto the
dorsal aspect of nude mice to reproduce functional skin-bearing HFs (Takagi
et al., 2016). Of note, the HFs showed a normal HF subtype ratio, spacing, and
hair cycle. Establishment of methods for in vitro production of 3D-IOS comprising
full-size human HFs generated from hiPSCs will transform regenerative medicine.
In a recent seminal work, an organoid culture method adopting stepwise TGFb
and FGF signaling modulation was used to reproduce complete skin structure,
including HFs and sebaceous glands, exclusively from hiPSCs (Lee et al., 2020).
6.Strategies for treating hair loss disorders using hiPSCs
6.1 Development of pathophysiologically specific therapeutic
approaches
Hair loss disorders are common dermatological problems. Hair loss can be caused
by external insults, e.g., burn or trauma, which can be irreversible (Ohyama,
2012). The more common hair loss conditions are caused by immuno- or
infection-mediated destruction of HFs or by hair cycle dysregulation. The former
is represented by AA, in which the bulbar portion of anagen-stage HFs is damaged
by an autoimmune reaction (Fig. 1.2) or scarring alopecia caused by destruction of
HF stem cells (Strazzulla et al., 2018a;Ohyama, 2012). The latter is typified by
MPHL and FPHL, which are characterized by acceleration of the hair cycle with
HF miniaturization (Jung et al., 2014;Adil and Godwin, 2017;Manabe et al.,
2018)(Fig. 1.1). Unlike the hair loss caused by physical HF damage, these subtypes
of hair loss cannot be cured without resolving the underlying condition. Importantly,
in most hair loss conditions, the HF structures are not destroyed (Ohyama, 2019).
Accordingly, the most straightforward regenerative approach, i.e., bioengineering
of complete HFs from hiPSCs and transplantation may not always be required
(Ohyama, 2019)(Fig. 1.10).
6.2 Cell-based therapies to reverse HF miniaturization
MPHL and FPHL are frequently encountered dermatological problems clinically
characterized by a decrease in hair density within a defined scalp area and
6. Strategies for treating hair loss disorders using hiPSCs 15
histologically by HF miniaturization (Jung et al., 2014;Adil and Godwin, 2017;
Manabe et al., 2018)(Fig. 1.1). For MPHL, pharmacotherapiesdincluding topical
application of minoxidil and oral administration of anti-5areductases or autologous
HF transplantation of androgen less-sensitive occipital HFsdare effective but some-
times unsatisfactory (Manabe et al., 2018). In addition, in part because the pathogen-
esis of FPHL is unclear, the available treatment options for this condition are limited
(Manabe et al., 2018;Fabbrocini et al., 2018). Because HF structures are miniatur-
ized in these conditions, their enlargement by means of cell-based therapies is a
feasible and relatively inexpensive option (Ohyama, 2019)(Fig. 1.10).
DPCs and DSCCs have the ability to induce hair growth (Ohyama et al., 2010;
Yang and Cotsarelis, 2010;Ohyama and Veraitch, 2013). Of note, transplanted
DSCCs but not DPCs induced HFs in a human subject, indicating that DSCCs
can regenerate do novo DPs (Reynolds et al., 1999). DSCCs migrated into DP struc-
tures in vivo in experimentally regenerated human hairy skin (Yoshida et al., 2019).
Because the hair shaft width is positively correlated with DP size (Chi et al., 2013),
these observations suggest that hair loss can be reversed by DSCC-based therapies.
Indeed, Tsuboi et al. demonstrated that injection of autologous DSCCs into areas
affected by MPHL and FPHL improved the hair density and diameter without any
serious adverse events (Tsuboi et al., 2020). Although DPCCs adjacent to DPCs
are regarded as DPC precursors, the molecular signatures of the two cell populations
are different, suggesting both a close relationship and biological distinctness
(Niiyama et al., 2018). Because some DPC properties can be induced in hiPSC-
derived mesenchymal cells, differentiation of hiPSCs into cells functionally equiv-
alent to DSCCs would enable cell-based therapy of hair loss disorders involving hair
cycle dysregulation and hair miniaturization.
Telogeneanagen conversion can be accelerated by MSC-derived extracellular
vesicles and human HF growth can be promoted by adipose-derived stem cells
and their secretory factors (Yamao et al., 2015;Rajendran et al., 2017;Won et al.,
2017). Therefore, hiPSCs may ameliorate hair loss by inducing the secretion of
similar factors by hiPSC-derived differentiated cells.
6.3 Suppression of immune responses involving HFs by hiPSC-
derived cells
Hair loss can result from unwanted immune responses involving HFs. In AA, peri-
follicular inflammation as a consequence of an autoimmune reaction to an HF
component is mainly observed around the bulb of anagen HFs (Strazzulla et al.,
2018a;Whiting, 1995)(Fig. 1.2). Because HF stem cells reside in the bulge area,
which is not affected in AA, HF regeneration can be expected when inflammatory
changes are sufficiently suppressed. In primary scarring alopecia, such as lichen
planopilaris or chronic cutaneous lupus erythematosus, immune responses involve
the bulge area and lead to the loss of stem cells (Ohyama, 2012;Harries and
Paus, 2010). In the latter form of alopecia, permanent hair loss can be observed
and so HF regeneration could be beneficial (Ohyama, 2019). Obviously, simple
16 CHAPTER 1 Strategies to utilize iPS cells for hair
HF regeneration cannot be a treatment option for such conditions, because trans-
planted HF structures will be damaged unless inflammatory changes involving
HFs are suppressed.
Mouse iPSCs can be differentiated into functional autoantigen-specific regulato-
ry T (T reg) cells, which inhibit the activity of pathogenic immune cells in a murine
model of type 1 diabetes (Haque et al., 2019), implying that iPSC-derived cells can
immunomodulate pathogenic responses. In this regard, hiPSCs may provide novel
therapeutic approaches for immune-mediated hair loss disorders, e.g., AA and pri-
mary scarring alopecia. Indeed, hiPSC-derived MSCs exhibited immunosuppressive
activity in vitro and repopulated IL-10-producing FoxP3-positive Tregs in vivo in
immune-deficient NSG mice (Roux et al., 2017), suggesting the therapeutic poten-
tial of hiPSC-derived immunosuppressive cells in an inflammatory subset of hair
loss diseases (Fig. 1.10).
Another possible but rather chimerical approach is the regeneration of immuno-
suppressive HFs. HFs, by producing chemokines, play pivotal roles in the local mod-
ulation of immune responses (Nagao et al., 2012). Transplantation of hiPSC-derived
HFs producing immunosuppressive cytokines may be effective for intractable auto-
immune scarring alopecia.
7.Conclusion
A technological basis for human HF regeneration using hiPSCs has been estab-
lished. In addition, hiPSC-derived HFs may facilitate dissection of the complex
pathophysiology of a variety of hair loss disorders, enabling discovery of drugs
effective against currently intractable hair loss diseases.
List of abbreviations
3D-IOS 3D integumentary organ system
AA alopecia areata
DP dermal papilla
DPC dermal papilla cell
DS dermal sheath
DSCC dermal sheath cup cell
EMI epithelial mesenchymal interaction
EPC ectodermal precursor cell
FPHL female pattern hair loss
HF hair follicle
hiPSC human iPSC
iDPC iMSC-derived dermal papilla-like cell
iMSC hiPSC-derived mesnchymal stem/stromal cell
iPSC induced pluripotent stem cell
KC keratinocyte
List of abbreviations 17
MPHL male pattern hair loss
MSC mesenchymal stem/stromal cell
ORS the outer root sheath
RA retinoic acid
Acknowledgment
This author would like to thank Ms. Aki Tsukashima, Momoko Kimishima, Yoshimi Yama-
zaki, and Dr. Masaharu Fukuyama for their technical assistance. The writing of this manu-
script and the generation of preliminary data presented in the manuscript are supported by
JSPS KAKENHI Grant Number JP 16H05370.
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22 CHAPTER 1 Strategies to utilize iPS cells for hair
iPSCs and cell therapy for
Parkinson’s disease 2
Jeffrey S. Schweitzer
1
, Bin Song
2
, Kwang-Soo Kim
2
1
Department of Neurosurgery, Massachusetts General Hospital, Harvard Medical School, Boston,
MA, United States;
2
Molecular Neurobiology Laboratory, McLean Hospital, Harvard Medical
School, Belmont, MA, United States
Chapter outline
1. Introduction ................................................................................................ .........25
2. PD as an established target for cell therapy........................................................... 26
3. Developing a cell therapy approach for PD ............................................................ 27
3.1 Disease models for tests of efficacy ...................................................... 27
3.2 Sources of transplantable tissue ........................................................... 27
3.3 Fetal cell transplantation as early proof-of-concept studiesdlessons and
challenges .......................................................................................... 27
3.4 hESC cell sources for PD therapy.......................................................... 28
3.5 Non-iPSC autologous cell sources ......................................................... 29
3.6 iPSC-derived DA cells .......................................................................... 29
3.7 Identification and generation of clinical grade hiPSCs ............................ 30
3.8 Genomic stability of hiPSCs and epigenetics ......................................... 31
3.9 Generation of transplantable mDA progenitors from patient-derived
hiPSCs ............................................................................................... 31
3.10 Safety of hiPSC-derived cell grafts........................................................ 33
4. Clinical factors .................................................................................................... 33
4.1 Variable reprogramming potential and differentiation specificity .............. 34
4.2 Variable response of different forms of the disease ................................. 34
4.3 Expression of the disease within the graft.............................................. 34
4.4 Implantation target site and cell number ............................................... 35
4.5 Graft survival and outgrowth ................................................................. 35
4.6 Surgical technique .............................................................................. 35
5. Toward clinical implementation of iPSC-based therapy for PD ................................. 37
5.1 Production of clinical-grade iPSCs ........................................................ 37
5.2 Optimization and standardization of in vitro differentiation protocols ....... 37
5.3 Patient selection and follow-up monitoring criteria ................................. 38
5.4 Regulatory and socioeconomic acceptance ............................................ 38
6. Perspective .........................................................................................................38
Abbreviations............................................................................................................ 39
CHAPTER
23
Recent Advances in iPSCs for Therapy, Volume 3. https://doi.org/10.1016/B978-0-12-822229-4.00005-X
Copyright ©2021 Elsevier Inc. All rights reserved.
Acknowledgments ..................................................................................................... 39
References ............................................................................................................... 39
Abstract
Parkinson’s disease is a common degenerative disorder of the brain for which the
cellular basis is in large part the loss of dopaminergic neurons from the midbrain,
making it an attractive target for cell replacement therapy. Available medical and
surgical treatments have significant drawbacks, further encouraging such efforts.
Attempts to achieve this with various sources of dopaminergic and catecholamin-
ergic tissue have been undertaken over several decades, and the use of human fetal
midbrain, although associated with numerous problems, has suggested the possi-
bility of significant benefit from cell replacement therapy. iPSC technology has now
provided a possible solution to many of these problems. We present the history of
cell replacement therapy for Parkinson’s disease and the various current methods
and strategies to create, differentiate and develop iPSC as a clinically applicable
therapeutic option for both autologous and allogeneic use.
Keywords:
6-OHDA model; Autologous transplantation; Cell reprogramming; Cell therapy; Deep
brain stimulation; Dopamine; Dyskinesia; Embryonic stem cells; Fetal tissue transplantation;
Immunogenicity; In vitro differentiation; Induced pluripotent stem cells; Levodopa therapy; Midbrain
dopaminergic progenitors; Parkinson’s disease.
Parkinson’s disease (PD) is the most common movement disorder and the second
most prevalent neurodegenerative disorder (de Lau and Breteler, 2006). As the pop-
ulation in the developed world ages, this creates an escalating burden on society both
in economic terms and in quality of life for these patients and for the families that
support them. Although currently available pharmacological or surgical treatments
may significantly improve the quality of life of many PD patients, these are symp-
tomatic treatments that do not slow or stop the progressive course of the disease.
Because motor impairments in PD largely result from loss of midbrain dopamine
neurons in the substantia nigra pars compacta (SNpc), PD has long been considered
to be one of the most promising target diseases for cell-based therapy. Indeed,
numerous clinical and preclinical studies using fetal cell transplantation have pro-
vided proof of concept that cell replacement therapy may be a viable therapeutic
approach for PD (Sonntag et al., 2018). However, the use of human fetal cells as
a standardized therapeutic regimen has been fraught with fundamental ethical, prac-
tical, and clinical issues, prompting scientists to explore alternative cell sources. A
decade ago, Shinya Yamanaka and his colleagues reported the groundbreaking
finding that somatic cells can be reprogrammed to early embryonic-like induced
pluripotent stem cells (iPSCs) (Takahashi and Yamanaka, 2006). Since that time,
such iPSCs have been the subject of extensive research, leading to tremendous
advancement in our understanding of this novel class of stem cells and promising
great potential for regenerative medicine. In this review, we will discuss the pros-
pects and challenges of stem cell-based cell therapy for PD.
24 CHAPTER 2 iPSCs and cell therapy for Parkinson’s disease
1.Introduction
Parkinson’s disease (PD) is a common neurodegenerative disorder, affecting about
w1% of the population over the age of 60 (de Lau and Breteler, 2006). This number
will increase as the aging population grows. PD is characterized by the loss of
midbrain dopamine (mDA) neurons, and dopamine (DA)-replacement therapy
(e.g., L-dopa and/or DA agonists) has for decades remained the gold standard treat-
ment for the motor symptoms of the disease. While drug therapy can significantly
improve the quality of life, significant side effects such as dyskinesia, hypotension,
and gastrointestinal symptoms limit its usefulness in many patients (Kang and Fahn,
1988;Weiss et al., 1971).
A number of surgical interventions have also been used to treat symptoms of PD.
These interventions developed out of serendipitous observations and used radiofre-
quency energy (Kopyov et al., 1997), cryogenic means (Rand, 1968), or radiosurgery
(Friedman et al., 1996) to create lesions interrupting control loops in the basal
ganglia. Such a strategy has gained some renewed currency with the availability
of real-time MRI monitored ablation techniques such as focused ultrasound (Hariz,
2013;Schlesinger et al., 2015). Such techniques create permanent new pathology to
counterbalance symptoms of the existing dysfunction. Deep brain stimulation
(Okun, 2012) is a less destructive and more reversible alternative with increasingly
sophisticated flexibility, but is nonetheless a palliative procedure that carries surgical
risk and a permanent dependence on implanted hardware.
Augmentative gene therapies represent another area of ongoing therapeutic
research. Genes the expression of which may modify disease symptoms (e.g.,
GAD to produce GABA in the subthalamic nucleus or AADC to increase DA pro-
duction in the striatum) are stereotactically delivered using viral vectors (Bartus
et al., 2014;Kordower and Bjorklund, 2013;LeWitt et al., 2011). Gene strategies
used to date are also palliative and do not prevent or replace the progressive loss
of mDA neurons; their long-term usefulness remains uncertain.
An ideal treatment would clearly involve early diagnosis and intervention to pre-
vent the pathophysiology that underlies the disease; but once cell loss has progressed
to the point where motor symptoms are apparent, restoration and long-term recovery
of this impaired motor function may better be accomplished by replacing the
missing cell population.
In 2006, Yamanaka et al. reported the ability to induce pluripotency in termi-
nally differentiated mouse somatic cells using unique reprogramming factors
(Takahashi and Yamanaka, 2006). They and other workers soon thereafter demon-
strated that human somatic tissues could be reprogrammed into human induced
pluripotent stem cells (iPSCs) using similar methods (Takahashi et al., 2007;Yu
et al., 2007;Park et al., 2008). This technique, the focus of this volume, allowed
for the creation either of standard stem cell lines or of individualized, autologous
patient-specific stem cells, either of which could then be treated with appropriate
1. Introduction 25
differentiation protocols to produce any desired cell type. This approach circum-
vents the ethical and logistical issues involved with the use of human fetal tissue
or embryonic stem cells. It potentially allows the production of an unlimited source
of cells for differentiation. When used to produce autologous iPSC, it may reduce
or eliminate the need for immunosuppression inherent in the use of embryonic stem
cells or xenogeneic sources.
In this chapter, we review the history and current status of cell therapy for PD,
including the use of fetal tissue, embryonic stem cells (hESCs), and hiPSCs. We
introduce the potential of iPSC to provide a basis for autologous, personalized
cell therapy as well as standardized cell lines for more general use and the necessary
considerations to achieve this potential.
2.PD as an established target for cell therapy
While PD involves both motor and nonmotor, non-DA-responsive symptoms, the
motor symptoms including resting tremor, rigidity, bradykinesia, and postural
instability are arguably the more debilitating aspects and those which correlate
best with histopathological changes related to the loss of A9-type mDA neurons
in the substantia nigra pars compacta (SNpc) (Kalia and Lang, 2015). Decreased
dopaminergic input to the striatum as a result of this loss is believed responsible
for the motor expression of the disease. This understanding led to various ap-
proaches to replacing the lost innervation with catecholaminergic cells including
autologous adrenal medulla, autologous carotid body, retinal pigment epithelium
as well as mDA neurons from human fetal or xenogeneic fetal sources (Allan
et al., 2010;Barker et al., 2015a,b;Bjorklund and Kordower, 2013;Lindvall,
2016). The largest body of accumulated data exists for the human fetal mDA
transplants, placed into the striatum (normally the innervation target of the
mDA neurons). This work, though with mixed results, served to establish proof
of concept (Piccinietal.,1999;Hagell et al., 1999;Kefalopoulou et al., 2014),
as benefit was clearly seen in a subgroup of patients. Problems with surgical tech-
nique, specificity, and handling of the fetal tissue, as well as obvious ethical and
logistical problems contributed to a moratorium on this work in the first decade
of this century. The availability of induced stem cell technology, among other fac-
tors, has led to a current resurgence of interest in the concept. Current reprogram-
ming technology may permit the production of iPSC of sufficient quality and safety
for clinical use (Takahashi and Yamanaka, 2006). Differentiation protocols to pro-
duce in vitro specific desired dopaminergic cell populations from such iPSC have
now demonstrated safety and efficacy in animal models of PD. This may allow
iPSC to become the basis for both standard allogeneic and personalized autologous
cell lines and for the production of therapeutic mDA cell transplantation sources
from them.
26 CHAPTER 2 iPSCs and cell therapy for Parkinson’s disease
3.Developing a cell therapy approach for PD
3.1 Disease models for tests of efficacy
There exists no completely satisfactory animal model for PD in which potential cell
therapy can be fully examined for its effects on all aspects of the disease, especially
nonmotor symptoms. Most commonly used models have involved neurotoxic lesion-
ing (e.g., 6-OHDA in rodents or MPTP in nonhuman primates), which adds some
complexity and may alter the host environment in ways not dissimilar to the disease.
Thus, outcome studies of any tissue transplantation need to be considered with
caution, particularly with regard to nonmotor symptoms and to symptoms such as
tremor that have proven difficult to reproduce in such models.
3.2 Sources of transplantable tissue
Among tissues that have been used historically to reintroduce dopaminergic inner-
vation to the motor striatum include adrenal medulla (Goetz et al., 1990), carotid
body (Arjona et al., 2003), and retinal pigment epithelium (Gross et al., 2011). Initial
reports of benefit with the adrenal medulla work later appeared problematic (Ahl-
skog et al., 1990), and such autologous sources of catecholaminergic tissue have
not excited much further research interest.
3.3 Fetal cell transplantation as early proof-of-concept
studiesdlessons and challenges
The use of human fetal midbrain tissue transplantation into Parkinson’s patients
showed promise in early open-label trials pioneered first in Sweden in the 1980s
(Lindvall, 1989). Long-term follow-up showed that some patients gained symptom
relief lasting two decades or more (Piccini et al., 1999;Hagell et al., 1999;Kefalo-
poulou et al., 2014;Kordower et al., 2008;Li et al., 2008;Mendez et al., 2008).
Follow-up data included postmortem demonstration of grafted tissue that had inte-
grated and innervated the host striatum up to 24 years posttreatment (Li et al., 2016).
However, NIH-funded double-blind, sham-controlled clinical trials failed to show
clear benefit overall and resulted in the development of undesirable side effects
such as dyskinesia in some patients (Freed et al., 2001;Olanow et al., 2003), the eti-
ology of which is not clear. Nonetheless, these trials suggested possible benefit for
certain subpopulations, and the reasons for the overall lack of success involved many
potentially remediable technical and logistical factors (Barker et al., 2015a,b;Bjor-
klund and Kordower, 2013;Lindvall, 2016;Freed et al., 2001;Olanow et al., 2003).
Thus, although these unsatisfactory clinical trial outcomes resulted in decreased in-
terest in the approach in the years following those studies, continued long-term
follow-up of some of the original fetal transplant patients showed that some derived
persistent and significant benefit, maintaining the hope that the cell therapy approach
3. Developing a cell therapy approach for PD 27
still had validity in principle. Interestingly, long-term autopsy results also showed
a-synuclein-containing Lewy bodies in 10%e15% of graft-derived DA neurons
(Kordower et al., 2008;Li et al., 2008,2010,2016;Kurowska et al., 2011;Chu
and Kordower, 2010;Kordower and Brundin, 2009), with implications for etiology
and long-term treatment.
3.4 hESC cell sources for PD therapy
A source of dopaminergic cells for PD therapy that is safe, efficacious, logistically
simple to access, and is free of ethical constraints would clearly justify a reexami-
nation of the possible benefits of cell therapy. The arrival of stem cell therapy offered
a new approach to the treatment of PD that would overcome a number of the prob-
lems related to human fetal tissue sources (Parmar et al., 2020). The first hESC lines
were established In 1998 by Thompson et al., and these lines proved readily adapt-
able to the production of mDA cells using various protocols including cocultures,
monolayers, and suspensions (Sonntag et al., 2018;Arenas et al., 2015). These pro-
tocols utilized mDA-specific morphogens and growth factors including SHH, FGF8,
BDNF, GDNF, and dcAMP (Wagner et al., 1999;Roy et al., 2006). Further research
has moved toward the identification of small molecules and peptides to manage spe-
cific developmental pathways in order to optimize mDA induction (Kirkeby et al.,
2012;Morizane et al., 2011;Fasano et al., 2010;Chambers et al., 2009), focusing
on two critical regulatory loops (WNT1-LMX1A and SHH-FOXA2) (Kittappa
et al., 2007;Chung et al., 2009;Joksimovic et al., 2009). To replace the beneficial
effect of the coculturing used in earlier protocols, dual inhibition of SMADs (target-
ing BMP and TGFbsignaling) plus dual activation of WNT and SHH pathways ap-
pears to optimize the efficiency and specificity of mDAN differentiation (Kirkeby
et al., 2012,2017;Morizane et al., 2011;Xi et al., 2012;Kriks et al., 2011;Grealish
et al., 2014;Kikuchi et al., 2011, 2017a,b; Samata et al., 2015,2016;Sundberg et al.,
2013;Chen et al., 2016). Current differentiation protocols developed with the use of
hESC are capable of producing in vitro neurons that express tyrosine hydroxylase
(TH) (the rate-limiting enzyme for DA synthesis) as well as other defining charac-
teristics of mDA neurons including LMX1A, PITX3, FOXA2, NURR1, EN-1, and
the DA transporter (DAT). mDA neurons comprise multiple populations with
distinct locations, connections, and electrophysiology (Vogt Weisenhorn et al.,
2016). These include markers such as GIRK2 (A9 DA population phenotype) or cal-
bindin (A10 population phenotype), with physiological properties similar to equiv-
alent fetal tissueederived neurons (Grealish et al., 2014). Of these subtypes, the A9
DA cells are preferentially lost in PD and are also characterized by spontaneous
firing, while A10 cells of the ventral tegmentum are less affected by PD degenera-
tion. At present, in vitro differentiation protocols that efficiently produce A9 and not
A10 have not been described. Whether improved purity of this type would improve
clinical results is not known.
28 CHAPTER 2 iPSCs and cell therapy for Parkinson’s disease
3.5 Non-iPSC autologous cell sources
Direct use of endogenous adult cell populations such as mesenchymal stem cells to
treat PD has achieved little success (Venkataramana et al., 2010), despite an unfor-
tunate popularization in the pseudo-scientific arena. Other adult stem cell sources
from the CNS itself have been proposed (Xu et al., 2013;Bloch et al., 2014;Cave
et al., 2014;Wang et al., 2012) but present obvious logistical barriers to routine use.
Recent efforts have shown some success in direct conversion of adult somatic
cells such as glia or transplanted fibroblasts into other cell lineages without interme-
diate generation of PSCs, e.g., induced neurons (iNs) (Yoo et al., 2017;Rivetti di Val
Cervo et al., 2017;Grealish et al., 2016;Tanabe et al., 2015;Vierbuchen et al.,
2010). This has produced human induced dopamine neurons (iDNs) with phenotypic
and functional characteristics promising clinical utility (Caiazzo et al., 2011;Del-
l’Anno et al., 2014), including long-term survival when grafted into 6-OHDA treated
animal models and resulting rescue of motor function. The technique has thus far
been less effective than PSC-based strategies, and safety and stability are still works
in progress (Kim, 2011;Mertens et al., 2016;Ang and Wernig, 2014;Xu et al.,
2015).
3.6 iPSC-derived DA cells
Despite progress with differentiation protocols, the use of hESCs still requires at
some level the fertilized eggs from donors and the destruction of early embryos
and does not obviate these problems. Cell lines ultimately derived from these sour-
ces such as H7 or H9 (Thomson et al., 1998) alleviate some of these concerns and are
under development as a therapy option (Tabar, 2016). A clear drawback of alloge-
neic tissue sources for cell therapy is the potential for immunologically mediated
host rejection of the grafted tissue. The traditional view that the brain is an immu-
nologically privileged area is overly simplistic, and the role of and need for immu-
nosuppression in the fetal tissue studies were not determined (Wenker et al., 2016).
In the generally older and more debilitated population at which PD cell therapy is
aimed, any need for a significant period of immunosuppression carries appreciable
risk. The use of hiPSC offers an opportunity to circumvent this issue using autolo-
gous cells to manufacture the therapeutic product. A source that could also circum-
vent the use of immunosuppression would be yet more advantageous. Research in
nonhuman primates suggests that mDANs differentiated from either autologous
iPSC or from MHC-matched phenotypes indeed showed significantly reduced
immunogenicity (Morizane et al., 2013,2017), though certain cell types may differ
in this respect and more evidence is needed (Liu et al., 2017;Aron Badin et al.,
2019).
An iPSC cell source holds the advantages of ESCs without the ethical issues and
allows for the options to create either autologous or allogeneic sources for mDA pro-
duction. There is some suggestion of improved symptomatic efficacy using DA neu-
rons derived from autologous versus allogeneic iPSC (Morizane et al., 2013), in
3. Developing a cell therapy approach for PD 29
addition to the theoretical advantages of reduced immunogenicity. The cost in time
and financial resources required to individualize iPSC production, however, is a ma-
jor consideration, as are potential differences in the reprogramming efficiency and/
or differentiation potential of cells from different individuals. Thus, use of both
autologous and allogeneic iPSC for the treatment of PD may have clinical relevance.
Compromise approaches have been described, such as the establishment of banks of
HLA-matching hESC or hiPSC lines (Jacquet et al., 2013;Bravery, 2015;Solomon
et al., 2015) to cover the majority of a population, as is in progress in Japan at the
time of this writing (Morizane et al., 2017). This strategy may be less practical
for regions with ethnically diverse populations, and the extent to which it obviates
the need for immunosuppression remains unclear (Aron Badin et al., 2019). There-
fore, the goal of developing efficient methods to generate unlimited autologous stem
cell sources that would allow truly personalized cell therapy for PD remains
appealing (Sonntag et al., 2018)(Fig. 2.1).
3.7 Identification and generation of clinical grade hiPSCs
Any product intended for clinical use as a long-term tissue implantation therapy, as
would be required for cell therapy for PD, requires a level of safety much greater
than for cells used for developmental and mechanistic studies only; principally,
avoiding the use of potentially harmful viral agents, genetic material that can inte-
grate or disrupt host physiology, and cells with any appreciable neoplastic potential.
Thus, methods to generate hiPSCs must be used that avoid these fundamental
FIGURE 2.1
Schematic illustration of an iPSC-based strategy for the treatment of Parkinson’s disease.
Fibroblasts or other readily obtainable cells are harvested from a patient and used as an
autologous source for reprogramming to become pluripotent stem cells. These are then
differentiated in vitro into midbrain dopaminergic progenitors, the committed precursors
of mature dopaminergic neurons. Safety and quality control testing is performed in vitro
and both safety and efficacy may be tested in animal models prior to implantation.
Optionally, cells may be frozen at the iPSC or mDAP stages for banking and later use.
30 CHAPTER 2 iPSCs and cell therapy for Parkinson’s disease
problems. Some such options have included the use of nonviral and/or nonintegrat-
ing vectors, e.g., adenovirus (Zhou and Freed, 2009), Sendai virus (Fusaki et al.,
2009), or temperature-sensitive Sendai virus (Ban et al., 2011); the use of episomal
vectors (Yu et al., 2009;Okita et al., 2011), RNAs (Warren et al., 2010) or self-
replicative RNAs (Yoshioka et al., 2013) or miRNA (Miyoshi et al., 2011); or direct
protein delivery methods (Kim et al., 2009). Moreover, the good manufacturing pro-
cess (GMP)-compatible conditions that are required for clinical application necessi-
tate feeder-free and xeno-free culture conditions (Oliveira et al., 2014;Silva et al.,
2015;Wang et al., 2015).
3.8 Genomic stability of hiPSCs and epigenetics
As emphasized earlier, to establish novel hiPSCs-based therapy as a viable therapeu-
tic option for human diseases, a considerable margin of safety must be demonstrated,
and the issue of genomic integrity is critical. Mutations and genetic damage that
could result in uncontrolled proliferation, tumor formation, or unstable differentia-
tion must be demonstrably absent from a potential cell therapy product. In fact, such
issues became a factor in the first clinical trial involving hiPSCs in Japan when mu-
tations were identified in one of the two patients initially enrolled (Garber, 2015;
Mandai et al., 2017). Chromosomal variations in hiPSCs may be present in the orig-
inal donor cells or may occur and increase during reprogramming, passaging,
culturing, or differentiation. Methods to screen for such abnormalities include kar-
yotyping, SNP genotyping, mRNA, whole exome and genome sequencing, and
genome-wide methylome analysis (Schapira and Jenner, 2011;Redmond et al.,
2008;Clarkson et al., 1998;Schweitzer et al., 2020). As evident in the first iPSC-
based clinical trial (Mandai et al., 2017), safe clinical use will require the identifica-
tion and exclusion of hiPSC lines with functionally significant traits of this type
including cancer-causing mutations or disabling epigenetic modifications.
3.9 Generation of transplantable mDA progenitors from patient-
derived hiPSCs
While the protocols for mDA differentiation in vitro developed for ESC may be
adapted directly to hiPSC lines, these cells exhibit variable and in some cases rela-
tively inefficient differentiation potential compared to hESC lines (Hu et al., 2010;
Feng et al., 2010;Ebert et al., 2009), and it is therefore essential to establish the prac-
ticality of differentiating specific hiPSC lines into mDA neurons with therapeutic ef-
ficacy as well as with the safety factors already discussed. These mDA neurons must
be proved to effectively reinnervate the host brain and rescue motor behavior deficits
in established animal models of PD (Kriks et al., 2011;Song et al., 2020;Nolbrant
et al., 2017;Kikuchi et al., 2017a,b). Optimization and standardization of protocols
to create such cells are currently a topic of active research (Fig. 2.2).
The in vitro differentiation process that creates mDA neurons is not directly syn-
chronous with in vivo embryology. Furthermore, the host environment into which
3. Developing a cell therapy approach for PD 31
the cells are injected is not an embryonic, but an adult one. Therefore, the optimal
stage of in vitro differentiation at which cells should be harvested for in vitro use
must be determined. Less differentiated cells may raise the risk of teratoma forma-
tion (Bjorklund et al., 2002) while completely mature, differentiated neurons may be
less able to survive the transplantation process (Hedlund et al., 2008;Jonsson et al.,
2009;Moon et al., 2013). Thus, mDA progenitor cells, a stage at which development
is committed to a single fate but not yet fully expressed, may represent the best cell
source. Transplantation of hiPSC-derived mDA progenitors FACS-sorted by CORIN
or LRTM1 has been shown to improve significantly motor behavior in 6-OHDA
lesioned rats without any teratoma formation (Samata et al., 2016;Doi et al.,
2014), and CORIN-positive DA progenitors can be expanded in vitro to produce
mDA neurons with minimal serotonergic contamination (Sundberg et al., 2013).
FIGURE 2.2
(A) Schematic timeline for a typical differentiation protocol. Such protocols were generally
developed using embryonic stem cells and have been applied to the functionally
equivalent iPSCs. The “dissociation” step indicates passaging of the cells at this time
point. AA, Ascorbic acid; b-mer, beta-mercaptoethanol; BDNF, Brain-derived
neurotrophic factor; CHIR, CHIR99021; dbcAMP, dibutyryl cyclic adenosine
monophosphate; FGF-8, Fibroblast growth factor 8; GDNF, Glial cell lineederived
neurotrophic factor; KSR, knock out serum replacement; LDN, LDN193189; L-Glu,L-
glutamine; NEAA, Nonessential amino acid; PMN, Purmorphamine; QC, Quercetin; SB,
SB431542; SHH, Sonic Hedgehog; TGF-b3, transforming growth factor beta 3. Numbers
represent concentrations in ng/mL and those in parentheses represent in mM. (B)
Selected markers of neuronal and dopaminergic differentiation on day 28 of the
differentiation protocol illustrated in (A), a time point within the window proposed for
clinical transplantation. Insert box in each panel shows Hoechst staining displaying all
nuclei in the field, of which the panel reveals the specifically labeled subset.
From J Clin Invest. 2020 Feb 3; 130(2):904e920.
32 CHAPTER 2 iPSCs and cell therapy for Parkinson’s disease
In a nonhuman primate model, CORIN-selected DA neurons differentiated from
iPSC sourced from both PD patients and controls achieved long-term survival and
correction of motor deficits (Kikuchi et al., 2017a,b). DA progenitors derived
from either human or mouse iPSC may be expanded and cryopreserved without
loss of functional efficacy, providing an important logistical advantage (Chung
et al., 2011;Wakeman et al., 2017).
3.10 Safety of hiPSC-derived cell grafts
As previously discussed, for hiPSC-based cell therapy to achieve clinical usefulness,
it is vital to employ safe and effective mechanisms to remove from the transplantable
cell product all residual undifferentiated cells with any teratoma potential. Positive se-
lection of differentiated cells by cell sorting strategies is one potential mechanism as
outlined earlier, but additional strategies for active removal of undifferentiated cells
may also prove necessary. Gamma irradiation represents one such strategy (Katsu-
kawa et al., 2016). At least two methods have been published that employ small mol-
ecules to eliminate hPSCs while sparing progenitor and fully differentiated cells. In
one such approach, a large screening study identified compounds that eliminated
hPSCs selectively, apparently by inhibiting oleic acid biosynthesis (Ben-David
et al., 2013). A second approach leverages the finding that hPSCs contain proapopto-
tic pathways that are actively inhibited by specific genes such as BCL10 and survivin
and that inhibition of the expression of these genes using small molecules such as
quercetin of YM155 results in selective apoptosis of hPSC while leaving differenti-
ated cells including mDA progenitors unaffected (Lee et al., 2013).
Thus, while success has been demonstrated with both positive and negative se-
lection strategies to reduce tumorigenic potential from implantable mDA progenitor
cell populations, the reduced handling and lesser risk of genetic damage involved in
small moleculeebased elimination of hPSCs may favor this approach. However,
attainment of the mDA progenitor cell stage during the differentiation process spans
a significant period from about day 16 onward depending on the differentiation pro-
tocol(Sonntag et al., 2018;Kriks et al., 2011;Song et al., 2020;Nolbrant et al., 2017;
Kikuchi et al., 2017a,b). Even after elimination of cells with tumorigenic potential,
cells may remain alongside the desired mDA progenitors that retain the capacity to
form other neural structures such as rosettes, and the long-term implications of such
“neutral” contaminants are not fully understood.
4.Clinical factors
To fully realize the potential of iPSC-derived mDA implantation for PD, the safe
production of the therapeutic cell product is not the only consideration. Rather,
host factors need to be considered, both with respect to the patient as a donor of
the originating cells for the reprogramming process and as a recipient of the
transplant.
4. Clinical factors 33
4.1 Variable reprogramming potential and differentiation
specificity
There is evidence of significant variability in reprogramming efficiency between
adult cells derived from different individuals (Kyttala et al., 2016). This factor
may greatly affect the practicality and cost of autologous, personalized therapy,
and not every PD patient might be a candidate. Decisions on the use of autologous
or allogeneic cell sources will need to weigh these considerations against those of
immunogenicity and differences in efficacy as discussed previously. Differentiation
protocols for ESC and iPSC are based in large part on specification of location along
the neuraxis and do not result in uniform populations of the desired A9 mDA cells.
Although the presence of “neutral” or uncharacterized cells in these cultures has not
been shown to adversely affect outcome in animal models, the presence of contam-
inating serotonergic neurons may be a clinically significant variable because these
cells are thought to play a role in graft-induced dyskinesia (Shin et al., 2012;Politis
et al., 2011).
4.2 Variable response of different forms of the disease
PD arises from multiple etiologies and its expression varies widely in both severity
and predominance of particular symptoms (Schapira and Jenner, 2011). These var-
iations are likely to affect responsiveness to mDA implantation as a therapy (Schies-
ling et al., 2008). The iPSC-based mDA implantation described in this chapter is
aimed at the motor expression of the disease, which is often but not always, and
never exclusively, the most disabling set of symptoms. Moreover, the optimal patient
age, symptoms profile, and disease stage (early vs. late) for favorable results from
transplantation need to be further defined (Piccini et al., 2005).
4.3 Expression of the disease within the graft
Use of autologous cells entails the risk, for individuals in whom the disease is genet-
ically determined, that the pathophysiology of the disease may eventually be
expressed in the graft itself. When the genetic defect is well understood, the use
of autologous iPSC-derived cells may eventually allow in vitro correction of genetic
alterations that have led to the host disease state through gene editing approaches
(e.g., CRISPR/Cas9), thus opening an entirely new approach to therapy (Yang
et al., 2014). This approach will be particularly important for early-onset and
more aggressive familiar forms of PD. Clearly this adds a layer of complexity
that must necessarily be weighed against the use of allogeneic cells. Even for the
sporadic PD, the disease may in some instances involve prion-like cell-to-cell trans-
mission that would affect allogeneic sources as has been observed in fetal tissue
grafts (Li et al., 2016). These considerations are especially important for younger
onset PD patients.
34 CHAPTER 2 iPSCs and cell therapy for Parkinson’s disease
4.4 Implantation target site and cell number
The optimal location for graft implantation has not been systematically explored.
Fetal tissue studies mainly used the striatum, which is the innervation target of
mDA neurons rather than their normal cell body location in the substantia nigra,
although reports of midbrain implantation exist as well (Mendez et al., 2002).
The extent to which the equivalent of fetal DA cells is able to locate and innervate
their targets in adult tissue is not fully understood, nor is the role of presynaptic
innervation of the graft cells (or lack thereof) in treatment efficacy. The number
of cells implanted and the form in which the cells are introduced (cell suspension
or solid tissue (Redmond et al., 2008;Clarkson et al., 1998)) are also likely to affect
outcome.
4.5 Graft survival and outgrowth
Significant differences exist between the controlled environment of the culture dish
and the host environment into which hiPSC-derived mDA cells are introduced. The
presence of TH
þ
cells in the culture dish and that of other markers of dopaminergic
fate do not correlate directly to the ultimate number of DA neurons in the mature
graft. Rather it is the maturation of dopaminergic progenitor cells that ultimately
give rise to the neurons that serve to replace dopaminergic function. Injury to the
cultured cells as well as disruption that may lead to apoptosis is likely to occur dur-
ing the harvesting and processing involved in the preparation for surgery. The injury
and inflammation of surrounding host tissue and the lack of direct vascular supply
resulting in relative ischemia of the newly deposited grafts may all have deleterious
effects on outcome. Whether these effects are general or selective is not known, but
the overall result is relatively modest numbers of the desired cells in the mature
graft, necessitating large excesses of implanted cells (Kirkeby et al., 2017;Kikuchi
et al., 2017a,b). Direct control of the differentiation process is lost once the cells are
implanted, which may also adversely affect the proportion of the graft that success-
fully forms the desired dopaminergic neurons. Nonetheless, maturation of appro-
priate functional neurons and robust reversal of symptoms have recently been
demonstrated in animal PD models (Song et al., 2020)(Fig. 2.3).
4.6 Surgical technique
Surgical implantation techniques used for fetal tissue recipients varied widely, with
no consensus on number or direction of implantation sites or trajectories or on the
equipment and process used. It is certainly possible that tissue damage and inflam-
mation that are a necessary part of the implantation process may affect subsequent
graft growth and function (Wenker et al., 2016). Techniques designed to minimize
these factors have been described but not systematically compared (Schweitzer
et al., 2020;Barker &consortium, 2019;Mendez et al., 2000;Wenning et al.,
1997;Nikkhah et al., 1994). The choice of the striatum as the target site is based
on its historical use for adrenal medullary and fetal tissue transplantation using
4. Clinical factors 35
the technology of the 1980s and where the volume of the implant was relatively large
(Lindvall et al., 1987). The normal location of the A9 DA cell bodies lost in PD is in
the SNpc of the midbrain, which although surgically accessible using modern imag-
ing and neuronavigational techniques remains a location with significantly higher
risk from surgery as well as from mass effect or potential inflammation surrounding
FIGURE 2.3
Graft functionality and appearance and in the rat 6-OHDA model of Parkinson’s disease.
(A) Correction of amphetamine-induced rotation behavior in 6-OHDA lesioned
immunodeficient rats by hIPSC-derived mDAP grafts (100,000 or 300,000 cells). By
4 weeks after implantation, behavior had returned to the lack of rotations expected from
unlesioned animals, or was even reversed. Data are presented as mean SEM.
** indicates P<.01, *** indicates P<.001. (B) Immunohistochemistry for hNCAM, a
specific marker for the grafted cells, reveals extensive fiber outgrowth into multiple areas
throughout the host brain in successive coronal sections. (C) Histological analysis using
immunohistochemistry for TH þdopaminergic neurons at 6 months posttransplantation
of grafts produced by D28 DA progenitors. Note A9-like neuronal morphology with large,
angular cell somata (D) as well as smaller spherical A10-like neurons (E).
From J Clin Invest. 2020 Feb 3; 130(2):904e920.
36 CHAPTER 2 iPSCs and cell therapy for Parkinson’s disease
the graft. Though holding the potential advantage of native presynaptic innervation
and host environment, higher complication rates with surgical implantation into this
region as well as potential harm to the residual host population of dopaminergic neu-
rons have dissuaded most groups from utilizing this target. Although a recent animal
study showed a promising result (Adler et al., 2019), it remains unclear whether
mDA progenitor cells introduced here would successfully innervate targets in the
adult, pathological environment of the PD human brain. Nonetheless, this issue
may deserve renewed examination once cell therapy becomes a more established
therapeutic strategy.
5.Toward clinical implementation of iPSC-based therapy
for PD
To summarize the considerations outlined in this chapter, the advantages of avail-
ability and potential reduced immunogenicity of an iPSC-derived dopaminergic
cell therapy for PD have the potential to overcome drawbacks of alternative proced-
ures and build on a basic proof of concept that is now several decades old. The
remaining issues that must be solved to realize this potential include:
5.1 Production of clinical-grade iPSCs
iPSCs for use in the in vitro differentiation process, whether autologous or alloge-
neic, must be available with a quality that exceeds mandated clinical (GMP) stan-
dards. The quantities of such cells required for use need to be produced in an
appropriate time frame. Recent advances in the technology of producing such cells
have been described that achieve the goal of making them less subject to epigenetic
memory and free of footprints of the reprogramming process (Song et al., 2020).
Better understanding of both the genetic and the metabolic changes that accompany
the reprogramming process holds the promise of improved efficiency and safety in
producing these cells (Song et al., 2020;Cha et al., 2017).
5.2 Optimization and standardization of in vitro differentiation
protocols
Current differentiation protocols to produce A9 mDA-like progenitor cells in vitro
have been based on work with hESC lines such as H9, which are in fact themselves
in the pipeline for use as a therapeutic product (Tabar, 2016). In applying these pro-
tocols to multiple different lines as would be required to develop a bank of HLA-
matched autologous iPSC-derived cells, or even more so personalized, autologous
cells, variability of the iPSC and varying susceptibility to the differentiation process
will likely require extensive safety testing of multiple lines in contrast to that
required for a “one size fits all” hESC-based strategy. Although safety is a primary
concern, efficacy is obviously the primary goal, and as described earlier, current
5. Toward clinical implementation of iPSC-based therapy for PD 37
differentiation protocols are inefficient and/or variable at producing high percent-
ages of dopaminergic neurons. Progress in this area is necessary and expected.
5.3 Patient selection and follow-up monitoring criteria
A standardized clinical protocol for patient selection, screening, surgery, and follow-
up monitoring should be established. Initially, this protocol will be based on the clin-
ical experience with human fetal midbrain tissue transplantation. Little information
exists on the expected time course of clinical improvements, the scope of any non-
motor effects, and the durability of the improvements. Concern remains about the
possibility of irreversible dyskinesias as occurred in some fetal transplant patients.
Imaging criteria for successful or unsuccessful growth and innervation by the graft
have not been established. Although animal models may be useful in this regard,
they are imperfect analogs of the disease. Ultimately, the criteria for therapeutic
success will come only from well-constructed, double-blinded clinical trials.
5.4 Regulatory and socioeconomic acceptance
Initial costs of an iPSC-derived therapy for PD are likely to be high. Although such
costs generally improve with time, the magnitude of the disease burden of PD is
large and growing, and comparisons with other therapies including medical and sur-
gical (Armstrong and Okun, 2020) will involve complex calculations of efficacy and
cost efficiency that are difficult to predict at this time. It is the author’s belief that
general acceptance of cell therapy for PD, and in particular of autologous, person-
alized cell therapy as opposed to ESC-based therapy, will require that costs and risks
reach levels at least as favorable as those for organ transplant procedures for chronic
disease, but as there will be no need for a waiting list, volumes may be considerably
higher. Success will depend greatly on the outcome of clinical trials, and both the
medical community and the general public are likely to follow the outcomes of
such trials with close attention to this common degenerative disease.
6.Perspective
In 2017, Mandai et al. reported the first use in a human patient of iPSC-derived,
in vitro differentiated cells to treat age-related macular degeneration. Several
intended clinical trials have been announced but as of the time of writing of this
chapter, no studies comparable to the sham controlled study of fetal tissue trans-
plantation in PD is known to have been completed (Barker et al., 2015a,b;Barker
&consortium, 2019).
Although PD has been an attractive target for cell therapy because of the well-
established pathophysiology of loss of particular cell populations with clear motor
effects, it remains a complex disease with significant symptomatology that is incom-
pletely understood. In this context, iPSC-based cell therapy is likely to become one
38 CHAPTER 2 iPSCs and cell therapy for Parkinson’s disease
of a menu of options that will require tailoring to individual patient needs and cir-
cumstances. Combined use of better pharmaceuticals with fewer side effects,
improved deep brain stimulation strategies with new targets and closed-loop strategy
(aDBS) (Swann et al., 2018), and neuroprotective therapies arising from greater
understanding of the multiple etiologies and pathophysiologies of PD will all
contribute to improved management of this debilitating disease. We expect iPSC-
based cell therapy to play a key role in this promising future.
Abbreviations
CNVs copy-number variations
DA dopamine
EB embryoid body
fVM fetal ventral mesencephalon
hESCs human embryonic stem cells
hiPSCs human induced pluripotent stem cells
iDNs induced dopamine neurons
iNs induced neurons
iPSCs induced pluripotent stem cells
mDA midbrain dopamine
NPCs neural progenitor cells
PCs progenitor cells
PD Parkinson’s disease
PSCs pluripotent stem cells
SCNT somatic cell nuclear transfer
Shh Sonic hedgehog
SN substantia nigra
SNVs single nucleotide variations
Acknowledgments
The authors’ research was in part supported by NIH grants (NS084869 and NS070577) and by
private charitable donations. We also thank members of the Molecular Neurobiology Labora-
tory for their discussion and suggestions.
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Induced pluripotent stem
cells as a potential
treatment for Huntington’s
disease 3
B. Srinageshwar
1,2,3
, G.L. Dunbar
2,3,4,5
, J. Rossignol
1,2,3
1
College of Medicine, Central Michigan University, Mt. Pleasant, MI, United States;
2
Field
Neurosciences Institute Laboratory for Restorative Neurology, Central Michigan University, Mt.
Pleasant, MI, United States;
3
Program in Neuroscience, Central Michigan University,
Mt. Pleasant, MI, United States;
4
Department of Psychology, Central Michigan University, Mt.
Pleasant, MI, United States;
5
Field Neurosciences Institute, St. Mary’s of Michigan, Saginaw, MI,
United States
Chapter outline
1. Huntington’s disease ............................................................................................ 50
2. HTT gene and protein function .............................................................................. 51
3. Epigenetics and HD .............................................................................................. 51
4. Signs and symptoms of HD .................................................................................... 52
5. Stem cell therapy for HD....................................................................................... 53
5.1 Mesenchymal stem cells (MSCs) and HD................................................. 54
5.2 Neural stem cells (NSCs) and HD ........................................................... 54
5.3 Embryonic stem cells (ESCs) and HD ...................................................... 55
6. Induced pluripotent stem cells (iPSCs) ..................................................................55
6.1 Induced pluripotent stem cells (iPSCs) and HD........................................ 55
6.2 iPSC-based therapy for HD..................................................................... 56
6.2.1 Transplantation of undifferentiated iPSCs for HD............................... 56
6.2.2 Transplantation of differentiated iPSCs for HD .................................. 57
7. Stem cellsebased HD clinical trials ...................................................................... 59
8. Summary and conclusions .................................................................................... 61
Acknowledgments ..................................................................................................... 62
References ............................................................................................................... 62
Abstract
Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder
that usually affects middle-aged individuals, the median age of symptom onset is 42
years. HD is invariably fatal and the drugs that are currently available are only
palliative. Of the many promising therapeutic approaches, genetic therapies and
CHAPTER
49
Recent Advances in iPSCs for Therapy, Volume 3. https://doi.org/10.1016/B978-0-12-822229-4.00002-4
Copyright ©2021 Elsevier Inc. All rights reserved.
especially reintroduction of genetically altered stem cells have gained considerable
attention. Studies using stem cell therapies have evolved from using mesenchymal
stem cells (MSCs), neural stem cells (NSCs), and embryonic stem cells (ESCs) to
induced pluripotent stem cells (iPSCs). Since iPSCs have the ability to differentiate
into any of the three mammalian germ layers, their use has become a promising new
approach for cell replacement therapy, including their use for treating HD. Two
therapeutic approaches using iPSCs for cell replacement include transplantation of
undifferentiated iPSCs or using those that have undergone partial differentiation
into a desired cell lineage. Very little is known about the relative efficacy of
transplanting these two different types of iPSCs, especially with regard to treating
HD. This chapter gives a brief overview of stem cellebased transplantation therapy
in HD, with a major focus on what is currently known about the efficacy of un-
differentiated versus partially differentiated iPSCs transplant therapy in HD. In
addition, the novel idea of transplanting reprogrammed (i.e., gene-corrected)
autologous cells from HD patients as a source of iPSCs is explored.
Keywords:
Autologous transplantation; Cell differentiation; Degeneration; Differentiated iPSCs;
Fibroblast; Gene correction; HD models; HD patients; HTT; Huntington’s disease; Induced plurip-
otent stem cells; Medium spiny neurons; Reprogramming; Transplantation; Undifferentiated iPSCs.
1.Huntington’s disease
Huntington’s disease (HD) is a fatal neurodegenerative disease that is inherited in an
autosomal dominant pattern, affecting both males and females. HD is found globally
with an incidence of six per 100,000 individuals. HD is a late-onset disease, usually
occurring at the age of 40 years or above. The major underlying cause of the overt
phenotypes of the disease first comes from neuronal degeneration in the caudate and
putamen regions of the human brain. Other brain regions that undergo degeneration
include the cortex, thalamus, and cerebellum (Ross and Tabrizi, 2011). The neuro-
degeneration in these regions primarily affects dopaminergic (DA), gamma amino-
butyric acid (GABA), and glutamatergic neurotransmitter systems (Frank, 2014).
The degeneration results from the aggregation of mutant Huntingtin protein
(mHTT), formed by a polyglutamate tract encoded by cytosineeadenineeguanine
(CAG) repeat expansions in exon 1 of the gene. The number of CAG repeats are
negatively correlated with the age of onset. mHTT is insoluble, forming aggregates
in cells that eventually become toxic, specifically mHTT accumulation in medium
spiny neurons (MSNs) of the basal ganglia is the suspected cause of neuronal degen-
eration in this brain region. Patients with more than 37 CAG repeats are diagnosed as
having HD (Roos, 2010). The elongated CAG repeats lead to toxicity in a gain-of-
function manner. However, there is considerable evidence that there is a significant
loss of function that also contributes to, and exacerbates, HD pathology (Paine,
2015).
Currently, there is no cure or effective treatment for HD. However, there are
many potential drugs being considered, several of which are being tested in clinical
trials. The only FDA-approved drug is tetrabenazine (TBZ), which is used to combat
50 CHAPTER 3 Induced pluripotent stem cells as a potential
the aberrant motor symptoms of HD. Although TBZ is effective in controlling the
chorea-like symptoms of HD, some of the patients experience adverse side effects
emanating from intolerance of TBZ. In addition to TBZ, new drugs such as deute-
trabenazine, riluzole, and amantadine are being tested for controlling HD symptoms
(Frank, 2014;Dean and Sung, 2018).
2.HTT gene and protein function
The human HD gene (HTT; formally known as IT-15) is one of the largest genes
(325 kb) in humans and is located at chromosomal loci 4p16.3. HD exhibits an auto-
somal dominant inheritance pattern with 100% penetrance, equally affecting males
and females (Myers, 2004). The HTT gene encodes for the 3144 amino acid HTT pro-
tein (348 kDa). The HTT protein is expressed ubiquitously, with the highest expression
in such brain regions as the striatum, cerebellum, cortex, and hippocampus. Due to the
association of the N-terminal region of the gene with HD, an area that encodes a spe-
cific toxic protein fragment, the N-terminus has been thoroughly characterized and
studied. Nonetheless, the exact function of the entire HTT protein has yet to be fully
elucidated, although some of the most commonly attributed functions include:
(1) Interaction with huntingtin-associated protein (HAP) to aid anterograde and
retrograde transport of organelles and cargo in the axons and dendrites of the
neurons;
(2) Cell division, as HTT is found in the mitotic spindle fibers in dividing cell types
required for cell division;
(3) Cellular processes such as endocytosis, endosomal trafficking, and vesicle
recycling are also some of the major functions of HTT, following their in-
teractions with certain proteins; and
(4) Regulation of autophagy, leading to self-degradation of the aggregated mHTT
in the cells (Saudou and Humbert, 2016;SeeFig. 3.1).
3.Epigenetics and HD
Wild-type (WT) HTT, as well as mHTT, has a major role in upregulation and down-
regulation of genes by interacting with gene activators and repressors. Therefore,
differential expression of certain genes leads to neuronal dysregulation, as well as
the triad of symptoms (motor, cognitive, and psychiatric) that characterize HD.
We have described the epigenetic basis of HD in detail in a previous publication (Sri-
nageshwar et al., 2017), so this is not extensively covered in this chapter.
A key epigenetic interaction that deserves special attention is the role of HTT and
mHTT in activating or deactivating the gene encoding brain-derived neurotrophic
factor (BDNF). In general, BDNF plays a major role in maintaining neuronal activ-
ity and integrity, cellecell signaling, and facilitating neurotransmitter release.
3. Epigenetics and HD 51
BDNF is also involved in different cell signaling pathways that are required to main-
tain normal cellular functions and metabolism. BDNF is dysregulated in HD as well
as in Parkinson’s disease (PD), Alzheimer’s disease (AD), and multiple sclerosis
(MS) leading to neuronal degeneration (Bathina and Das, 2015). Specifically, in
HD, there are various histones (such as H3 and H4) that are either hypoacetylated
or methylated at the BDNF promoter region, leading to reduced expression of the
BDNF protein (Chen and Chen, 2017). Therefore, introduction of a BDNF-based
therapy has been very popular and successfully used in HD translational research
performed by many researchers, including us (Dey et al., 2010).
4.Signs and symptoms of HD
HD has a triad of symptoms including motor, cognitive, and psychiatric disturbances
that characterize the disease (Fig. 3.2). The motor symptoms include chorea (dance-
like, uncontrollable movements), jerkiness, rigidity, loss of coordinated voluntary
movement and coordination. Choreic movement is considered the hallmark overt
symptom of HD and its occurrence is commonly used to define the onset of HD.
In HD, cognitive dysfunction emerges around 15 years prior to the onset of overt
symptoms of the disease and worsens as the disease progresses. The cognitive symp-
toms include memory loss, attention and learning deficits, deficits in the speed of
psychomotor processing, perseverative thinking, impairment in olfaction, and lan-
guage difficulties. As these symptoms get worse, there is concomitant brain atrophy
and decrease in key neurotrophic factors and growth hormones. Usually the cogni-
tive assessment for HD is based on the Unified Huntington Disease Rating Scale
(UHDRS); however, measures of mild cognitive impairment (MCI) have been devel-
oped to assess cognitive impairment in prodromal HD patients. Psychiatric
FIGURE 3.1
A representation of the general function of HTT gene.
52 CHAPTER 3 Induced pluripotent stem cells as a potential
symptoms of HD are categorized into two types: (1) affective and (2) non-affective
(Smith et al., 2000;Paulsen, 2011;Paoli et al., 2017). Affective symptoms include
increasingly larger shifts in mood and can include behavioral outbursts and bouts of
depression. Nonaffective symptoms often include disorders of thought, paranoia,
and/or episodes of psychotic-like behavior.
5.Stem cell therapy for HD
Although early experimental transplant therapies for HD utilized fetal or embryonic
stem cells (ESCs), more recent studies shifted to the use of adult stem cells, such as
mesenchymal stem cells (MSCs), neural stem cells (NSCs), and induced pluripotent
stem cells (iPSCs; Fig. 3.3).
Due to the ethical controversies, propensity for rejection, and lack of availability,
the use of ESCs for transplants has been slowly supplanted by the use of MSC,
NSCs, or iPSCs. The degree to which any of these stem cells differentiate into the
types of neurons needed to produce a significant cell replacement therapy is still
controversial, although there is growing evidence that iPSCs show considerable
promise to do so (Medvedev et al., 2010).
In any case, it is well documented that these stem cells have therapeutic potential,
by either supporting or maintaining the existing healthy neurons through secretion of
trophic factors. A brief description of how these different types of stem cellebased
therapies are being used in translational research for treating HD is presented as
follows.
FIGURE 3.2
Representation of the triad of symptoms associated with HD.
5. Stem cell therapy for HD 53
5.1 Mesenchymal stem cells (MSCs) and HD
Mesenchymal stem cells/stromal cells (MSCs) are adult multipotent stem cells that
have been widely used as a potential therapy for HD. The degree to which the MSCs
can differentiate into functional neurons is highly debated, but their ability to provide
critical trophic factors has been shown repeatedly (Rossignol et al., 2015). Given
that the HD brain contains abnormally low levels of BDNF, making the host neurons
vulnerable to apoptosis and excitotoxicity, it was hypothesized that MSCs transplants
might restore BDNF levels and provide critical neuroprotection (Zuccato et al., 2011).
MSCs are known to secrete BDNF, which helps to create an optimal microenvi-
ronment for maintaining the neuronal integrity of the brain. We and others have
transplanted MSCs derived from rodent bone marrow (BM) and umbilical cord
(UC) into several rodent models of HD. In addition, we have also shown that
MSCs, when genetically modified to overproduce BDNF, are highly therapeutic in
transgenic YAC128 HD mice by alleviating the signs and symptoms of the disease
(Dey et al., 2010;Fink et al., 2013). There are a variety of studies that have
confirmed the therapeutic effects of MSCs and BDNF in HD (Olson et al., 2012;
Rossignol et al., 2015;Pollock et al., 2016). A detailed description of MSC trans-
plantation in HD can be found in our recent review (Srinageshwar et al., 2020).
5.2 Neural stem cells (NSCs) and HD
NSCs can be obtained from a specific region of the brain and can be easily expanded
in culture and induced to differentiate into neurons. Transplantation of NSCs in
different HD rodent models has proven to be highly advantageous, leading to very
promising outcomes. The transplanted NSCs are able to integrate into the host tissue
at the site of degeneration in the HD brain and either by repopulating the area of the
lost neurons and/or preventing further neuronal loss and revitalizing vulnerable neu-
rons so that motor function are improved, as well as enhancing the long-term
FIGURE 3.3
A representation of different types of stem cell transplantation for treating HD
54 CHAPTER 3 Induced pluripotent stem cells as a potential
survival of the transplant recipient. There is growing evidence that the transplanted
NSCs are capable of differentiating into neurons and glia at the lesion site (McBride
et al., 2004;Ryu et al., 2004;Roberts et al., 2006). However, the extent to which
non-autologous NSCs, or the cells derived from them, survive in the host tissue re-
quires more research before definitive conclusions can be made.
5.3 Embryonic stem cells (ESCs) and HD
Although ethical issues continue to surround the use of ESCs, scientists have suc-
cessfully used them to reduce symptoms in HD, and there is considerable evidence
that ESCs are capable of differentiating into neural cells in the HD brain. One of the
major advantages of using ESCs over other types of stem cells is that ESCs are not
readily rejected by the immune system and are relatively safe, in terms of not
inducing uncontrolled proliferation (Volarevic et al., 2018). Previous studies using
the neurotoxic quinolinic acid (QA) rat model of HD showed that transplanting hu-
man ESCederived neural stem cells into HD rats was therapeutic; the transplanted
cells differentiated into neurons at the site of the QA-induced lesion in rats, leading
to the reduction of behavioral deficits (Song et al., 2007). Similar results were ob-
tained by transplanting human ESCederived GABAergic neurons in QA-lesioned
mice, leading to recovery from locomotor deficits (Ma et al., 2012). However, the
ethical issues and availability of human embryos, as well as some residual concerns
surrounding the long-term efficacy and safety of ESC transplants, have prompted re-
searchers to explore using iPSCs transplants for HD.
6.Induced pluripotent stem cells (iPSCs)
As the name suggests, iPSCs are pluripotent stem cells that are capable of self-
renewal, as well as differentiating into multiple lineages, giving rise to endoderm,
ectoderm, or mesoderm germ layers. iPSCs were first cultured in 2007 by reprog-
ramming somatic cells (skin fibroblasts) using specific factors, Oct4, Klf-4, Sox2,
and c-Myc, popularly known as Yamanaka factorsdOKSM. iPSCs are very similar
to ESCs in terms of cell differentiation, proliferation, characterization, and genetic
and epigenetic markers. The discovery of the means to produce iPSCs has enhanced
stem cellebased research, and iPSCs are rapidly replacing ESCs in the pursuit of
potential therapies for a number of diseases, including PD, AD, and HD, because
researchers can now circumvent the ethical concerns associated with ESCs through
the use of iPSCs, which still retain the ability to produce multiple cell lineages
(Takahashi et al., 2007;Romito and Cobellis, 2016).
6.1 Induced pluripotent stem cells (iPSCs) and HD
iPSCs have critical advantages compared to any other cell types for stem cellebased
therapy, as well as for disease modeling. Perhaps the most significant advantage of
6. Induced pluripotent stem cells (iPSCs) 55
iPSCs over other types of stem cells in cell replacement therapies is that the patient
receiving the transplant can donate the iPSCs being transplanted. This ensures
greater immunocompatibility and reduces the risk of rejection. Of course, in the
case HD and other genetic disorders, an additional step of editing the mutated
gene is required before the autologous transplant can be done. With the use of
new gene-editing tools, such as CRISPR-Cas9, this procedure is now feasible and
represents a novel concept and approach to iPSC transplantation for HD. The added
advantage of iPSCs over ESCs (besides circumventing many of the ethical issues
surrounding the use of human embryonic tissue) is the greater availability of iPSCs,
since these can be produced readily from the patient’s own cells. Another major
advantage of iPSCs over MSCs and NSCs is that iPSCs, like ESCs, have a greater
propensity to utilize the biochemical cues of the transplant site environment and
to differentiate into the type of region-specific neurons that are most needed for
functional recovery (Liu et al., 2016).
6.2 iPSC-based therapy for HD
Use of iPSCs-based therapy for HD includes (1) transplantation of undifferentiated
iPSCs or (2) transplantation of iPSCs that have been partially differentiated into the
specific cell type most needed to compensate for the cells that have degenerated in
the HD brain (Fig. 3.4). The following section will summarize the different strate-
gies of iPSCs transplantation in HD and the therapeutic outcomes.
6.2.1 Transplantation of undifferentiated iPSCs for HD
Recently, Mu et al. (2014), (2016) transplanted iPSCs into the ipsilateral ventricle of
a QA rat model of HD. The iPSCs migrated from the lateral ventricles to the lesioned
FIGURE 3.4
A representation of iPSC transplantation in HD.
56 CHAPTER 3 Induced pluripotent stem cells as a potential
striatum and differentiated into region-specific neurons. To assess the motor out-
comes following treatment, behavioral tests were performed, which showed that
the QA-lesioned rats receiving iPSC transplants recovered from the QA-induced
motor deficits when compared to the untreated controls (Mu et al., 2014). A series
of immunohistochemistry and protein assays revealed changes in the expression of a
number of protein markers that correlated with the therapeutic outcome following
iPSC transplantation. Some of the proteins that were upregulated include glia-
derived neurotrophic factor, basic fibroblast growth factor, leptin, and other hor-
mones known to regulate glucose. Most importantly, the inflammatory marker,
cytokine-induced neutrophil chemoattractant-3, was downregulated, indicating
reduced inflammation following iPSC transplantation (Mu et al., 2016).
Work in our lab utilized the reprogramming of rat tail-tip fibroblasts into iPSCs
and then transplanted them into the striatum of rats that had been given the neurotoxin,
3-nitropropionic acid (3-NP; Fink et al., 2014), which induces HD-like neuropa-
thology. Intrastriatal transplantation was done at different time points, corresponding
to the early, middle, and late stages of HD. The accelerated rotarod task was used to
assess the degree of recovery from 3-NP-induced motor deficits following transplan-
tation. This study indicated that the iPSC transplants alleviated the motor deficits in
rats receiving iPSC treatment at all stages. The rats that received the iPSCs at the early
and intermediate stages of HD showed behavioral sparing on the rotarod task and the
preservation of striatal metabolic activity, as well as protection against 3-NP-induced
ventricular enlargement in the brain compared to untreated rats and those that received
iPSC treatment at the later stages of HD. Importantly, the rats that received iPSC trans-
plants during the period that mimicked the later stages of HD showed functional re-
covery on the accelerated rotarod task, in which their 3-NP-induced motor deficits
were reversed by the iPSC treatment. In addition, some of the transplanted iPSCs
differentiated into astrocytes around the transplanted region. Treatment at early and
intermediate stages in this study also prevented much of the 3-NP-induced neuronal
death compared to the rats that did not receive treatment and those receiving iPSC
at a later time point. Impressively, immunohistochemical analysis provided evidence
that some of the transplanted iPSCs differentiated into region-specific neurons. These
results suggest that transplanting iPSCs during the early stages of HD may serve a
neuroprotective role and preserve behavioral functioning and that iPSC transplants
at later stages can actually restore some of the lost functions. However, the degree
to which restoration of function is achieved may depend on the ability of the
iPSCs to differentiate into region-specific neurons. This role of iPSC-induced recov-
ery needs to be studied further (Table 3 . 1).
6.2.2 Transplantation of differentiated iPSCs for HD
Given that MSNs are a specific striatal cell population that degenerate early in HD,
some researchers have focused on differentiating the iPSCs into MSN-like cells prior
to transplantation. Carri et al. (2013) differentiated human iPSC cells into MSN pro-
genitor cells that express the markers FOXG1, OTX2, and GSX2. Following termi-
nal differentiation, these cells expressed DARPP-32, a protein marker that is
6. Induced pluripotent stem cells (iPSCs) 57
expressed by 95% of MSNs in the striatum. Further electrophysiological analysis
showed that 57% of these cells achieved repeated firing and action potentials estab-
lishing their functionality in vitro. These cells were transplanted into the striatum of
the QA-lesioned HD rats. The tissue was collected and analyzed at different time
points showing the presence of the graft. During early stages following transplanta-
tion, the cells expressed only a few neuronal markers. However, by week 9 following
transplantation, the transplanted cells expressed the DARPP-32 specific marker,
indicating that the grafted cells were adopting the MSN cell fate. Further, the rats
with the iPSCs transplant showed reduced apomorphine-induced rotations compared
to the untreated QA rats (Carri et al., 2013).
Jeon et al. (2014) partially differentiated HD patientederived iPSCs (72 CAG
repeats) toward an medium spiny neuronal lineage by co-culturing them with PA6
stromal cells. Following this partial differentiation, these cells were bilaterally trans-
planted into the striatum of 12-month-old YAC128 mice and the rotarod task was
used to assess functional outcome. Interestingly, even though the transplanted iPSCs
retained their mHTT aggregates, the cells were therapeutic and improved the motor
activity of the HD mice. Moreover, tissue analysis showed that the transplanted cells
expressed neural precursor markers, including those that give rise to GABAergic
cells and MSNs. However, the origin of the mHTT aggregates throughout the tissue
is unclear, since mHTT is produced by both the transplanted iPSCs and the host tis-
sue (Jeon et al., 2014).
Table 3.1 A summary showing the outcomes of transplantation of naı
¨ve
iPSCs in HD.
Study Source
Agents/genes
for
reprogramming HD model Study outcome
Mu
et al.
(2014)
Healthy
mouse
fibroblast
Lentivirus Quinolinic acid
(QA) treated rats
Sparing of motor
behavioral deficits
Mu
et al.
(2016)
Healthy
mouse
fibroblast
Lentivirus Quinolinic acid
(QA) treated rats
Increased expression of
growth factors and
neurotrophic factors that
helps in maintaining
neurons.
Downregulation of
inflammatory markers
Fink
et al.
(2014)
Healthy
rat tail-tip
fibroblast
Adenovirus
containing Oct4,
Sox2, Klf-4, and
c-Myc
3-nitropropionic
acid (3-NP)
treated rats
Sparing of motor
behavioral deficits and
loss of neurons in the
early and intermediate
stages. Functional
recovery after the late-
stage intervention.
58 CHAPTER 3 Induced pluripotent stem cells as a potential
Another study from our lab used iPSCs that were derived from healthy adult mice
and which were partially differentiated into induced neural stem cells (iNSCs).
These iPSCs-derived iNSCs were then characterized using markers that are specific
for cell differentiation into a neuronal lineage, including Nestin, Sox2, b-tubulin-III,
and NeuN. Upon confirming the neuronal differentiation, the cells were transplanted
bilaterally into the striata of YAC128 HD mice and measures of tissue pathology and
behavioral improvements were made. Our results revealed that the HD mice given
iPSC-iNSCs showed behavioral sparing, relative to vehicle-treated HD mice. More-
over, the histological analyses showed the presence of the transplanted cells in the
striatum containing the phenotypic biomarker, DARPP-32, suggesting that they
had differentiated into region-specific MSNs. There was also a significant increase
in production of BDNF in the iPSC-iNSC-transplanted HD to vehicle-treated HD
mice (Al-Gharaibeh et al., 2017).
As far as clinically relevant use of iPSCs therapy for HD, a critically important
study was conducted by An et al. (2012), who used HD-iPSCs-derived NSCs as a
cell replacement therapy. In this study, the HD cells underwent gene correction
before partial differentiation into NSCs and MSNs lineages. These cells were trans-
planted into the striatum of QA-treated HD mice and the gene-corrected NSCs sur-
vived and fully differentiated into MSNs (An et al., 2012).
Similarly, Cho et al. (2019) differentiated WT- and HD monkeyederived iPSCs
into neural progenitor cells (NPCs), which were then gene-corrected. Specifically,
the HTT gene in the HD-iPSC-NSC cells was targeted using sh-RNA, which sup-
presses expression of the HTT protein and consequent aggregate production.
Following this, the HD HTT gene-modified cells or WT cells were transplanted
into N171-82Q HD mice. The study showed that the normal and the gene-
corrected NPCs derived from iNSCs were able to survive in the striatum following
transplantation and differentiated into site-specific neural cells and astrocytes. More-
over, these cells ameliorated the motor deficits as evidenced by performance on the
rotarod task. In terms of survival, the mice that received the gene-corrected cells sur-
vived longer than the control groups. This study clearly shows that the mice
receiving transplants of gene-corrected NSCs derived from iPSCs were able to sur-
vive and differentiate in the HD mouse brain at higher rates compared to the non-
gene-corrected cells. Furthermore, mice given transplants of gene-corrected iPSCs
exhibited preserved grip strength, motor performance on the rotarod test, and longer
survival times than mice that were vehicle-treated and those transplanted with WT-
derived iPSCs (Cho et al., 2019)(Table 3.2).
7.Stem cellsebased HD clinical trials
Stem cells have been used in clinical trials for HD with some success (see Srinagesh-
war et al., 2020, for detailed descriptions). Most of the clinical trials in HD used fetal
stem cells, but these studies produced mixed results, along with some adverse side
effects. However, no clinical trials using pluripotent stem cells, specifically iPSCs,
7. Stem cellsebased HD clinical trials 59
Table 3.2 Summary of outcomes of studies using transplantation of differentiated iPSCs in animal models of HD.
Study Source
Agents/genes
for
differentiating
iPSCs Differentiated cell type HD model Study outcome
Carri et al.
(2013)
HD patients Neural induction
media
iPSCs differentiated to medium
spiny neuron progenitor cells
Quinolinic
acid (QA)-
treated HD
rats
Transplanted cells survived,
differentiated into MSNs, and
reduced apomorphine-induced
rotations
Jeon et al.
(2014)
HD patient Neural induction
media
HD iPSCs differentiated into
neural precursor
YAC128 HD
mice
Transplanted cells survived,
differentiated, and reduced
behavioral deficits
Al-
Gharaibeh
et al.
(2017)
Healthy mouse
tail-tip
fibroblast
Neural induction
media
iPSCs differentiated into neural
stem cells
YAC128 HD
mice
Transplanted cells survived,
differentiated, and reduced
behavioral motor deficits
An et al.
(2012)
Commercially
purchased
Embryoid body
method
Gene-corrected HD iPSCs
differentiated into neural stem
cells
Quinolinic
acid (QA)-
treated HD
rats
Transplanted cells survived,
differentiated into MSNs
Cho et al.
(2019)
HD and WT
monkeys
Neural induction
media
HD (corrected with sh-RNA)
and WT iPSCs differentiated
into neural progenitor cells
N171-82Q
HD mice
Transplanted cells survived and
reduced behavioral deficits and
increased longevity
60 CHAPTER 3 Induced pluripotent stem cells as a potential
have been conducted for HD. A persistent fear is that production of iPSCs often in-
volves oncogenes, such as c-Myc, which could potentially result in the production of
tumors by the transplanted cells. Another potential drawback for clinical testing on
HD patients involves whether the advantages of autologous iPSCs transplantation
would be lost because such transplants would entail the use of iPSCs that contain
mHTT. However, as discussed in this review, these shortcomings may be surmount-
able as there is growing evidence that iPSCs do not appear to produce tumors and the
use of gene-corrected iPSCs would circumvent the risk of initiating more problems
by autologous transplants in HD patients.
8.Summary and conclusions
Although only a few studies have been conducted using iPSCs as a potential cell
replacement therapy for HD, there are two emerging approaches: (1) transplanting
undifferentiated iPSCs or (2) transplanting partially differentiated iPSC that may
readily give rise to the specific type of neurons or other cells needed to enhance ther-
apeutic outcomes. Although the use of partially differentiated iPSCs makes sense
when it is known what the specific type of replacement cells are needed to be, the
use of undifferentiated iPSC may be more appropriate for situations in which the
specific type of replacement cell that is needed is unknown or when several cell
types need to be replaced so that relying on the brain microenvironment to signal
which types of cells are needed may work best. Clearly, further research is needed
to better understand the complexities of the brain microenvironment, but as this
knowledge emerges, inducing a specific defining cell lineage prior to transplantation
may provide greater efficacy in terms of promoting recovery. As further research us-
ing iPSCs to treat HD unfolds, the promise of safely using iPSCs to effectively treat
HD may soon be realized.
Critical to the use of iPSCs for treating HD is whether autologous transplantation
of the cells obtained from HD patients is feasible. Although early work in this area is
encouraging, whereby no adverse effects following transplantation of human HD
cells in HD mice were observed (Jeon et al., 2014), autologous transplantation in a
clinical setting may require correction of the mHTT gene of the iPSCs prior to trans-
plantation (Fig. 3.5). However, unlike for some other neurological or CNS injuries in
which time is a critical factor for generating sufficient amounts iPSCs, use of iPSCs
for treating HD is relatively less time-sensitive. For time-sensitive treatments, allo-
geneic transplantation may be a viable option, especially as the iPSC-based technol-
ogy for cell replacement therapy improves (Ohnuki and Takahashi, 2015).
As new research on the use of iPSCs for HD emerges, a clearer understanding of
whether transplanting undifferentiated or partially differentiated iPSCs would pro-
vide more optimal outcomes or whether transplanting gene-corrected autologous
or allogeneic cells would be more effective should be easier to address. Encourag-
ingly, the early work in this emerging field suggests that stem cell treatment for HD
is a very promising approach to combat this devastating disease.
8. Summary and conclusions 61
Acknowledgments
The authors would like to thank Dr. Robert Petersen for his help in proof reading this chapter.
This research was supported by the Field Neurosciences Institute, the John G. Kulhavi Profes-
sorship, the CMU Program in Neuroscience, and the CMU College of Medicine.
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Present and future of adult
stem cells and induced
pluripotent stem cells
therapy for ischemic
stroke
4
Ana Bugallo-Casal
1
, Marı
´aPe
´rez-Mato
2
, Francisco Campos
1
1
Clinical Neurosciences Research Laboratory (LINC), Health Research Institute of Santiago de
Compostela (IDIS), Santiago de Compostela, A Corun
˜a, Spain;
2
Neuroscience and
Cerebrovascular Research Laboratory, Department of Neurology and Stroke Center, La Paz
University Hospital, Neuroscience Area of IdiPAZ Health Research Institute, Universidad
Auto
´noma de Madrid, Madrid, Spain
Chapter outline
1. Introduction ................................................................................................ .........68
2. Protective and recovery approaches for ischemic stroke ........................................ 69
3. Adult stem cells: NSCs and mesenchymal stem cells.............................................. 74
3.1 Protective effects of NSCs in the treatment of stroke ................................ 74
3.2 Therapeutic potential of MSCs in stroke: immune modulation,
neuroprotection, and neurorepair ............................................................ 75
4. Induced pluripotent stem cells: a multifaceted role in the study of neurological
diseases..............................................................................................................81
4.1 iPSCs in stroke ..................................................................................... 83
4.2 iPSCs and CADASIL .............................................................................. 84
5. Conclusion and future perspectives ....................................................................... 87
Glossary ................................................................................................................... 89
References ............................................................................................................... 90
Abstract
Stroke, which is caused by an obstruction of the blood supply to the brain, is a
leading cause of mortality and morbidity in developed countries. Unfortunately, its
incidence is increasing due to the progressive aging of the population. Despite the
great effort invested in treating stroke, to this day, therapies able to promote re-
covery after stroke have not yet been consolidated. In this regard, studies with stem
cells have shown a remarkable potential in cell replacement processes, since they
are an important source of neurotrophic molecules with the ability to stimulate
CHAPTER
67
Recent Advances in iPSCs for Therapy, Volume 3. https://doi.org/10.1016/B978-0-12-822229-4.00014-0
Copyright ©2021 Elsevier Inc. All rights reserved.
damaged neuronal tissue. Preclinical and clinical studies with embryonic and adult
stem cells have allowed researchers to further their studies on a multitude of
neurological diseases. Although further breakthroughs are yet to be made to better
understand the mechanisms underlying the beneficial effects of stem cell therapy,
results seem to point to its therapeutic advantages. Currently, the emergence of
induced pluripotent stem cells as a tool for disease modeling, drug discovery, and
new therapeutic targets has laid the foundation for a promising avenue in the
context of personalized cell therapy alongside new opportunities for regenerative
medicine, including for the treatment for stroke.
Keywords:
Adult stem cells; Cell modeling; Cell reprogramming; Cell therapy; Drug screening;
Embryonic stem cells; Hemorrhagic stroke; Immune modulation; Induced pluripotent stem cells;
Ischemic stroke; Neurological disorders; Neuroprotection; Neurorecovery; Neurorepair; Outcome
stroke models.
1.Introduction
According to the American Stroke Association/American Heart Association, stroke
is defined as “a neurological deficit attributed to acute focal injury to the central
nervous system (CNS) from a vascular cause, including cerebral infarction, intrace-
rebral hemorrhage, and subarachnoid hemorrhage and is a leading cause of
disability and death worldwide”(Sacco et al., 2013). The Global Burden of Disease
reports that stroke (ischemic and hemorrhagic) affects about 14 million people
worldwide each year and is recognized as the second leading cause of death, incur-
ring 5.5 million deaths per year (Collaborators et al., 2018). The incidence of stroke
is normally similar among men and women under 55 years of age, but is signifi-
cantly higher in men than in women aged 55e75 years (Collaborators, 2019).
Improvements in the management of stroke patients in primary care and hospi-
tals, especially in developed countries, have contributed to the reduction in mortal-
ity rates observed in recent decades. However, the incidence of stroke has not
followed the same trend, given the continuous increase in developed countries
and an especially worrying increasing trend in underdeveloped and developing
countries. Demographic changes, the rise of hypertension and obesity cases, and
the emergence of other comorbidities could explain the increase in stroke inci-
dence (Tabl e 4.1 ). In addition, the demographic change expected in Europe over
the next 50 years suggests that this situation will only worsen (Ovbiagele and
Nguyen-Huynh, 2011).
Depending on a number of parameters, such as the nature or size of the lesion,
and the progression or characteristics obtained on neuroimaging, among others,
stroke may be divided into two categories: (i) ischemic stroke, induced by a decrease
in cerebral blood flow caused by the presence of an occluding thrombus in a brain
artery; and (ii) hemorrhagic stroke, defined as all nontraumatic events caused by
subarachnoid or intracerebral hemorrhage due to extravasation of blood followed
by rupture of a vessel, artery, or vein (Amarenco et al., 2009)(Fig. 4.1).
68 CHAPTER 4 Present and future of adult stem cells
From a therapeutic point of view, the control of bleeding and reduction of pres-
sure in the brain represent the main therapeutic strategies used to prevent neuronal
damage after hemorrhagic stroke (Hemphill et al., 2015), while during the acute
phase of ischemic stroke, the main goal is to remove the thrombus, either by throm-
bolysis with recombinant tissue plasminogen activator (rtPA) or by mechanical
thrombectomy (Fig. 4.2), in order to restore the blood flow to the injured area
(Campbell et al., 2019). Indeed, reperfusion therapy is the only treatment currently
available for ischemic stroke; however, less than 20% of patients are suitable for this
treatment option due to the short therapeutic window and limited availability outside
of referral hospitals (Tomsick et al., 2010).
2.Protective and recovery approaches for ischemic stroke
In light of the fact that the treatments for stroke are restricted to recanalization ap-
proaches during the acute phase of stroke, there is a need to develop alternative ther-
apeutic options to limit the severity of the ischemic injury (neuroprotective
approaches) or stimulate the recovery of the brain region affected (neurorecovery
approaches). With regard to neuroprotective approaches, a considerable amount
of research has focused on the development of novel treatments capable of protect-
ing the brain from damage after a stroke, with special attention to the penumbra re-
gion (Fig. 4.3). The penumbra is a region where the affected brain tissue is
susceptible to recovery, so it could be a potential therapeutic target for reducing
Table 4.1 Risk factors in stroke.
Nonmodifiable risk
factors Modifiable risk factors
(i) Ischemic stroke Age Hypertension
Sex Hyperlipidemia
Race Smoking habits
Genetic causes Diabetes mellitus
Cardiac causes
Apolipoprotein B to A1
Diet
Physical inactivity
Genetic causes
a
(ii) Hemorrhagic stroke Age Hypertension
Sex Current smoking
Race Alcohol consumption
Genetic causes Diet
Genetic causes
a
a
Geneeenvironment interactions.
2. Protective and recovery approaches for ischemic stroke 69
the sequelae of the ischemic injury (as long as the alterations derived from the
ischemic cascade have not generated irreversible damage leading to tissue death).
Numerous neuroprotective treatments have been identified that show great potential
in animal models of stroke, such as glutamate antagonists, calcium blockers, stress
oxidative free radicals, or immunomodulators. Unfortunately, nearly all of them
have failed to provide protection in human trials (Castillo et al., 2016).
In the context of neurorepair in stroke pathology, two related processes occur,
neurogenesis and angiogenesis (Kaur et al., 2013). Neurogenesis is the process of
producing new functional neurons from neural stem/progenitor cells (NSCs). This
FIGURE 4.1 Types of stroke.
An ischemic stroke occurs when the blood flow through the artery that supplies oxygen-
rich blood to the brain gets blocked. It can be caused by two types of blockages: in
cerebral thrombosis, the thrombus forms inside the brain vessel, while in cerebral
embolism, a thrombus is formed somewhere in the circulatory system, enters the
bloodstream, and reaches the blood vessels in the brain, effectively blocking them.
A hemorrhagic stroke occurs when a brain vessel loses blood or ruptures. This leaking
blood increases the brain pressure levels, damaging the brain cells, leading to loss of
proper function. Hemorrhagic stroke may be caused due to arteriovenous malformations
or aneurysms.
This image was created using Servier Medical Art.
70 CHAPTER 4 Present and future of adult stem cells
process also includes the proliferation of these endogenous NSCs, their migration
and their differentiation into mature functional neurons. It is now well accepted
that neurogenesis occurs in two distinct regions in the intact human brain throughout
life: the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone
in the dentate gyrus of the hippocampus. In pathological conditions such as ischemic
stroke, enhanced neurogenesis has been reported in animal models of stroke and
even in stroke patients, pointing to a potential avenue for the treatment of ischemic
stroke. However, it is now apparent that neurogenesis is not a stand-alone consider-
ation in the fight for complete functional recovery following a stroke. Angiogenesis,
defined as new microvessel formation via branching off from preexisting vessels, is a
multistep biological process, including the proliferation and sprouting of endothelial
cells, formation of tube-like vascular structures, branching and anastomosis.
It has been reported that both neurogenesis and angiogenesis occur in the brain of
stroke patients and a positive correlation was seen between patient survival and the
density of microvessels. Several findings in addition to this prove that neurogenesis
and angiogenesis are coupled processes after an insult such as ischemic stroke and
should be acknowledged and pursued as concurrent and nonmutually exclusive
events to further develop neurorestorative therapies (Ruan et al., 2015).
FIGURE 4.2 Therapeutic approaches in acute ischemic stroke.
Pharmacological treatment (A) is applied by means of intravenous (IV) administration of
rtPA within the first 4.5 h after symptoms appear. This thrombolytic procedure may also
be applied in a wider therapeutic window when rtPA is administered via intraarterial route
(IA), in combination with mechanical devices, to increase the efficiency of dissolving the
thrombus. In case of an intervention with physical procedures on the occluded vessel (B),
it can be performed by direct rupture of the thrombus, by mechanical thrombectomy, or
by aspiration of the thrombus with a catheter.
This image was created using Servier Medical Art.
2. Protective and recovery approaches for ischemic stroke 71
In order to restore areas of the CNS damaged as a result of brain injury, prom-
ising strategies have been developed that focus on stem cell therapy (Lindvall and
Kokaia, 2011). Stem cells are immature cells with self-renewal capacity that are
able, to various degrees, to differentiate into different lineages. Stem cells present
a series of properties that are central to their therapeutic potential: they migrate to
areas of injury, secrete neuroprotective compounds, and differentiate into other types
of functional and specialized cells (Fortier, 2005). Stem cells can be classified
according to their capacity for differentiation (Zakrzewski et al., 2019)(Fig. 4.4):
(i) Totipotent: they are found at the beginning of embryonic development and can
differentiate into embryonic tissue (endoderm, ectoderm, and mesoderm) and
extraembryonic tissue (placenta, amnion, yolk sac, allantoid, and chorion),
being able to create embryos and form a complete organism.
(ii) Pluripotent: they differentiate into any type of cell or tissue derived from the
three embryonic lineages and sex cells.
(iii) Multipotent: also known as organ-specific cells because they can generate a
complete organ in both embryonic and adult stages; they are obtained from
sources such as bone marrow or umbilical cord blood. In human adult tissues,
they are found in different regions such as the skin, brain, retina and pancreas.
FIGURE 4.3 Illustration of the penumbra concept.
Infarct core (brown): death tissue. Penumbra (yellow): salvageable tissue at risk for
infarction in case of persistence vessel occlusion.
This image was created using Servier Medical Art.
72 CHAPTER 4 Present and future of adult stem cells
(iv) Unipotent: also known as oligopotent; they lack significant potential since they
can only specialize into a single cell lineage.
In addition, stem cells may also be classified according to their origin (Chagas-
telles and Nardi, 2011):
(i) Embryonic stem cells (ESCs): these are pluripotent cells isolated from the in-
ternal mass of an embryonic blastocyst implanted in the uterus. ESCs have the
ability to self-renew and become any type of specialized cell, except for
extraembryonic tissues.
(ii) Adult stem cells (ASCs): this is a large population of undifferentiated cells
housed in the body with the ability to self-renew as well as differentiate into
the mature cell type of the tissue in which they reside. Their progeny is in
charge of cell replacement guaranteeing the homeostasis of the tissue.
(iii) Induced pluripotent stem cells (iPSCs): these are stem cells generated from
adult somatic cells through cell reprogramming. This cell type shares the same
properties as ESCs and is able to differentiate into any cell type in the body.
They are considered to be promising candidates for cell therapy as their
procurement circumvents the ethical issues surrounding the acquisition of
ESCs. In addition, their versatility allows for their differentiation into cellular
phenotype required for a particular treatment or disease.
ESCs are obtained from the internal mass of the blastocyst 4e5 days after fertil-
ization and also from surplus embryos from assisted reproduction clinics (Thomson
FIGURE 4.4 Potential-based hierarchy throughout stem cell development.
Stem cells can be classified according to their plasticity/regenerative potential into
totipotent, pluripotent, multipotent, oligopotent, and somatic cells. Induced pluripotent
stem cells (iPSCs) can be generated from somatic cells and have similar properties to
embryonic stem cells.
This image was created using Servier Medical Art.
2. Protective and recovery approaches for ischemic stroke 73
et al., 1998). They are in the early stages of development and can be differentiated
into any of the three embryonic lineages (Lee et al., 2008). This characteristic has
garnered great interest in the field of cell therapy as a source for the treatment of neu-
ral diseases, among others (Castillo-Melendez et al., 2013). ESCs have been shown
to be able to differentiate into neurons and glia cells after transplantation (Wichterle
et al., 2002). In addition, they can provide trophic support by secreting neurotrophic
factors in the damaged area (Nagai et al., 2010). Preclinical stroke studies of intra-
cerebral murine ESCs transplantation in rats subjected to a middle cerebral artery
occlusion model show that they appear to be responsible for reducing infarct sizes
and improving motor and sensory function in animals (Tae-Hoon and Yoon-Seok,
2012). However, the use of ESCs has always been limited not only by ethical issues
(King and Perrin, 2014), but also due to their high rate of tumor transformation, thus
limiting their application in the study of this disease (Hentze et al., 2009).
3.Adult stem cells: NSCs and mesenchymal stem cells
Cell therapy based on ASCs has become the core of a key approach in the study of
diseases such as diabetes mellitus (Path et al., 2019), heart disease (Katarzyna,
2017), autoimmune diseases (Cipriani et al., 2013), and liver and kidney failure
(Nicolas et al., 2016;Rota et al., 2019). In the case of CNS diseases, cell therapy
has been focused on two types of ASCs: NSCs and mesenchymal stem cells
(MSCs) (Bang et al., 2016). NSCs are a type of multipotent stem cells located in
the brain that generate most of the cells of the CNS. They have the ability to migrate
to the injured area, where they proliferate and mature into well-differentiated and
functional neurons. This cell type is considered the most optimal for cell therapy
treatment in neurological diseases because it has the same cellular origin as the
damaged cells. In addition, they play an important therapeutic role due to their
capacity for the secretion of trophic molecules, immunomodulation and cellular
replacement (Tuazon et al., 2019). On the other hand, MSCs are a type of stem cells
that are housed in the bone marrow. Despite having a limited capacity to differentiate
in comparison to other types of stem cells, they exert a number of beneficial effects
such as participating in the immune response, migrating to affected brain areas and
exerting a paracrine function mediated by the secretion of bioactive molecules that
appear to regulate neurogenic processes (Wagers and Weissman, 2004).
3.1 Protective effects of NSCs in the treatment of stroke
NSCs are located in two regions: the SVZ and the hippocampus (Ihunwo et al.,
2016). They present the ability to self-renew and differentiate into neurons and glial
cells (astrocytes and oligodendrocytes) (Baker et al., 2019). Under different condi-
tions, the cell division of NSCs may be symmetric, in which one cell gives rise to
two new NSCs; or asymmetric, in which at least one of the resulting cells is a neural
progenitor cell (NPC), which has a more limited capacity for self-renewal and whose
74 CHAPTER 4 Present and future of adult stem cells
fate will be more committed to giving rise to precursors of mature cells. Both types
of cells are responsible for the continuous production of new neurons (Vishwakarma
et al., 2014). These cells respond to cell death caused by brain injuries such as Hun-
tington’s disease (HD), Alzheimer’s disease (AD), and stroke (Ul Hassan et al.,
2009). In the context of brain injury, they play a key role in the generation of new
neurons trying to replace those lost during the ischemic process (Koh and Park,
2017). Currently, several studies in animal models of ischemic stroke demonstrate
neurofunctional improvement as a consequence of the transplantation of exogenous
NSCs that compensates for the deficit of endogenous NSCs and a reduction in the
volume of infarct due to the improvement of the inflammatory microenvironment
of the injured regions (Zhang et al., 2019). However, a limitation of these newly
differentiated neurons is their poor ability to migrate to the cortex, resulting in an
inadequate repair of the dead tissue (Kaneko et al., 2011). To address this limitation,
different strategies have been tested to improve the recovery effect of the NSCs used
as supplement to trophic or angiogenic factors (Hsu et al., 2007).
Another key property that makes NSCs a promising tool in cell therapy is their
ability to release neurotrophic factors and extracellular vesicles containing bioactive
molecules. With regard to this ability, recent studies have shown that in an in vitro
preconditioning medium, the NSCs cause the release of a series of neurotrophic fac-
tors and extracellular vesicles with significant therapeutic efficacy when they are
administered in different stroke animal models. Indeed, interest in this approach
has increased in the last years for the treatment of brain injuries and neurological
diseases (Uzun et al., 2010;Vogel et al., 2018). Despite current preclinical findings
that seem to demonstrate the recovery effect of NSCs in stroke, there remains no
consensus opinion about the routes of administration, dose, and therapeutic window,
which is critical information for future clinical applications (Krause et al., 2019;
Huang et al., 2014;Luo et al., 2017;Mine et al., 2013;Yang et al., 2018;Obernier
and Alvarez-Buylla, 2019;Rodriguez-Frutos et al., 2016). In addition, although
NSCs would be ideal in their neurorepair function due to their strong role in neuro-
genic differentiation, the ethical complications involved in obtaining them remain a
very limiting factor (Lindvall and Kokaia, 2006). Currently, the most promising al-
ternatives to this cell type are MSCs and iPSCs. Both of these cells types are capable
of performing the functions stem cells have in the treatment of stroke (Fig. 4.5),
including neuroprotection, cell replacement, immunomodulation, and the stimula-
tion of angiogenesis in the regions of the brain that have been damaged
(Fernandez-Susavila et al., 2019).
3.2 Therapeutic potential of MSCs in stroke: immune modulation,
neuroprotection, and neurorepair
MSCs are multipotent stromal progenitor cells and precursors of the bone, cartilage,
and adipose tissue (Bianco et al., 2008). They have also the ability to transdifferen-
tiate into other lineages such as neurons and glia (Bianco et al., 2008;Scuteri et al.,
2011). They are mainly housed in the bone marrow, where they coexist with another
3. Adult stem cells: NSCs and mesenchymal stem cells 75
stem cell population, such as the hematopoietic stem cells (Feng et al., 2011)
(Fig. 4.6).
They can be obtained mainly from the bone marrow (BM-MSCs), but also from
placenta, umbilical cord (UC-MSCs), and adipose tissue (AT-MSCs) (Dulamea,
2015). Although these are all MSCs, it should be noted that the sources of cellular
FIGURE 4.5 Mechanisms of action of cell therapies based on MSCs and iPSCs for the treatment
of stroke.
The microenvironment generated after suffering an ischemic stroke can be modulated
through multiple pathways. MSCs and iPSCs stimulate the migration of endogenous NSCs
into the damaged region to promote differentiation into new neurons contributing to the
replacement of lost cells. They also have an immunomodulatory role in the regulation of
the microglia through the blockage of type 1 macrophages (M1) and the activation of type
2 macrophages (M2). M1 is a proinflammatory effect or phenotype, while M2 is involved
in the control of inflammation and tissue repair. In order to avoid an aggravation of the
ischemic region by the formation of toxic molecules, these cells inhibit cell degradation as
a neuroprotective mechanism. In addition, the formation of new blood vessels in the
ischemic region has a positive effect on synaptic plasticity. All these effects are mostly
produced by the soluble factors released by the stem cells, although they may also be due
to the interaction between cells.
This image was created using Servier Medical Art.
76 CHAPTER 4 Present and future of adult stem cells
origin cause variations in the properties and functions of these cells (Wegmeyer
et al., 2013;Kozlowska et al., 2019). Compared to other types of SCs, BM-MSCs
have a limited differentiation capacity (Pittenger et al., 2019) but present enhanced
beneficial properties compared to the other SCs, such as migration to sites of inflam-
mation, low levels of immunogenicity, a higher rate of secretion of bioactive mole-
cules, and neurogenic properties (Pittenger et al., 2019;Li et al., 2000;Chenet al.,
2001,2002). A study on AD observed that MSCs contributed to the reduction of
b-amyloid deposits and improved synaptic transmission in animal models (Bae
et al., 2013). In the case of an animal model of Parkinson’s disease (PD), the use
of MSCs achieved a cellular replacement of dopaminergic neurons (Parmar et al.,
2020). In general, animal ischemic stroke models showed improved neurofunctional
performance compared to untreated groups (Vahidy et al., 2016). The MSCs seem to
exert a neuroprotective effect through the modulation of the inflammatory response
(Wang et al., 2018). Regarding their ability to differentiate into neural lineages,
when these cells are cultured in vitro, they maintain their ability to differentiate
into these neural lineages and express neuronal and glial markers (Hernandez
et al., 2020), but, once they are transplanted in vivo, the survival and differentiation
rate of these cells are compromised (Li et al., 2008). These results suggest that
although MSCs are able to differentiate into neural lineages, their therapeutic effects
rely more on their ability to secrete trophic factors, which promote and enhance pro-
cesses of neuroprotection, angiogenesis, and endogenous neurogenesis, and
contribute to diminishing the immune and inflammatory responses (Li et al.,
2002,2008). The results of the clinical trials proposed for the treatment of stroke
with MSCs are quite variable (Zheng et al., 2018). Some of these trials do not appear
to demonstrate a clear beneficial effect, while others show some clinical
FIGURE 4.6
MSC differentiation into different cell types.
This image was created using Servier Medical Art.
3. Adult stem cells: NSCs and mesenchymal stem cells 77
improvement (Chang et al., 2013). However, all these studies agree on the safety and
absence of adverse effects following MSC treatment. It is also important to note that
MSCs have been widely studied in ischemic stroke, but less so in hemorrhagic stroke
(Turnbull et al., 2019).
Despite the special attention paid to MSCs as a promising therapeutic candidate
for stroke, parameters such as administration route or cell dosage remain under
discussion.
Relatively few studies have compared the different possible routes of administra-
tion of stem cells. The first studies that used stem cells for cerebrovascular diseases
were aimed at neuronal replacement, so they chose intraparenchymal injection as the
most direct route for cell engraftment. These studies showed that the stem cells not
only survived, but also migrated to the affected zone (Darsalia et al., 2007). Howev-
er, this choice is not the most suitable due to the need to create a cranial window as
well as the brain parenchyma damage involved, which is not convenient for stroke
patients.
The main alternatives to this route of administration are the vascular routes,
either intraarterial (i.a.) or intravenous (i.v.), which are currently the most widely
used for cell delivery. Intravenous injections are minimally invasive, and cell
tracking studies following this route have shown that most administered cells remain
trapped in the lungs, liver, and spleen, indicating that a reduced number of cells
reach the brain. On the other hand, i.a. administration is a promising strategy to
direct the majority of injected cells to the brain, but remains a risky administration
route, with the fate of injected cells following this route remaining unknown due to
inconsistencies across reported results.
Whether one route is more efficient than the other is unclear and depends on the
cell type used. Thus, in some studies, it was found that the injection of NPCs by i.a.
through the carotid artery presented a higher migration rate and a wider distribution
pattern than by i.v. administration. Nevertheless, the mortality rate for this i.a. deliv-
ery was significantly higher (41%) than with i.v. injection (8%) (Li et al., 2009).
However, in other studies with B-MSCs and bone marrow mononuclear cells, there
was no greater mortality or greater recovery of infarct volume of one route with
respect to the other (Yang et al., 2013;Vasconcelos-dos-Santos et al., 2012).
The size of the stem cells is also a critical parameter when passing through the
lungs and should be taken into consideration when selecting the best route of admin-
istration. As an example, when using MSCs, the majority of them get trapped in the
lungs, while the NSCs have a pass-through rate that is two-fold higher (Fischer et al.,
2009).
In an attempt to clarify the discrepancies with regard to the best cell administra-
tion route in the treatment stroke, an experimental study aimed at investigating
whether MSCs were able to reach the brain following i.a. or i.v. administration after
transient cerebral ischemia in rats, while evaluating the therapeutic effects of both
routes, was recently carried (Argibay et al., 2017).
78 CHAPTER 4 Present and future of adult stem cells
These findings showed that MSCs were found in the brain following i.a. but not
i.v. administration in ischemic rats (Fig. 4.7). However, the i.a. route increased the
risk of cerebral lesions (microstrokes) (Fig. 4.8) but did not improve functional re-
covery, while the i.v. delivery produced functional recovery and was safe, though
MSCs did not reach the brain tissue. This implies that the benefits of treatment
are not a result of brain MCS engrafting after stroke (Argibay et al., 2017).
Cell dose is another issue to be considered for both i.a. and i.v. administration,
which has not yet been well elucidated. In line with other studies, it was esti-
mated that doses higher than 1e2.5 10
5
cell/mL administered i.a. as a bolus
infusion significantly increased the risk of arterial occlusion (Argibay et al.,
2017;Hosoda et al., 2014)(Fig. 4.6), while other studies have estimated that
doses of up to 3 10
7
cells are safe (Yang et al., 2013;Vasconcelos-dos-Santos
et al., 2012).
In summary, MSCs have demonstrated their advantageous properties regarding
functional recovery in animal models of stroke, and these effects have also been veri-
fied in the clinical practice (Lee et al., 2010). Despite these promising results, more
studies are warranted to understand the cellular mechanisms underlying their thera-
peutic effects and to determine the dose, route, and time of administration.
FIGURE 4.7
Magnetic resonance image (MRI) of one brain slice of a rat, 4 h after i.a. administration of
MSCs labeled with magnetic particles. Labeled cells can be observed as black punctate
patterns in the brain’s right hemisphere. Scientific Reports. 2017; 7:40,758.
3. Adult stem cells: NSCs and mesenchymal stem cells 79
FIGURE 4.8
Upper figure: Multifocal ischemia is observed all along the brain after intraarterial (i.a.)
administration of 1 10
6
MSCs (indicated with yellow arrows). Lower figure: Electron
transmission micrograph of the rat brain cortex 4 h after i.a. delivery of MSCs with
magnetic particles, showing the arterial occlusion caused by the cell administration. (A)
Dilated brain vessel surrounded by neuropil (Scale bar 5 mm). (B) Magnification of vessel
dilation in (A) Red blood (RB) corpuscles are on the right, and platelets (PTs) and MSCs
can be observed inside the vessel (Scale bar 2 mm). (C) Magnification of the upper vessel
expansion in (B) (Scale bar 1 mm). (D) Longitudinal section of a brain vessel in which two
labeled cells can be observed. (Scale bar 2 mm). Scientific Reports. 2017; 7:40,758.
80 CHAPTER 4 Present and future of adult stem cells
4.Induced pluripotent stem cells: a multifaceted role in the
study of neurological diseases
The iPSCs were discovered by two researchers who were awarded the Nobel Prize in
Physiology or Medicine in 2012 for their findings in this field (Yamanaka, 2012).
The first researcher was John B. Gurdon, who, by using a technique called cell
nuclear transfer, succeeded in transplanting the nucleus of a somatic frog cell into
an enucleated frog egg, which was able to give rise to tadpoles, suggesting that
the nucleus contained all the information necessary to form a new individual,
even in the nucleus of the somatic mature cells (Gurdon, 1962). The second
researcher was Shinya Yamanaka. This researcher and his colleagues, based on
the conclusions of the Gurdon experiments, managed to turn a somatic cell into a
cell with embryonic characteristics by a method called cell reprogramming. This
technique required the use of four essential factors: Oct4, Sox2, Klf4, and c-Myc,
widely known as the four Yamanaka’s factors. With this technique, a somatic differ-
entiated cell would be converted into a pluripotent cell (Takahashi and Yamanaka,
2006). To obtain these iPSCs, a biological sample from the donor (somatic cells
or biopsy) is required and the introduction of these reprogramming factors is done
by using (i) viral vectors (which have minimal technical requirements and allow
for the relatively stable reproducibility of reprogramming, but are not yet amenable
to clinical adaptations); (ii) episomal DNA vectors (which have low manufacturing
costs and no viral derivatives that could harm the patient, but which incur a risk that
the cells will identify the episomes as fragments of their own DNA, integrating them
into their genomes, becoming aberrant cells, and thus making them difficult to trans-
late); (iii) RNA-based methodologies (which have been proven to have broad
methodological advantages, but also a number of limitations, such as low integration
in the number of donor cells and inefficiency in the reprogramming of blood cells)
(Seki and Fukuda, 2015;Cherkashova, 2020)(Fig. 4.9).
Over the last few years, several advances unraveling the behavior of these iPSCs
have been made, opening up the possibility to develop in vitro models (known as
“disease-in-a-dish” models) of several diseases (Doss and Sachinidis, 2019). With
these models, by cotransfecting the key pluripotency-associated factors (OCT3/4,
SOX2, C-MYC, and KLF4), adult somatic cells can be reprogrammed to iPSCs
that have the potential to differentiate into the cell type of interest. iPSC-derived
cells carry the genetic information of the donor and therefore represent patient-
specific disease models to be used to recapitulate the human pathology and to model
multiple diseases (including neurological diseases) for cell or molecular analysis, for
in vitro high-throughput drug screening (Bahmad et al., 2017;Ellis and Bhatia,
2011), or to test drug candidates directly in the patients’ own cells for diseases
that lack effective therapies (Farkhondeh et al., 2019)(Fig. 4.10). This novel cell
strategy has already been in use in different neurological pathologies. For example,
dermal fibroblasts from PD patients were successfully reprogrammed into iPSCs and
were differentiated into dopaminergic neurons, which were later implanted in PD
4. Induced pluripotent stem cells: a multifaceted role in the study 81
models in rats, resulting in an improvement in functional deficits (Kriks et al., 2011).
In HD disease, the generation of HD-specific iPSCs that conserve the expression of
the CAG nucleotides sequence, which is the underlying cause of this pathology, was
reported for the first time and is useful for drug screening (Park et al., 2008). In the
case of amyotrophic lateral sclerosis (ASL), several authors have obtained iPSCs
from fibroblasts of patients with the disease, which were successfully differentiated
into motor neurons, since this is the cell type lost as a consequence of the disease
(Dimos et al., 2008). Generating an ASL model would also usher in progress in
the study of the pharmacological efficacy of riluzole, the only drug approved by
the American Food and Drug Administration for the treatment of this disease
(Jaiswal, 2017). In AD, viable neurons from AD patients were generated, which
is a challenge in this pathology due to its great limitations in achieving a model
that approximates the sporadic form of the disease (Yang et al., 2016).
In addition, with the recent development of gene editing technologies such as
CRISPR/CAS9, it is possible to repair mutations with high efficiency in human
FIGURE 4.9 Overview of reprogramming methods.
Depending on the needs of the research project, the form of cell reprogramming may vary.
If the objective is cell therapy, the method of choice should not leave any residual
transgenic sequences from the viral vectors. The researchers using iPSCs for drug
screening may use a less strict methodology. In addition, the type of cell used as a starting
point must be considered, as this will lead to methods that are more successful in terms of
reprogramming efficiency.
This image was created using Servier Medical Art.
82 CHAPTER 4 Present and future of adult stem cells
mutant iPSCs, useful for the investigation of the molecular and cellular mechanisms
underlying the pathology and providing a new vision of cell therapy for many human
genetic diseases (Huang et al., 2019). For example, in iPSC lines originated from
patients with juvenile HD, CRISPR techniques have been used to correct the gene
disorder in these iPSCs, offering a new perspective for treatment based on the use
of autologous corrected cells (Doss and Sachinidis, 2019).
Finally, recent studies have shown promising results with 3D cellular models
generated from iPSCs, known as organoids (Lancaster and Knoblich, 2014). With
this approach, interactions between cells and cell organization and signaling are
more accurately represented in these systems (Skardal et al., 2016), which would
imply a more translational and accurate approach, better reflecting the biology of
the disease in question (Lancaster et al., 2013). To date, work continues to be carried
out to strengthen the inherent weaknesses to working with iPSCs (Rehakova et al.,
2020).
4.1 iPSCs in stroke
In the first few years following Yamanaka and Takhashi’s iPSC research, the exper-
iments and preclinical studies on stroke assessed the effect of a direct injection of
iPSCs into the affected region. Several studies reported improvements both in infarct
FIGURE 4.10 Applications of iPSCs for drug screening, disease modeling, and cell therapy.
IPSCs obtained from a biopsy or somatic cells of a patient can serve as in vitro disease
models in vitro that allow for the study of molecular mechanisms as well as the detection
of drug candidate molecules. In addition, with the genetic editing tools currently available,
it is possible to correct mutations in patient-derived iPSCs and use them as material for
cell transplantation.
This image was created using Servier Medical Art.
4. Induced pluripotent stem cells: a multifaceted role in the study 83
volume reduction and in functional recovery (Jiang et al., 2011;Chen et al., 2010;
Tornero et al., 2013). In addition, improvements in the neurological function and
survival rate in hemorrhagic stroke were described (Arumugam et al., 2015). How-
ever, most of these improvements attributed to the iPSCs were really due to a differ-
entiation of these iPSCs into different ASCs in the affected region, which were the
cells that actually performed the repairing process and incurred the beneficial effects
(Buhnemann et al., 2006;Takagi et al., 2005).
One of the main problems of these studies limiting clinical translatability has
been the formation of teratomas or tumorigenicity in the following weeks after
cell administration. This is due to the environmental effects of the niche in which
the iPSCs are implanted. Formation of teratomas can also occur by the transforma-
tion of residual iPSCs that remain in the implanted area and can develop into benign
teratomas after some time (Lee et al., 2013a,b). In this respect, some studies have
attempted to solve this issue. For example, a study published by Chen and colleagues
compared the formation of teratomas after the injection of iPSCs with and without
fibrin glue as a vehicular agent into the subdural region instead of injecting it right
into the cortex. Injecting iPSCs by themselves always incurred the formation of ter-
atomas after 4 weeks, while injecting iPSCs with fibrin glue did not. The authors
pointed out that this was not only due to the fibrin glue, but also due to the fact
that the subdural region was not an appropriate niche for inducing an uncontrolled
growth of the iPSCs (Chen et al., 2010). Furthermore, another study demonstrated
the possibility of inhibiting the formation of tumors due to residual iPSCs by treat-
ment with inhibitors of specific proapoptotic stem cell routes, inducing their
apoptosis and erasing them, while the derived differentiated cells survived and main-
tained their functionality (Lee et al., 2013b).
Currently, the proper differentiation of iPSCs to cell lines of interest (e.g.,
neuronal, epithelial .) with reduced division capacity seems to be the most conve-
nient way to address these limitations.
4.2 iPSCs and CADASIL
A total of 25%e30% of stroke cases are caused by cerebral small vessel diseases
(SVDs). Although stroke is a well-studied disease and its mechanisms and underly-
ing processes are well known, a number of diseases may be the cause of cerebral in-
farcts such as SVDs, which lack in treatment approaches and a deep understanding
(Joutel and Faraci, 2014). These diseases, due to their low incidence rate, are not
usually the object of much intense study, which is why knowledge remains limited.
This means that there are no solutions or treatments for them, even though they are
frequently a cause of stroke. The few studies that have been carried out on these dis-
eases have focused on monogenic variants of SVDs in order to provide valuable in-
sights into the molecular mechanisms underpinning idiopathic SVDs.
One of these is the cerebral autosomal dominant arteriopathy with subcortical in-
farcts and leukoencephalopathy, commonly known as CADASIL. CADASIL patients
develop primary symptoms, which include leukoencephalopathy, migraines with
84 CHAPTER 4 Present and future of adult stem cells
aura, recurrent ischemic strokes, motor disability, and dementia. There is currently
no treatment for this disease. These symptoms are caused by progressive weakness
in the small brain vessels, which spatially coincide with granular osmiophilic mate-
rial (GOM). This vessel weakness is due to the continuous aberrant accumulation of
the extracellular component of the Notch3 protein membrane in the GOM (Ayata,
2010). In CADASIL, the NOTCH3 gene has a mutation in one of its exons that leads
to a loss or a gain of a cysteine residue. This mutation leads to an incorrect metabolic
process in which Notch3 signal is triggered, resulting in the accumulation of the
extracellular domain of the protein in the GOM (Ayata, 2010;Coupland et al.,
2018)(Fig. 4.11).
Although there exist several mouse models of CADASIL (knockouts for several
different mutations), no solution has been found to address the progressive accumu-
lation of the extracellular domain of Notch3. As such, iPSCs may shed light on this
FIGURE 4.11
T2-weighted MRI images from the brain of a patient diagnosed with CADASIL. White
arrows indicate microangiopathies.
Images kindly provided by Prof. Jose
´Vivancos from the Neurology Department of the Hospital La Princesa
(Spain).
4. Induced pluripotent stem cells: a multifaceted role in the study 85
FIGURE 4.12 Characterization of an iPSC cell line derived from a blood sample from a patient
with CADASIL.
(A) Alkaline phosphatase test was used to detect pluripotent colonies. (B)
Immunofluorescence analysis of the expression of the membrane and nuclear
pluripotency markers (Tra-1-60, SSEA4, SOX-2, OCT-4, and NANOG.
86 CHAPTER 4 Present and future of adult stem cells
disease. The main cell types affected in CADASIL are vascular smooth muscle cells
(VSMCs), which express the mutation, and vascular endothelial cells (VECs), which
interact with the VSMCs. With cell modeling, it would be possible to generate these
cells from iPSCs reprogrammed from the adult somatic cells of a CADASIL patient
and perform mono and cocultures that would allow the study and better understand-
ing of the molecular mechanisms underlying this disease while clarifying the
confusing and sometimes contradictory information that the mouse models provide
(Coupland et al., 2018). In addition, drug screening may be carried out using these
cell cultures to see if any drug is able to slow down or even stop the accumulation of
the extracellular domain of Notch3. Lastly, it would also be possible, by genetic edit-
ing, to repair the mutation within the iPSCs and, once differentiated into VSMCs,
administer them in Notch3 knockouts mice to see if the healthy VSMCs replace
the damaged endogenous ones.
Currently, several studies are focused on the reprogramming of adult somatic
cells from a patient with CADASIL into iPSCs, proving that the NOTCH3 mutation
is not a limitation to the reprogramming (Ferna
´ndez-Susavila et al., 2018;Hamasaki
et al., 2012;Ling et al., 2019)(Fig. 4.12). VSMCs and VECs differentiated from
CADASIL-specific iPSCs showed gene expression changes associated with disease
phenotypes. All these studies have agreed that the generation of this iPSC line offers
an unprecedented opportunity for studying and modeling both CADASIL and other
pathologies related to the vascular risk of stroke (Ferna
´ndez-Susavila et al., 2018;
Hamasaki et al., 2012;Ling et al., 2019;Kelleher et al., 2019;Minakawa et al.,
2020)(Fig. 4.13).
5.Conclusion and future perspectives
In the last decade, numerous investigations have been developed that support the
enormous potential of stem cells as a fundamental axis in the treatment of stroke
by addressing both neurorepair and neuroprotection. In addition, they have served
to resolve certain aspects, such as the optimal number of cells or the most appro-
priate route of administration according to cell type, thereby consolidating the bases
for clinical translation. However, there remain many unsolved questions in the field
of stroke, largely because it is a pathology that is not defined as chronic and cannot
be treated over time. The therapeutic window in this disease continues to be a
=
(C) Immunofluorescence analysis of the three germ layers derived from iPSCs. (D)
Expression levels of genes encoding transcription factors essential to maintain pluripotent
embryonic stem cell phenotype (SOX2, OCT4, and NANOG). (E) Analysis of the presence of
the NOTCH3 mutation in iPSCs, previously diagnosed in the CADASIL patient. (F) Karyotype
of the new reprogrammed cell line showing a normal chromosomal profile in the iPSCs cell
line derived from a blood sample. Stem Cell Res. 2018; 28:16e20.
5. Conclusion and future perspectives 87
tremendous limiting factor, not only in finding effective therapies, but also in incor-
porating assessments and treatments into routine clinical practice. That is one of the
main reasons why drug treatments and mechanical procedures are still available only
to a small number of patients.
Cell therapy in stroke is based on two central axes, NSCs and MSCs. Knowl-
edge on the mechanisms of action of these cells has been growing over the years,
yet there is a need for a greater number of clinical trials to be undertaken to allow
us to better understand the suitability of stem cells for use in clinical practice.
However, cell therapy continues to be promising both for the protection of injured
neural tissue during the acute phases of stroke and for the replacement of lost tissue
either directly or by improving endogenous mechanisms in the chronic phase.
These effects seem to be exerted mainly by the extracellular vesicles that these
cells are able to release into the circulatory system, acting without having to enter
the brain.
FIGURE 4.13 Summary diagram of the cell reprogramming and cell differentiation process
used for CADASIL.
Human peripheral blood mononuclear cells (PBMCs) were obtained from a CADASIL
patient’s blood and reprogramming into iPSCs using the Sendai virus (nonintegrated
reprogramming system), to deliver the Yamanaka factors OCT4, SOX2, KLF4, and c-Myc.
After 15 days the first clusters of stem cells are observed. Once the iPSCs culture is
stabilized, the colonies can be used for differentiation to the cellular phenotypes of
interest, such as vascular smooth muscle cells (vSMCs) and vascular endothelial cells
(vECs) in case of CADASIL disease. In the left part of this image we can see the changes in
the morphologies of iPSCs until to generate vSMCs and vECs at 15 and 30 days.
88 CHAPTER 4 Present and future of adult stem cells
With regard to the other and more recently developed field of cell therapy in
stroke, the suitability of the use of iPSCs still remains unclear. The possibility
offered by iPSCs to obtain mature and functional neurons continues to provide a
hopeful approach to the repair of damaged tissue using endogenous resources
without the need for exogenous cells. In addition, research on iPSCs has improved
our understanding of the cellular and molecular mechanisms of neurological disor-
ders and in turn helped develop platforms for the discovery of new drugs and
therapeutic targets. Unfortunately, and despite the fact that there are many studies
that support the positive effects of iPSCs, there remain major limiting factors to
be overcome. The most worrying one is the risk of formation of a tumor that is
not necessarily ubiquitous in the area of cell transplantation but can form in any
other part of the body. However, while ongoing research is focused on ensuring
the clinical translation of iPSCs, this cell technology has provided considerable
advantages compared to other classical models for studying diseases, including
neurological diseases such as stroke. This new emerging application of iPSCs is
in vitro human disease modeling can significantly improve the never-ending search
for new pharmacological cures.
In conclusion, cell therapy is still met with a set of preclinical barriers coupled
with clinical challenges that need to be overcome to be more effective and efficacious.
Therefore, it is important to combine both basic and clinical efforts such that cell ther-
apy can become a clinical reality and ensure the successful treatment of stroke.
Glossary
AD Alzheimer’s disease
ASCs adult stem cells
ASL amyotrophic lateral sclerosis
AT-MSCs adipose tissue mesenchymal stem cells
BM-MSC bone marrow mesenchymal stem cells
CADASIL cerebral autosomal dominant arteriopathy with subcortical infarcts and
leukoencephalopathy
CNS central nervous system
ESC embryonic stem cells
HD Huntington’s disease
IC intracerebral
IN intranasal
iPSCs induced pluripotent stem cells
IV intravenous
MSC mesenchymal stem cells
NPCs neural progenitor cells
NSC neural stem cells
PD Parkinson’s disease
rtPA recombinant tissue plasminogen activator
SVZ subventricular zone
Glossary 89
UC-MSC umbilical cord
VEC vascular endothelial cells
VSMC vascular smooth muscle cells
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References 95
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Stem cell therapy in
Alzheimer’s disease 5
Milena Pinto, PhD, Christian Camargo, MD, Michelle Marrero, MD,
Bernard Baumel, MD
Department of Neurology, University of Miami Miller School of Medicine, Miami, FL,
United States
Chapter outline
1. Introduction ................................................................................................ .........98
2. Preclinical studies on the use of stem cells in dementias.....................................100
3. Endogenous approach ........................................................................................100
4. Exogenous approach ..........................................................................................101
4.1 Classification of stem cells for exogenous therapeutic approaches ........... 101
4.2 Embryonic stem cells .......................................................................... 102
4.3 Induced pluripotent stem cells ............................................................. 102
4.4 Neuronal stem cells............................................................................. 103
4.5 Mesenchymal stem cells ...................................................................... 104
4.5.1 Mechanisms of action ................................................................. 109
4.5.2 Homing and paracrine effects....................................................... 110
4.5.3 Neuroinflammation ..................................................................... 110
4.5.4 Neurogenesis ............................................................................. 112
4.5.5 Mechanisms of decreased Ab deposition ....................................... 112
4.6 MSCs and Alzheimer’s disease: preclinical experience ............................ 113
4.6.1 In vitro....................................................................................... 113
4.6.2 In vivo ....................................................................................... 113
5. Previous human experience with MSCs ............................................................... 114
5.1 Choice of stem cell for clinical studies .................................................. 114
5.2 Use of human MSCs in neurodegenerative diseases................................ 115
5.2.1 Safety: human phase 1 clinical trials in neurological disease............. 115
5.2.2 Efficacy: phase 2 clinical trials ...................................................... 115
5.2.3 Source of cells ............................................................................ 117
5.3 Route of administration considerations.................................................. 117
5.4 Cell dosage ......................................................................................... 119
5.4.1 Single versus multiple dosage ....................................................... 119
5.5 Concluding remarks ............................................................................. 121
References .............................................................................................................122
CHAPTER
97
Recent Advances in iPSCs for Therapy, Volume 3. https://doi.org/10.1016/B978-0-12-822229-4.00008-5
Copyright ©2021 Elsevier Inc. All rights reserved.
Abstract
This chapter presents the scientific rationale and evidence for using stem cells to
treat Alzheimer’s disease. There is sufficient evidence that these cells are effective
in reducing inflammation as well as in promoting regeneration. The authors outline
the preclinical work that justifies stem cell therapeutic trials in humans. The various
stem cell types used in preclinical studies are discussed. Their mechanism of action
in in vivo animal studies and the results of these studies are discussed. Then, the
human experience in using transplanted stem cells is reviewed. We examine what
cell properties are necessary to treat Alzheimer’s disease. We describe what cell
types have the most compelling evidence today for their safe use in clinical trials.
Some of these lessons have been learned from the experiences of treating other
neurological diseases with stem cells.
Keywords:
Alzheimer’s disease; Differentiation; MSC; Nervous system; SGZ; Stem cell.
1.Introduction
Neurodegenerative disorders are a heterogeneous group of diseases characterized by
the progressive destruction of neural cells within the nervous system. Decades of
arduous scientific research has been conducted in the pursuit of trying to slow
down the progression and ultimately cure these devastating diseases. Given their
innate capacity to differentiate into multiple cell lineages and to regenerate neurons
that have been destroyed by disease, stem cells provide hope and promise for future
therapeutic options. Stem cells have essential properties that make them unique:
they are capable of cell renewal, they have multilineage differentiation capacity,
and they can combat neuroinflammation.
For many decades, scientists had the belief that the nervous system had no capac-
ity of regeneration after cell death. However, research has shown that even though
the generation of new cells in the nervous system is reduced in adults, neurogenesis
persists in two major zones in the adult brain: the subventricular zone (SVZ), a
germinal zone surrounding the lateral ventricles, and the subgranular zone (SGZ)
in the hippocampus (Ghosh, 2019). Neural stem cells (NSC) inhabiting these zones
have the innate capacity to regenerate neurological injury by migrating to sites of
neurological damage leading to neurogenesis in these regions and by enhancing
neuronal communication through improved synaptic formation and transmission
(Bae et al., 2013).
For instance, research studies have shown that neurogenesis takes place in the
SVZ following ischemic stroke in nearby regions such as the striatum or overlying
cortex (Macas et al., 2006). Similarly, the rate of neural proliferation in the SGZ in-
creases during chronic neurodegenerative diseases such as Alzheimer’s disease
(AD) (Jin et al., 2004).
AD, the most common form of dementia, is characterized by the slow and pro-
gressive deterioration of cognitive function, with significant impairment in memory
98 CHAPTER 5 Stem cell therapy in Alzheimer’s disease
and orientation. We now know that AD is characterized by the accumulation of Ab
(amyloid-beta) plaques and neurofibrillary tangles leading to destruction of neurons
and synaptic loss. It has been well established that the hippocampus is one of the first
and most significant regions to be affected in the classic forms of AD. Given its role
in the formation and consolidation of new memories, the hippocampus is an essen-
tial part that allows mammals to remember new information. Once there is neuronal
loss caused by disease, patients present with the classical clinical features including
short-term memory loss and spatial disorientation.
Research studies have shown that there is neuronal dysfunction and downregu-
lation of neurogenesis in the SGZ of the hippocampus even in the early stages of
the disease (Oh et al., 2015). There are different mechanisms in which hippocampal
neurogenesis is inhibited, including neuroinflammatory pathways and downregula-
tion of essential transcription factors such as interferon-y and transcription factor nu-
clear factor-kB (Zheng et al., 2013). Mesenchymal stem cells (MSCs) may have
modulatory effects on Wnt signaling pathways, which may have neuroprotective ef-
fects and augmentation of hippocampal neurogenesis in patients with AD. Addition-
ally, it has been shown that MSCs may play an important role in reducing
neuroinflammation in the nervous system by suppressing the expression of proin-
flammatory molecules, such as interleukin IL-1, IL-6, and tumor necrosis factor-a
(Zhang et al., 2016). By suppressing the inflammation cascade, MSCs may have
an important role in targeting common neurodegenerative diseases such as AD
and frontotemporal dementia (FTD).
Research has shown that intravenous infusion of autologous human adipose
derived stem cells (hASCs) leads to improved cognitive function and decreased am-
yloid plaques and AB levels in mice with AD pathology (Kim et al., 2012b). Simi-
larly, Lee JK found significant reduction on tau phosphorylation and amyloid load as
well as prevention of cognitive decline in AD mice after intracerebral transplanta-
tion of bone marrowederived MSC (Lee et al., 2010b).
Tauopathies are a group of neurodegenerative diseases that are characterized
pathologically by the deposition of phosphorylated microtubule-associated protein
tau (MAPT) in the human brain. Tau protein is an essential component of microtu-
bule stabilization and once the protein becomes hyperphosphorylated, it forms insol-
uble aggregates known as neurofibrillary tangles, which are toxic to neurons. By
disrupting cell function and axonal transport, it leads to neuronal death and perpet-
uation of neuroinflammation. This abnormal tau deposition occurs in neurodegener-
ative diseases such as some types of FTD, corticobasal degeneration, progressive
supranuclear palsy (PSP), AD, among others. These diseases have a very complex
and diverse clinical presentation, which may include cognitive deterioration in mul-
tiple domains, motor symptoms as well as behavioral and psychiatric manifestations.
Given the common culprit that is present in these diseases (i.e., neuroinflammation
and cell death), stem cell therapy may target tauopathies.
By these complex mechanisms, stem cell therapy provides a novel therapeutic
option that may contribute to the treatment of several neurodegenerative diseases.
1. Introduction 99
2.Preclinical studies on the use of stem cells in dementias
Dementias are a group of debilitating disorders characterized by progressive cogni-
tive decline, behavioral disturbances, and loss of function of daily life. AD is the
most common cause of dementia, for which, as of today, there is no cure.
The use of cholinesterase inhibitors (donepezil, rivastigmine, and galantamine)
and an NMDA (N-methyl-D-aspartate) receptor antagonist (memantine) is the stan-
dard treatment for AD, but these agents are effective only on symptoms
management.
Over the past decades, clinical trials have focused on two main neuropatholog-
ical targets: extracellular Ab amyloid plaques and intracellular neurofibrillary tan-
gles of hyperphosphorylated tau (Liu et al., 2019). Drugs targeting Ab
accumulation and amyloid plaques (b- and g-secretase inhibitors, antibodies, or
peptides against Ab fragments) showed no or little effects (Cummings et al.,
2019) and results from anti-phospho tau therapy (LMTM, anti-tau antibodies) are
equivocal. Targeting neuroinflammation has also been tested in clinical trials
with disappointing results, probably due to the inhibitory effects on microglia
(Cummings et al., 2019).
In the last decade, preclinical studies on animal models suggested the use of stem
cells to treat dementias. Their transplantation has been proven to enhance neuro-
transmission, promote neurogenesis, and reduce neuroinflammation, as well as
decrease the accumulation of misfolded proteins (Wang et al., 2019).
Two main approaches have been tested in preclinical studies of stem cell therapy
for dementia: an endogenous approach, in which, in order to compensate for neuro-
degeneration, resident stem cells within the adult brain are stimulated pharmacolog-
ically or with gene therapy (Lopez-Toledano and Shelanski, 2007;Sun et al., 2006);
and an exogenous approach, in which stem cells are introduced from external sour-
ces. The introduction of exogenous stem cells aims to restore the physiological
neuronal circuits by either acting in a paracrine way or by an in situ action.
We will here review the preclinical studies on the use of exogenous stem cells in
dementias, focusing on AD.
3.Endogenous approach
Studies of stimulating adult neurogenesis in animal models of AD are contradictory,
probably due to the experimental conditions, such as the models used, their age, the
compound tested (Lazarov and Marr, 2010;Marlatt and Lucassen, 2010), Adult neu-
rogenesis can be stimulated through exercise and enriched environment (da Costa
Daniele et al., 2020), through the modification of physiological pathways (GSK-
3binhibitor B10(Shi et al., 2020)) or through the administration of growth factors
(erythropoietin, fluoxetine, G-CSF and AMD3100, BDNF, IGF-1, NGF, VEGF,
TGF-b)(Vasic et al., 2019).
100 CHAPTER 5 Stem cell therapy in Alzheimer’s disease
4.Exogenous approach
4.1 Classification of stem cells for exogenous therapeutic
approaches
In the last decades, stem cellebased brain transplantation therapies have been inves-
tigated and different lines of stem cells have been explored (Chan et al., 2014). The
choice of the appropriate cell type is an essential step in developing an adequate
stem cell therapy. The most utilized stem cells in preclinical studies for Alzheimer’s
therapies are ESCs, NSCs, iPSCs, and MSCs (Fig. 5.1)(Duncan and Valenzuela,
2017).
• ESCs (embryonic stem cells): pluripotent, derived from the undifferentiated inner
mass cells of a human embryo (embryonic day 5e6);
• iPSCs (induced pluripotent stem cells): pluripotent, derived from mature somatic
cells, genetically modified in vitro by small molecule treatment or viral
vector-delivered transcription factors, to become ESC-like in phenotype and
differentiation capacity;
ESCs NSCs MSCs
iPSCs
blastocist
Patients
in vitro differentiation
in vitro
reprogramming
somatic adult tissue
bone marrow
blood
adipose tissue
placenta
in vitro differentiation
neuronal tissue
FIGURE 5.1
Schematic diagram of stem cell sources for therapy in dementia patients. Four types of
stem cells are used for treatment of dementia: ESCs (embryonic stem cells) derived from
the undifferentiated inner mass cells of a human, iPSCs (induced pluripotent stem cells)
derived from mature somatic cells genetically reprogrammed, NSCs (neural stem cells)
present in the adult brain in neurogenic niches or derived from partially differentiated
ESCs and iPSCs, and MSCs (mesenchymal stem cells) derived from several sources such
as the bone marrow, umbilical cord, Wharton’s jelly, adipose tissue.
4. Exogenous approach 101
• NSCs (neural stem cells): multipotent, present in the adult brain in neurogenic
niches of the SVZ and the granular layer of the dentate gyrus in the hippocampus;
• MSCs (mesenchymal stem cells): multipotent, derived from several sources such
as the bone marrow, umbilical cord, Wharton’s jelly, adipose tissue. Phenotypic
expression and differentiation potential of MSCs can vary according to the tissue
of origin (Hass et al., 2011).
4.2 Embryonic stem cells
The use of embryonic stem cells, although very promising, implies a number of con-
cerns, not only ethical but also safety related (Volarevic et al., 2018). For research
purposes most ESCs are isolated from embryos derived from in vitro fertilization
and then donated to research, but the use of ESCs for clinical purposes remains a
controversy and is differentially regulated from country to country. The major
concern regarding safety of transplanting pluripotent stem cells, other than aberrant
immune reaction and rejection, is the possible formation of tumors, due to the
difficulty to control the cellular expansion after in vivo transplantation. Their clinical
translational potential is then limited. One way to prevent tumor formation is to
partially differentiate them in vitro before transplantation. Preclinical studies on
animal models of AD show how predifferentiated ESCs into basal forebrain cholin-
ergic neurons (BFCNs) can retain a neuronal phenotype after transplantation, can
functionally integrate into neuronal circuitry with no tumor formation, and can
improve animal impaired spatial memory and learning (Bissonnette et al., 2011;
Liu et al., 2013;Moghadam et al., 2009). ESCs have been also predifferentiated
into medial ganglionic eminence-like progenitors cells (MGE) giving comparable
results after transplantation into the hippocampus of a cognitive-impaired mouse
model (Liu et al., 2013).
4.3 Induced pluripotent stem cells
In the past years, an unprecedented opportunity arose from the study of iPSCs
derived from patients with AD or other forms of dementia (Tcw, 2019): patients’
derived cells can model a disease in vitro, retaining the genetic backgrounds of
patients. The iPSCs are mainly utilized in the study of dementia as a disease
modeling opportunity that allows to screen drugs and test personalized therapeutics.
Even if some limitations need to be considered (genetic reprogramming, for
example, resets aging signatures such as telomere shortening, mitochondrial
dysfunction, and cellular senescence), the value of this tool in the study of dementia
neuropathology is inestimable.
On the other hand, the use of iPSCs as therapeutic agent is still very immature
and more complicated. iPSCs can differentiate in functional neurons and neuronal
subtypes, able to form electrophysiological active synapses (Pang et al., 2011). Since
iPSCs derive from autologous somatic cells, their use would bypass the medical
problem of immune rejection, and it would also eliminate the ethical issues derived
102 CHAPTER 5 Stem cell therapy in Alzheimer’s disease
from ESCs alternative. However, autologous iPSCs would carry the patient’s genetic
defect that caused the pathological signs of AD or dementia in the first place,
including abnormal Ablevels, elevated tau phosphorylation, or reduced neurite
length. Moreover, also in this case, one major challenge is teratoma formation.
iPSCs’ safety in clinical routine has not been elucidated yet and only a few, although
very promising, preclinical studies are currently available. In animal models of
spinal cord injury, transplanted iPSCs survive and differentiate into functional neu-
rons, astrocytes, and oligodendrocytes (Tsuji et al., 2010). Transplantation of human
iPSC-derived cholinergic neuronal precursors into the hippocampus of a transgenic
AD mouse model showed how cells survived, differentiated into functional cholin-
ergic neurons, and reversed spatial memory impairment (Fujiwara et al., 2013,
2015).
Although there are promising preclinical studies, the use of iPSCs in clinical
routine is in a very premature phase. In most of the studies on other neurodegener-
ative diseases (Parkinson’s, Huntington), similarly to the ESCs, iPSCs are also pre-
differentiated into neuronal precursor cells.
4.4 Neuronal stem cells
NSCs can self-renew and differentiate into neurons and glial cells; they can be
collected from the brain tissue, but also genetically reprogrammed from somatic
cells or in vitro differentiated from ESCs and somatic cellsederived iPSCs.
In preclinical studies on rodent models of AD, transplantation of human or
mouse-derived NSC consistently showed improvements in AD pathologies and be-
haviors (Hayashi et al., 2020). The mechanism of action is believed to be multitar-
geted: once transplanted, NSCs can not only replace damaged neurons by generating
different cell types, but also promote endogenous neurogenesis (Feng et al., 2017)
and secrete neurotrophic factors (including BDNF, IGF, and GDNF) that have neuro-
protective functions (Kitiyanant et al., 2012).
Additionally, it has been shown that NSCs may play an important role in
reducing neuroinflammation in the nervous system by suppressing the expression
of proinflammatory molecules, such as interleukin (IL)-1, IL-6, and TNF-a(Zhang
et al., 2016).
NSCs do not seem to have a direct role in decreasing amyloid plaques or neuro-
fibrillary tangles. hNSCs transplanted into a triple transgenic mouse model (Oddo
et al., 2003) did not alter Abor tau pathology (Ager et al., 2015;Blurton-Jones
et al., 2009). However, it has been reported that NSCs can reduce plaque load by
increasing recruitment of activated microglia (McGinley et al., 2018).
Genetically modified NSCs have also been tested in animal models: NSCs over-
expressing neprilysin, insulin-degrading enzyme, plasmin, and cathepsin B, have
been used to reduce Ablevels (Hayashi et al., 2020).
Although promising, the use of NSCs shows some disadvantages: data on the dif-
ferentiation of NSCs in vivo after transplantation are not always reproducible,
because of the high dependence on the recipient’s brain microenvironment. Also,
4. Exogenous approach 103
NSCs trigger immune response upon allogeneic transplantation (Rossignol et al.,
2014). Moreover, to this day, the use of autologous NSCs is not clinically compatible
because current methods cannot isolate and generate sufficient amounts from the pa-
tient (Chan et al., 2014).
Finally, the use of NSCs in Alzheimer’s patients seems to be particular problem-
atic, as it has been shown in vivo how the expression of APP itself changes the fate of
NSCs leading to the generation of more astrocytes than neurons (Kwak et al., 2006).
4.5 Mesenchymal stem cells
MSCs hold great promise for therapeutic and regenerative medicine because of two
peculiar characteristics: (a) they are able to migrate to sites of injury, and (b) they are
considered “immune-privileged.” They do not express major histocompatibility
complex class II molecules, providing a mechanistic rationale for why they have
not been demonstrated to induce a rejection effect when implanted in a host (Klyush-
nenkova et al., 2005;Le Blanc et al., 2008). In animal models, the use of MSCs has
been demonstrated to be beneficial in various neurological pathologies such as epi-
lepsy and amyotrophic lateral sclerosis (ALS) (Shakhbazau and Potapnev, 2016),
multiple sclerosis (MS) (Ardeshiry Lajimi et al., 2013;Connick et al., 2011;Hou
et al., 2013), stroke (Bang et al., 2005;Lee et al., 2010c), demyelination, and spinal
cord injury (Uccelli et al., 2011a,b). Direct intracerebral transplantation of MSCs
has also been used to successfully treat mouse models of AD, leading to reduced
Abdeposits, reduced inflammation, and improved synaptic transmission (Babaei
et al., 2012;Bae et al., 2013;Lee et al., 2009,2010a,b,2012a) (see Table 5.1 for
list of references).
In contrast to embryonic stem cells and neural multipotent adult stem cells,
MSCs are a promising therapeutic option because they are relatively easy to isolate,
they do not undergo malignant transformation after patient transplantation (Patel and
Genovese, 2011), and they do not raise ethical concerns about their biological
source. Moreover, their high proliferative potential allows for rapid expansion
ex vivo, while maintaining multipotentiality.
MSCs were described for the first time in the 1960s (Friedenstein et al., 1966)as
stromal cells and bone-forming cells within the bone marrow. Bone marrowe
derived stem cells consist of hematopoietic stem cells (HSCs), endothelial progen-
itor cells (EPCs), and the MSCs, also called stromal stem cells.
MSCs can be defined by three main criteria: (a) adhesion to tissue culturee
treated plastic; (b) capacity to differentiate into mesodermal lineages (adipocytes,
osteoblasts, and chondrocytes); (c) expression of specific cluster of differentiation
(CD105, CD73, and CD90), as well as lack of expression of CD45, CD34, CD14,
CD11b, CD79a, CD19, and HLA-DR surface molecules.
These criteria are used to characterize the cells, although the combination of sur-
face markers is not definitive (Contreras-Kallens et al., 2017). Moreover, MSCs are
also capable, given specific in vitro conditions, of developing characteristics associ-
ated with neuronal cells (Dezawa et al., 2004).
104 CHAPTER 5 Stem cell therapy in Alzheimer’s disease
Table 5.1 List of studies involving administration of MSCs in animal models of Alzheimer’s disease.
References MSC source
Administration
route
Rodent
model Molecular effects
Behavioral
effects
Lee et al.
(2009, 2010a)
BM/UC blood IC Ab-
inoculated
mice
;Ab;
;inflammatory response
;glia activation, oxidative stress, and
apoptosis.
Learning and
memory [
Yun et al.
(2013)
Placenta IV Ab-
inoculated
mice
;Ab, APP, BACE1;
;activity of b- and g-secretase;
;neuroinflammation;
;neuronal death;
:neuronal differentiation
Prevent memory
impairment
Oh et al.
(2015)
Not mentioned IV Ab-
inoculated
mice
:WNT signaling;
:endogenous neurogenesis
Working
memory [
Zhang et al.
(2012)
BM; BDNF þMSCs IC Ab-
inoculated
rats
N/A Learning and
memory [
Sharma et al.
(2017)
MSCs þcerebrolysin IV Ab-
inoculated
rats
;Abdeposition;
;gliosis;
:regulation of NEP
NA
Shin et al.
(2014)
Not mentioned IV Ab-
inoculated/
APP/PS1
mice
:autophagy;
:Abclearance;
:hippocampal neuronal survival
NA
Lee et al.
(2010b),Wen
et al. (2011)
BM IC APP/PS1
mice
;Aband p-tau deposition;
;plaques
;inflammatory response;
:Ab-degrading factors
[behavioral and
spatial memory
Cognitive [
Continued
4. Exogenous approach 105
Table 5.1 List of studies involving administration of MSCs in animal models of Alzheimer’s disease.dcont’d
References MSC source
Administration
route
Rodent
model Molecular effects
Behavioral
effects
Lee et al.
(2012b),Bae
et al. (2013)
BM IC APP/PS1
mice
:Abclearance through active microglia
and :NEP
;Abdeposits
;inflammation by expression of CCL5
&IL-4;
Cognitive [
Prevented AD
related symptoms
in young mice
Garcia et al.
(2014)
BM (þVEGF) IC APP/PS1
mice
:neovascularization;
;plaques in hippocampus
Reduced
cognitive deficit
Liu et al.
(2015)
BM(Mouse) (As-MiR-
937MSCs)
IC APP/PS1
mice
;Abdeposits;
:BDNF
Cognitive [
Lee et al.
(2012a),Kim
et al. (2012a)
UC IC APP/PS1
mice
;Abdeposits;
:NEP;
Immunomodulatory effect on microglia
Cognitive [
neuronal survival
in vitro [
Yang et al.
(2013)
UC (predifferentiated) IC APP/PS1
mice
;Abdeposits;
:IL-4 ;IL-1b, TNF-a;
:IDE, NEP
Cognitive [
Ma et al.
(2013)
Adipose-derived IC APP/PS1
mice
;Abdeposition; regulation of activated
microglia;
:IL-4, ;IL-1b, TNFa;
:IDE, NEP, MMP9.
Restored learning
and memory
Yan et al.
(2014)
Adipose-derived IC APP/PS1
mice
;oxidative stress;
:neurogenesis in SVZ
Reduced
cognitive deficit
Zheng et al.
(2017)
AF IC APP/PS1
mice
;Abdeposition;
:regulation IDE, MPP9, NEP
:microglia;
:neurogenesis;
:synaptic plasticity
Spatial learning
and memory [
Naaldijk et al.
(2017)
BM (Mouse) IV APP/PS1
mice
;plaque size;
;TNF-a, IL-6, MCP-1, NGS;
;microglial numbers/size
NA
106 CHAPTER 5 Stem cell therapy in Alzheimer’s disease
Xie et al.
(2016)
UC IV APP/PS1
mice
;Abdeposition;
;IL-1b, TNFa;
:IL-10
Spatial learning [;
Alleviated memory
decline
Park et al.
(2016)
UC IV APP/PS1
mice
no change in the levels of Ab; no change
in NEP
No improvement
Harach et al.
(2017)
ischemia-tolerant
MSCs
IV/Intranasal APP/PS1
mice
;Ablevels;
;IL-10, TNF-a;
:regulation IDE, ECE, NEP
NA
Jiao et al.
(2016)
Placenta membrane IV APP mice ;plaque number;
;oxidative stress
Spatial learning
and memory [
Lee et al.
(2016)
UC IAc APP/PS1
mice
MSCs not detected in the brain NA
Boutajangout
et al. (2017)
UC IAc APP/PS1
mice
;plaque number Cognitive
function [
Kanamaru
et al. (2015)
BM IV DAL mice/
APP mice
;neuronal loss;
;Abdeposition
Memory [
Kim et al.
(2012b)
Adipose-derived IV/IC Tg2576 ;Abdeposits;
;plaques;
:regulation IL-10 and VEGF
Learning and
memory [
Kim et al.
(2013)
Amniotic membrane IV Tg2576 ;plaques;
:phagocytic microglial cells;
:IDE, MMP9;
:IL-10, TGFb;IL-1b, TNFa
Spatial learning [
Wu et al.
(2016)
BM þG-CSF IV after irradiation Tg2576/
Ab-
inoculated
mice
Pretreatment with G-CSF was
necessary for MSCs to reach the brain
and differentiate into neurons
NA
Cui et al.
(2017)
UC IV Tg2576
mice
No Abalteration
;oxidative stress;
:neurogenesis;
Cognitive
function [
Continued
4. Exogenous approach 107
Table 5.1 List of studies involving administration of MSCs in animal models of Alzheimer’s disease.dcont’d
References MSC source
Administration
route
Rodent
model Molecular effects
Behavioral
effects
Ruzicka et al.
(2016)
BM IC 3Tg-AD
mice
;Ab-56;
:regulation of GS levels;
:cellular proliferative capacity in the
SVZ
Preserved
working memory
Babaei et al.
(2012)
BM IC Aged and
Ibo-
induced
rats
N/A Learning and
memory [
Matchynski-
Franks et al.
(2016)
BM (Mouse) IC 5XFAD
mice
;Abdeposition Reduced learning
deficits
Son et al.
(2017)
UC (sRAGE- MSCs) IC 5xFAD mice ;Abdeposition; regulation of activated
microglia
NA
AF, Amniotic Fluid; BM, Bone Marrow; IAc, Intraarterial (carotid); IC, Intracerebral; IV, intravenous; UC, Umbilical Cord; :, Increased/Enhanced; ;, Decreased/
Reduced; [, improvement.
108 CHAPTER 5 Stem cell therapy in Alzheimer’s disease
4.5.1 Mechanisms of action
MSCs are considered a new and very promising option for the treatment of several
diseases because of their unique properties (Fig. 5.2):
(a) tissue-repairing abilities (da Silva Meirelles et al., 2006)
(b) multilineage differentiation capacity (Lv et al., 2014)
(c) broad immuneregulatory properties (Contreras-Kallens et al., 2017).
Multiple reports over the last decade showed improvement in various models of
neurodegenerative diseases or acute brain insults following MSCs transplantation in
multiple rodent models (See Table 5.1 for AD models).
apoptosys
angiogenesys
NSCs
synaptogenesis
neurogenesis
oligogenesis
astrogliosis and
microglia activation
X X
(
Mesenchymal Stem Cells
FIGURE 5.2
Mechanisms of neuroprotective and neurorestorative effects of MSCs (mesenchymal
stem cells). MSCs secrete a number of neurotrophic factors that induce angiogenesis and
promote neurogenesis, oligonenesis, and synaptogenesis. Inhibiting factors also act on
decreasing apoptosis and decreasing neuroinflammation by inhibiting astroglia growth
and activation.
4. Exogenous approach 109
Although not entirely defined, the mechanisms through which MSCs perform
their therapeutic action include neurogenesis, angiogenesis, antiinflammation
and immunomodulation (Fig. 5.2)(Furno et al., 2017). The immediate environ-
ment of MSCs is the most important factor determining their fate. The ultimate
function of an MSC is determined by environmental stimuli from other cells
and/or cytokines.
4.5.2 Homing and paracrine effects
When MSCs are administered intravenously, few cells reach the injury site as most
cells are sequestered in the lungs and in other organs. However, there are experi-
mental data that support that MSCs possess high migratory potential and the ability
to promote neural regeneration. There are data from animal models demonstrating
the homing of MSCs in different parts of the brain after IV injection. One study
by Panchenko et. al reports that MSCs can be found in hippocampus, dentate gyrus,
and temporal cortex of bulbectomized mice after IV injection (Panchenko et al.,
2014). Another study by Harach et. al found that after a single intravenous injection,
hMSCs were detected in APP/PS1 mouse brain one hour and one week following
administration. These cells migrated to the brain parenchyma and in particular to
the hippocampus (Harach et al., 2017). Nevertheless, there are studies that report
that human ucMSCs (umbilical cord derived) are not detected in the brain after intra-
arterial (IA) injection into the carotid artery (Lee et al., 2016). A potential limitation
to these findings may be that only two time points were evaluated, both very close to
the time of injection (5 minutes and 6 hours).
While MSCs moving across the bloodebrain barrier may not be an efficient pro-
cess, the presence of only few cells may still be therapeutic as a result of the para-
crine secretion of neurotrophic factors and cytokines (rather than by transformation
into various cell types themselves). Cytokines are able to modulate not only the im-
mune response, but also angiogenesis, apoptosis, oxidative stress, cell differentia-
tion, extracellular matrix composition, and misfolded protein aggregation
(Volkman and Offen, 2017).
Paracrine effects of MSCs include the production of a number of factors,
collectively known as the secretome, which includes several growth factors
such as VEGF (vascular endothelial growth factor), BDNF (brain-derived neuro-
trophic factor), NGF (nerve growth factor), BFGF (basic fibroblast growth factor)
as well as antiinflammatory cytokines (Linero and Chaparro, 2014). Upregulation
of genes associated with cell proliferation, neurogenesis, migration, and neuronal
survival has been also observed in the brain after injection with MSCs (Munoz
et al., 2005).
4.5.3 Neuroinflammation
MSCs have a potent immune regulatory potential as they are able to suppress the
function of various immune cells and to promote their regulatory immune functions.
MSCs are capable of interacting with various types of immune cells, including lym-
phocytes T and B, natural killer cells, macrophages, dendritic cells, neutrophils, and
110 CHAPTER 5 Stem cell therapy in Alzheimer’s disease
mast cells. These interactions can occur through direct cellecell contact or, more
importantly, through a paracrine effect: MSCs can secrete various growth and immu-
nomodulatory factors regulating inflammation remotely from the site of injury (Li
and Hua, 2017).
T cellemediated immunity is the key component of the adaptive immune sys-
tem. T cells protect against infections and malignancies but also mediate a number
of autoimmune diseases. One of the first observations made in vitro was that MSCs
can reduce T cell proliferation (Di Nicola et al., 2002). This is supported by the fact
that in vivo infusions of MSCs can control graft-versus-host disease following bone
marrow transplantation. TGF-band HGF are the molecules secreted by MSCs
responsible for the proliferation-inhibiting effect on T cells (Di Nicola et al.,
2002;Glennie et al., 2005). MSCs inhibit proliferation, can induce apoptosis
(Akiyama et al., 2012), and inhibit the differentiation of naı
¨ve T cells by production
of immunosuppressors such as nitric oxide (NO) and prostaglandin E2 (PGE2)
(Crop et al., 2010;Ren et al., 2008). Moreover, MSCs secrete chemokines and adhe-
sion molecules that are critical for lymphocyte recruitment to injured sites. The
expression of these soluble molecules is necessary for T cell inhibition (Ma et al.,
2014).
In addition to T cells, B cells are also effectors of the adaptive immune system.
B cells respond to pathogens by proliferating, differentiating, and ultimately produc-
ing antibodies that neutralize foreign bodies. MSCs are capable of suppressing the
proliferation, differentiation, and activation of B cells by effecting their cell cycle,
compromising their immunoglobulin-secreting ability, and reducing chemotactic
properties (Li and Hua, 2017;Tabera et al., 2008). These effects are not only
mediated by cellecell contact (Schena et al., 2010), but also by paracrine effectors.
For example, MSC-secreted CCL2 is of critical importance for B cell suppressive
function (Che et al., 2012;Rafei et al., 2008). Additionally, IL-1 receptor antagonist
(IL-1Ra) is essential for controlling B cell differentiation (Luz-Crawford et al.,
2016). MSCs can also induce the B reg-mediated secretion of anti-inflammatory
cytokine IL-10, resulting in the suppression of the immune response (Franquesa
et al., 2015;Peng et al., 2015).
MSCs have additional roles in suppressing various cells involved in the immune
system (reviewed in (Li and Hua, 2017)). One such example would be dendritic
cells, which are the most potent antigen-presenting cells. In addition, they suppress
M1 and M2 macrophages. M1 macrophages are proinflammatory and have antimi-
crobial abilities, while M2 have immunomodulatory capabilities and promote tissue
repair. Further, MSCs also suppress natural killers, which are the effector cells of the
innate immune system. Neutrophil suppression has also been demonstrated, which is
notable, as these polymorphonuclear leukocytes play a role in acute inflammation by
eliminating pathogens through phagocytosis and secretion of bactericidal molecules.
Moreover, mast cell suppression is important because these cells are major effectors
in allergic reactions, inflammatory diseases, and autoimmunity. Of particular inter-
est is the immunomodulation that MSCs show on microglial cells, the resident mac-
rophages in the brain (Zhou et al., 2013).
4. Exogenous approach 111
4.5.4 Neurogenesis
Adult neurogenesis is a complex process that includes proliferation, migration, and
differentiation of neural precursor cells (Ihunwo et al., 2016;Zou et al., 2010). It pre-
dominantly occurs in two areas of the central nervous system: the SVZ and SGZ of
the hippocampal dentate gyrus (Christie and Turnley, 2012;Ihunwo et al., 2016).
Given that adult neurogenesis has the potential to regenerate cortical neurons, it is
important to investigate whether MSCs can regulate or promote this process as it
may help in the specific AD pathology.
In AD there is a degeneration and death of neurons throughout the brain, mainly
in the basal forebrain, amygdala, hippocampus, and cerebral cortex (Crews and Mas-
liah, 2010). The treatments that are now approved temporarily improve symptoms
but they do not provide any protection against progressive cell degeneration.
One of the first proposed mechanisms of action for MSCs was transdifferentia-
tion, where MSCs reach the site of injury and adopt neural cell phenotypes (Munoz-
Elias et al., 2004). However, this mechanism occurs at a very low frequency and it
has been shown that the neural recovery observed in vivo after MSCs treatment re-
sults mainly from the release of soluble molecules by MSCs. Endogenous neuronal
precursors are stimulated to differentiate into neurons by MSC-derived growth fac-
tors and extracellular matrix components (Maltman et al., 2011). There is further
evidence from in vitro studies to support this mechanism: it has been demonstrated
that, when neuronal precursors are cocultured with MSCs, they differentiate into
mature neurons (Oh et al., 2015).
There are promising results from in vivo experiments on animal models that
support the therapeutic effect of MSCs-induced neurogenesys. Examples include
models of traumatic brain injury (Acosta et al., 2014;Tajiri et al., 2014a,b)andan-
imal models of Alzheimer’s disease (Kim et al., 2012b;Oh et al., 2015;Yan e t a l . ,
2014). Adipose-derived MSCs are able to improve cognitive function and induce
neurogenesis in APP/PS1 mice after transplantation in the SVZ (Ya n e t a l .,
2014) as well as in Tg2576 transgenic mice after either intracerebral injection or
IV injection (Kim et al., 2012b). Studies have further demonstrated that following
IV injection with hUC-MSCs, neurogenesis was detected in the hippocampus of
AD mice, probably by modulating Wnt signaling pathways (Oh et al., 2015;Cui
et al., 2017).
4.5.5 Mechanisms of decreased Ab deposition
Of particular interest for AD is the effect of MSCs on amyloid plaques and neuro-
fibrillary tangles. Different molecular mechanisms have been hypothesized and
studied to explain the reduction in plaque burden attributed to treatment with
MSCs. The most common and consistently proven mechanism for plaque burden
reduction is the regulation of degrading enzymes IDE (insulin degrading enzyme),
NEP (neprilysin), and MMP9 (matrix metallopeptidase 9). These enzymes show
enhanced expression in the brain of AD animal models after treatment with
MSCs (Zheng et al., 2017). Decreased activity of beta- and gamma-secretase,
enzymes involved in the processing of APP, has also been observed and proposed
112 CHAPTER 5 Stem cell therapy in Alzheimer’s disease
as a protective mechanism (Yun et al., 2013). Additionally, an increase of autophagy
has been observed and therefore proposed as a mechanism to explain the reduction
of plaques and Abfragment (Shin et al., 2014).
4.6 MSCs and Alzheimer’s disease: preclinical experience
4.6.1 In vitro
Studies show that MSCs have multiple roles in protecting neuronal cells from the
toxic effects of Abin vitro. Such protective effects of MSCs have been observed
when co-cultured with Ab-treated hippocampal neurons: the co-culturing resulted
in reduced neuronal apoptosis (Lee et al., 2010a). Other findings supporting a protec-
tive role for MSCs include the observation that MSCs increase expression of neuronal
factors in Ab-treated hippocampal precursor cells, otherwise attenuated by Ab, and
enhance neuronal differentiation (Oh et al., 2015). Decreased cell viability and
increased nuclear apoptosis of AD-derived neurons were delayed when co-cultured
with MSCs (Song et al., 2015). MSCs increased cellular viability and enhanced
LC3-II expression (marker of autophagy) in Ab-treated neuronal cell line. Further-
more, MSCs can markedly decrease intracellular Ablevels (Shin et al., 2014).
Also, MSC-derived soluble factors can modulate neuroprotective properties in
in vitro models of AD, confirming the hypothesis of a paracrine effect:
• hUCB-MSCs have a neuroprotective effect in vitro against Abtoxicity via
galectin-3 secretion (Kim et al., 2010).
• Adipose tissueederived MSCs secrete functional neprilysin, one of the enzymes
involved in the degradation of amyloid plaques (Katsuda et al., 2013).
• Coculture of hUCB-MSCs with microglia under amyloid-Abexposure induced a
reduction of Ab42 in the medium and overexpression of neprilysin in microglia
by release of soluble intracellular adhesion molecule-1 (sICAM-1) (Kim et al.,
2012a).
• MSCs and their secretome are able to decrease the toxicity of misfolded truncated-
Tau in a rat neuronal cell line (Zilka et al., 2011).
• Soluble factor CCL5 from BM-MSCs significantly increased microglia migration
in vitro and that the migration is markedly elevated after stimulation with Ab
(Lee et al., 2012b).
4.6.2 In vivo
A number of studies have demonstrated the efficacy of the intracerebral injection of
MSCs to treat murine models of AD. Bone marrowederived MSCs have been shown
to be effective in several transgenic mouse models of AD. In APP/PS1 double-
transgenic mice (expressing mutated forms of amyloid precursor protein and
presenilin-1), intracerebral transplantation of allogeneic bone marrowederived
MSCs significantly reduced amyloid-beta peptide (Ab) deposition (Lee et al.,
2010b). These beneficial effects were associated with increased Ab-degrading fac-
tors, decreased inflammatory response, alternatively activated microglial markers,
and release of chemoattractive factors (Lee et al., 2012b). Furthermore, tau
4. Exogenous approach 113
hyperphosphorylation was decreased, and the mice showed an improvement in
behavioral and spatial memory analyses (Lee et al., 2010b). Similarly, intracerebral
transplantation of bone marrow-derived MSCs was found to be effective at prevent-
ing Alzheimer’s related symptoms in young mice (Bae et al., 2013). In these studies,
APP/PS1 mice were treated at an age when they displayed the neuropathological, but
not the cognitive features of AD. Much like aged animals, these younger mice
showed a significant decrease in cerebral Abdeposition. This response was
sustained for at least 2 months, portending MSC transplantation as a potential ther-
apy for early intervention of AD. Importantly, intraventricular stereotactic injection
of MSCs preserved working memory in the triple transgenic mouse 3 Tg-AD
(expressing mutated forms of amyloid precursor protein, presenilin-1, and Tau)
(Ruzicka et al., 2016).
Favorable results have also been obtained from intravenously injected MSCs.
Intravenous administration of bone marrowederived mononuclear cells, which
contain a mixture of cells that included MSCs, was effective at improving memory
impairment and suppressing neuronal loss in AD mice models (Kanamaru et al.,
2015). Bone marrowederived MSCs also appeared to exert their beneficial effects
through intravenous injection, by enhancing autophagy to promote Abclearance
in a mouse preinjected with an Abfragment (Shin et al., 2014).
The following table (Table 5.1) lists the numerous studies involving administra-
tion of MSCs in animal models of AD.
In summary, the studies listed in the table, regardless of the mouse model, type of
administration, or source of MSCs, show that the therapeutic effects of MSCs
depend on three mechanisms: (1) the decrease of plaque burden, (2) regulation of
the inflammation, and (3) increased neurogenesis.
5.Previous human experience with MSCs
5.1 Choice of stem cell for clinical studies
The distinct properties and potential mechanisms of transplanted stem cells have
been appreciated ever since they were first investigated for their potential therapeutic
benefits (Hunsberger et al., 2016). As further consideration for their therapeutic use
in primary neurodegenerative diseases such as AD grows, the main questions that
arise include:
• What stem cell type has the ideal properties and mechanistic synergy with AD
such that one may reasonably hypothesize a therapeutic effect?
• What stem cell type has the most compelling evidence in existing clinical studies
in terms of safety and efficacy?
• What insights can the current body of clinical literature (in primary neurode-
generative disease as well as other clinical contexts) provide as it relates to route
of administration, dosing, and frequency of these stem cells?
114 CHAPTER 5 Stem cell therapy in Alzheimer’s disease
As we have detailed in the previous section, we believe the cellular and molec-
ular properties of MSCs, as demonstrated by in vitro studies, position them as an
ideal therapeutic consideration in neurodegenerative disease compared to other
stem cells. Therefore, we will now explore what precedent exists for the clinical
use of mesenchymal stem cells in humans.
5.2 Use of human MSCs in neurodegenerative diseases
5.2.1 Safety: human phase 1 clinical trials in neurological disease
The earliest studies to establish the safety of therapeutic administration of human
MSCs for neurologic conditions were conducted in non-AD contexts. For example,
Lee et al. (2008) sought to investigate the safety of intra-arterial and intravenous in-
jection of autologous MSCs for multiple systems atrophy (MSA), with exploratory
outcome variables for probing potential efficacy. This strategy has been utilized by
other studies as well. In fact, Table 5.2 summarizes a selection of peer-reviewed
articles that investigate the use of human MSCs for different neurologic conditions.
These studies investigated safety (and in some cases, explore efficacy) in Parkin-
son’s disease (PD), amyotrophic lateral sclerosis, multiple sclerosis, and even AD.
The results established safety, with mixed results concerning efficacy as a secondary
outcome. The consistency of the results in these different pathologies suggests
MSCs are safe for use in neurologic disease.
Additionally, another phase 1 study investigated and demonstrated safety of
intravenous MSC injections in the context of “aging frailty” (Golpanian et al.,
2016). The relevance of this study population to neurodegenerative disease is note-
worthy, as frailty is a common feature in patients with AD.
Finally, a recent case series of three individuals with AD, while not designed as a
safety clinical trial, demonstrated the safe administration of repeated doses of intra-
thecal mesenchymal stem cells. The injections were associated with increased cere-
bral glucose metabolism (Vaquero et al., 2019).
5.2.2 Efficacy: phase 2 clinical trials
As of the time of publication, there are no published phase 2 studies that were pri-
marily designed to investigate the therapeutic efficacy of MSCs in AD. In the setting
where MSCs were tested for efficacy in other neurologic conditions, the results have
been mixed. For example, in multiple systems atrophy, both phase 1 (Lee et al.,
2008) and phase 2 (Lee et al., 2012c) studies have looked at efficacy (secondarily,
in the phase 1 study.) While these phase 1 and phase 2 studies reported detection
of efficacy (defined as delaying progression of neurological deficits during the study
period), these results would need to be reproduced. Moreover, the choice of the most
appropriate efficacy outcome would need to be considered in subsequent studies.
Nevertheless, the lack of efficacy studies at this time prevents any conclusions
from being drawn regarding therapeutic effects of MSCs in AD or neurodegenera-
tive disease.
5. Previous human experience with MSCs 115
Table 5.2 Published MSC clinical trials in neurodegenerative and neuroinflammatory diseases.
Reference Disease Cell type Route
Cell quantity
(total dose) Safety Efficacy
Lee et al. (2008) MSA Autologous (BM) IA and IV 1.6 10
8
Yes Yes
Brazzini et al. (2010) PD Autologous (BM) IA NR Yes Yes
Mazzini et al. (2010) ALS Autologous (BM) Transplant (SC) At least 110 10
6
Yes No
Venkataramana et al.
(2010)
PD Autologous (BM) Transplant (SVZ)
a
110
6
(per kg) Yes Yes
Lee et al. (2012c) MSA Autologous (BM) IA 1.6 10
8
Yes Yes
Venkataramana et al.
(2012)
PD Allogeneic (BM) Transplant (SVZ)
b
210
6
(per kg) Yes Yes
Kim et al. (2015) AD Allogenic (UC) Transplant
c
Arm 1: 3 10
6
Arm 2: 6 10
6
Yes NA
Golpanian et al.
(2016)
Aging Frailty Allogeneic (BM) IV Arm 1: 2 10
7
Arm 2: 1 10
8
Arm 3: 2 10
8
Yes NA
Nikbin et al. (2007) MS Autologous (BM) Intrathecal 8.73 10
6
(mean) Yes Mixed
Yamout et al. (2010) MS Autologous (BM) 5 mL intrathecally and 5 mL
intracisternally
3.5 10
7
Yes No
Mazzini et al. (2012) ALS Autologous (BM) Transplant (SC) NR Yes No
Karussis et al. (2010)
(ALS arm)
ALS Autologous (BM) IV and Intrathecal (LP) 78.1 10
6
Yes NA
Karussis et al. (2010)
(MS arm)
MS Autologous (BM) IV and Intrathecal (LP) 87.7 10
6
Yes NA
BM, Bone Marrow; hMSC, Human Mesenchymal Stem Cells; NA, not applicable; NR, not reported; SC, Spinal Cord; SVZ, Subventricular Zone; UC, Umbilical
Cord.
a
Unilateral transplantation.
b
Bilateral transplantation.
c
Bilateral hippocampi and right precuneus.
116 CHAPTER 5 Stem cell therapy in Alzheimer’s disease
5.2.3 Source of cells
The various studies investigating MSCs for their clinical effects in neurodegenera-
tive disease utilize different sources for harvesting MSCs. For example, several of
the reported studies in Table 5.2 utilized autologous, bone marrowederived MSCs
(Brazzini et al., 2010;Golpanian et al., 2016;Karussis et al., 2010;Lee et al.,
2008,2012c;Mazzini et al., 2010,2012;Nikbin et al., 2007;Venkataramana
et al., 2010;Yamout et al., 2010). Only one (Venkataramana et al., 2012) utilized
allogeneic bone marrowederived MSCs. All of these trials, however, demonstrated
safety. Thus, published clinical trial results of MSCs in neurodegenerative disease
suggest there is no difference in safety between allogeneic and autologous MSCs.
In ongoing studies specifically looking to treat AD with intravenously delivered
MSCs as reported on ClinicalTrials.gov (Table 5.3), however, nearly all utilize allo-
geneic MSCs. In these studies, the majority derive their cells from umbilical cord
blood, followed by bone marrow, adipose tissue, and placenta.
Notably, there is emerging evidence for the safety of a novel, specific subset of
umbilical cordederived cells (originating from the epithelial layer) (Patel et al.,
2013). In a series of studies, these intravenously delivered MSCs were utilized to
treat patients with heart failure. The results of these studies demonstrated safety
of patients with heart failure, as well as cardiac findings suggesting a clinical benefit
based on their reported outcome measures (Bartolucci et al., 2017;Tuma et al.,
2016).
5.3 Route of administration considerations
Another point of distinction when comparing MSC studies is the route of delivery,
which is variable even in studies intervening in the same condition. This lack of
consensus reveals the difficulty of determining the ideal route, which may also
impact decisions regarding the dosing of the MSCs. For example, in one group of
PD studies, the cells were directly transplanted to the subventricular zone (SVZ),
similar to what has been done in animal models (See Table 5.2)(Venkataramana
et al., 2010,2012). Additionally, the intervention in a phase 1 study of AD partici-
pants was bilateral transplantation of MSCs to their hippocampus (Kim et al., 2015).
Both of these studies demonstrated safety of direct transplantation.
Among studies utilizing an intravascular approach, some chose intraarterial (IA)
injections (Brazzini et al., 2010) while other studies have opted for intravenous (IV)
administration (Jarocha et al., 2015)(Golpanian et al., 2016), or combination of both
(Lee et al., 2008,2012c). In all cases, safety was established in the respective neuro-
logical condition of interest.
Some additional insight can be gained from considering the route of administra-
tion in nonneurologic contexts. In the field of hematology and oncology, there is a
well-established body of literature demonstrating the safety and efficacy of both
autologous and allogeneic IV-administered MSCs, particularly in pediatric popula-
tions. In refractory/relapsed aplastic anemia, IV-administered allogeneic cells were
demonstrated to be safe, but efficacy was not established in a phase 2 study
5. Previous human experience with MSCs 117
Table 5.3 Ongoing clinical trials where MSCs are used on Alzheimer’s
patients.
Study Disease Cell type Route
Total
dose (in
cells) Country
Longeveron LLC
NCT02600130
AD Allogeneic
hMSCs
IV Arm1:
210
7
Arm2:
110
8
USA
Stemedica Cell
Technologies Inc.
NCT02833792
AD Allogeneic
hMSCs
IV 1.5 10
7
USA
Medipost Co Ltd.
NCT02054208
AD Allogeneic
hUBCB-
MSC
Ommaya Arm1:
310
7
Arm2:
910
7
Korea
Affiliated Hospital
to Academy of
Military Medical
Sciences
NCT01547689
AD Allogeneic
hUBCB-
MSC
IV 1.6 10
8
China
Nature Cell Co.
Ltd.
NCT03117738
AD Autologous
(adipose)
IV Not
Reported
USA
Medipost Co Ltd.
NCT03172117
AD Allogeneic
hUBCB-
MSC
Ommaya Arm 1:
110
7
Arm 2:
310
7
Korea
South China
Research Center
for Stem Cell and
Regenerative
Medicine
NCT02672306
AD Allogeneic
hUBCB-
MSC
IV 1.6 10
8
China
CHABiotech CO.,
Ltd NCT02899091
AD Allogeneic
Human
Placenta e
MSC
IV 4 10
8
Korea
Hope Biosciences
NCT04228666
AD Autologous
(adipose)
IV 2 10
8
USA
Stemedica Cell
Technologies
NCT02833792
AD Allogeneic
hMSCs
IV 1.5 10
6
per kg
USA
University of Miami
NCT04040348
AD Allogeneic
hMSCs
IV Arm 1:
410
8
Arm 2:
810
8
USA
Medipost Co Ltd.
NCT01297218
AD Allogeneic
hUBCB-
MSC
Ommaya Arm 1:
310
6
Arm 2:
610
6
Korea
118 CHAPTER 5 Stem cell therapy in Alzheimer’s disease
(Cle et al., 2015). IV-administered allogeneic MSCs have also demonstrated safety
and efficacy in refractory Crohn’s disease (Forbes et al., 2014). Similarly, IV-
administered allogeneic MSCs were administered in active ankylosing spondylitis
patients (refractory to NSAIDs), where it was found to be both safe and effective
(Wang et al., 2014).
Therefore, the use of an intravenous route of administration for MSCs has a basis
in this body of evidence, supporting its safety in neurological and other diseases. In
fact, ongoing studies listed on clinicaltrials.gov also appear to overwhelmingly favor
an intravenous route, and less so intracerebrospinal fluid (e.g., via Ommaya reser-
voir) administration (Table 5.3).
5.4 Cell dosage
The dosing of stem cells has been variable in the clinical literature. In our list of
selected studies (Table 5.2), the absolute number of cells administered (independent
of route) range from 3 10
6
cells to 2 10
8
cells. The lower figure, however, cor-
responds to the amount of cells administered via direct transplantation. The doses
administered intravenously range from 2 10
7
cells to 2 10
8
cells (Golpanian
et al., 2016). Thus, safety has been demonstrated for MSC intravenous injections
up to a total of 2 10
8
cells in a population that closely resembles the target
population of a cohort with neurodegenerative disease. Further studies are being
conducted to test the safety of higher total doses, specifically in AD. For example,
Table 5.3 shows that the University of Miami is testing the safety of up to 8 10
8
cells injected intravenously (albeit divided into several doses). At this time, the
optimal dosage required for therapeutic effect is unknown.
5.4.1 Single versus multiple dosage
While the dose of intravenously administered mesenchymal stem cells has been
established to be safe up to 2 10
8
cells, evidence favoring their administration
as single dose or across multiple doses is not established. AD, like other neurological
conditions such as Multiple Systems Atrophy (MSA), is a primary neurodegenera-
tive disease with expected progression over time. None of the proposed mechanisms
of action for MSCs would suggest that a one-time dose would lead to a resolution of
underlying pathophysiology. Therefore, we hypothesize that multiple doses admin-
istered as the disease progresses may confer a sustained or cumulative benefit. We
reviewed the literature to investigate if any published clinical trials have specifically
sought to address the role of multiple MSC injections in neurodegenerative disease
(Table 5.4).
An approach utilizing a single IA and three subsequent monthly IV injections
was employed in two studies (phase 1 and phase 2) in MSA (Lee et al., 2008,
2012c). The total amount of administered cells in these studies was 1.6 10
8
or
160 million cells. As previously mentioned, both safety and efficacy were demon-
strated in these studies. In fact, six of the ongoing clinical trials queried on clinical-
trials.gov utilize a repeated-dosing strategy, suggesting the shared belief that a single
5. Previous human experience with MSCs 119
Table 5.4 Clinical studies in neurodegenerative and neuroinflammatory disease utilizing a multiple-dosing strategy.
Reference/
Study Phase
#
Subjects Disease # Doses
Dose
frequency Route 1
Dose
a
of
route 1 Route 2
Dose
a
of
route 2
Total
Dose
a
Lee et al.
(2008)
I-II 29 MSA 4 (1 þ3) (1) IA once
(2) IV
Monthly
IA 4 10
7
IV 4 10
7
16 10
7
Lee et al.
(2012c)
II 33 MSA 4 (1 þ3) (1) IA once
(2) IV
Monthly
IA 4 10
7
IV 4 10
7
16 10
7
Mazzini et al.
(2012) (ALS
arm)
I-II 9
b
ALS 2 NR Intrathecal
b
54.7 10
6
(17.4 10
6
)
IV 23.4 10
6
(6.0 10
6
)
78.1 10
6
Mazzini et al.
(2012),
Karussis et al.
(2010) (MS
arm)
I-II 5
b
MS 2 NR Intrathecal
b
63.2 10
6
(2.5 10
6
)
IV 24.5 10
6
(2.5 10
6
)
87.7 10
6
NCT02054208 I-IIa NA AD 3 Monthly Ommaya Arm 1:
110
7
Arm 2:
310
7
ee Arm 1:
310
7
Arm 2:
910
7
NCT01547689 I-II NA AD 8 Every
2 weeks
IV 2 10
7
ee 16 10
7
NCT03117738 I-II NA AD 10 Every
2 weeks
IV NR ee NR
NCT03172117 I-IIa NA AD 3 Monthly Ommaya Arm 1:
110
7
Arm
2: 3 10
7
ee Arm 1:
310
7
Arm 2:
910
7
NCT02672306 I-II NA AD 8 Every
2 weeks
IV 2 10
7
ee 16 10
7
NCT02899091 I-IIa NA AD 2 Monthly IV 2 10
8
ee 410
8
NCT04040348 I 10 AD 4 Every
13 weeks
IV Arm 1:
110
8
Arm 2:
210
8
ee Arm 1:
410
8
Arm 2:
810
8
a
Dose given in absolute number of cells.
b
Refers to the number of patients within the study arm that received both intrathecal and IV (the rest received only Intrathecal).
treatment may not optimally confer therapeutic benefit and that using only single
treatments may limit our ability to detect true therapeutic efficacy (Table 5.4).
Additionally, the use of multiple IV-injected allogeneic MSCs in nonneurologic
disease suggests that the practice is not only established but preferred in some
cases. To the authors’ knowledge, the largest body of literature supporting multiple
IV-injected allogeneic MSCs is in treating refractory acute graft-versus-host
disease (aGVHD). Early literature (reviewed in Nitkin and Bonfield, 2016) found
that participants responded to MSCs and were more likely to benefit from addi-
tional doses in the case of aGVHD recurrence. These findings have been further
supported by clinical trials, such as a study of 75 patients that found that eight
twice-weekly IV infusions of 2 10
6
hMSCs/kg for 4 weeks were enough to
confer an objective clinical benefit (Kurtzberg et al., 2014). The authors further
discuss several reasons for multiple infusion strategies. They suggest a high-
inflammation environment may impede MSCs, therefore multiple doses of antiin-
flammatory MSCs may diminish the inflammatory response and potentiate the
effect of the MSCs. Further, they discuss that there may be improved tolerance
of MSCs in subsequent doses as a result of prior MSC exposure. Finally, their
review of the prior literature supports that multiple doses may lead to prolonged
therapeutic effect.
Among other nonneurologic studies utilizing a multiple dosing strategy, one
study on active ankylosing spondylitis (refractory to NSAIDS) administered
MSCs intravenously at 1 10
6
MSCs/kg body weight at four time points, each
separated by 7 days (Wang et al., 2014). The authors noted improvements in clinical
symptoms, decrease in serologic and radiologic markers of inflammation, and
clinical effectiveness greater than 7 weeks. They further speculated that increasing
the quantity of MSCs and frequency of treatments could improve the response. In
another study looking at aplastic anemia, allogeneic MSCs were administered intra-
venously in multiple, weekly doses (ranging from two to five doses of
1.3e4.5 10
6
MSCs/kg each). While efficacy was not demonstrated, safety was
established for this multiple dosing protocol (Cle et al., 2015). In luminal Crohn’s
disease, a phase 2 study demonstrated safety and efficacy with a multiple dose
regimen of allogeneic MSCs as well, delivered as four-weekly intravenous doses
of 2 10
6
cells/kg body weight (Kurtzberg et al., 2014;Nitkin and Bonfield, 2016).
5.5 Concluding remarks
The preclinical studies cited in this chapter demonstrate compelling evidence that
mesenchymal stem cells directly affect the pathophysiology of AD by reducing am-
yloid beta plaque burden, regulating inflammation, and promoting neurorestoration
by increasing neurogenesis. Some of these mechanisms are directly relevant to the
pathophysiology of other primary neurodegenerative diseases. Therefore, based on
the existing preclinical literature, we believe there is a mechanistic argument for the
use of mesenchymal stem cells in AD and in other primary neurodegenerative
conditions.
5. Previous human experience with MSCs 121
Moreover, mesenchymal stem cell transplantation has been assessed for safety in
several medical conditions, primary neurodegenerative disease, and specifically in
AD. In all cases, safety was established across all administration routes reported
(intravascular, direct transplantation to the brain, and intrathecal). Multiple, repeated
intravenous dosing of human pooled allogeneic stem cells was found to be safe in
AD, as well as across various disease processes.
In conclusion, we believe the present body of literature supports the safety of
autologous and allogeneic mesenchymal stem cell use in human study subjects.
Whether or not these stem cells provide a therapeutic benefit will require consistent
clinical trial evidence in larger studies specifically designed to address efficacy.
Nevertheless, the strong mechanistic arguments of the preclinical literature and early
efficacy trends in phase I studies and small case series provide a basis for researchers
to design and conduct definitive trials in AD and other neurodegenerative disorders.
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132 CHAPTER 5 Stem cell therapy in Alzheimer’s disease
Stem cell therapies for
glaucoma and optic
neuropathy 6
Ziming Luo
1
, Michael Nahmou
1
, Kun-Che Chang
1,2
1
Spencer Center for Vision Research, School of Medicine, Stanford University, Palo Alto, CA,
United States;
2
Department of Ophthalmology, Louis J. Fox Center for Vision Restoration,
University of Pittsburgh School of Medicine, Pittsburgh, PA, United States
Chapter outline
1. Introduction ................................................................................................ .......134
2. Retinal cell fate specification .............................................................................134
2.1 Regulatory mechanisms of retinal ganglion cell differentiation ................ 134
2.2 Stem cell to retinal ganglion cell differentiation ..................................... 136
2.3 Retinal organoid differentiation ............................................................ 137
3. Retinal cell transplantation .................................................................................139
3.1 Primary retinal ganglion cell transplantation .......................................... 139
3.2 Stem cellederived retinal ganglion cell transplantation .......................... 140
3.3 Retinal organoid transplantation ........................................................... 140
3.4 iPSCs for cell replacement therapy ....................................................... 143
4. iPSCs for modeling of familial glaucoma .............................................................143
5. Summary and future directions............................................................................146
References .............................................................................................................146
Abstract
Glaucoma and optic neuropathy cause permanent blindness due to loss of retinal
ganglion cells (RGCs) and their axons, which are both important components of
visual circuits. RGC axons neither regrow nor are they replaced after injury and
death, for which cell transplant is a potential therapeutic strategy. In this review we
discuss the molecular mechanisms of RGC differentiation and compare the pros
and cons of stem cellederived RGC differentiation from 2D and 3D protocols.
Then, we summarize the progression of cell transplantation in rodents and its
prevision in nonhuman primates. We further point out the benefits of using induced
pluripotent stem cell (iPSC) for familial glaucoma, focusing on its potential as a
disease model for studying an inherited genetic mutation and for drug screening.
Keywords:
3D culture; Cell fate specification; Cell transplant; CRISPR/Cas9; Glaucoma; HESC;
HiPSC; Intraocular pressure; Nonhuman primate; Optic neuropathy; PACG; POAG; Retinal ganglion
cell; Retinal organoid; Tissue engineering scaffold.
CHAPTER
133
Recent Advances in iPSCs for Therapy, Volume 3. https://doi.org/10.1016/B978-0-12-822229-4.00010-3
Copyright ©2021 Elsevier Inc. All rights reserved.
1.Introduction
Glaucoma is a major cause of blindness, estimated to affect 80 million by 2030
worldwide. Primary open-angle glaucoma (POAG) is the most common form of
the disease, resulting in trabecular meshwork blockade but leaving open the space
between the cornea and iris. Primary angle-closure glaucoma (PACG) is another
type of glaucoma that is caused by a narrowing or blockade of the drainage angle
between the cornea and iris. Elevation of intraocular pressure (IOP) is a common
symptom for diagnosing both POAG and PACG, as high IOP causes irreversible
retinal ganglion cell (RGC) damage and subsequent vision loss. However, some
patients still suffer from glaucoma even with normal-range IOP, called normal-
tension glaucoma (NTG). Patients diagnosed with NTG develop optic nerve damage
gradually. The exact mechanisms responsible for glaucoma remain unclear, but
many studies have shown that glaucoma can be inherited due to genetic mutations
(Wiggs and Pasquale, 2017).
Although reducing IOP can slow down the progression of glaucoma, vision loss
remains irreversible because RGCs do not regrow axons after damage and are not
replaced after death. Stem cell differentiation and transplantation provide potential
therapeutic strategies for RGC replacement. Here, we review the mechanisms of
RGC differentiation in vivo, the advances of stem cellederived RGC differentiation,
and the progress of RGC transplantation into adult host eyes. We also review the po-
tential gene/loci mutations that contribute to familial glaucoma. Lastly, we discuss
CRISPR/Cas9 as a tool to identify the gene/loci for familial glaucoma and as a
candidate approach to repair genetic defects in the future.
2.Retinal cell fate specification
How different retinal cell types are generated during visual circuitry development
has interested scientists for decades (Wu et al., 2018). Foundational studies showed
that the retina is composed of many cell types including photoreceptors (rods and
cones), glia (Mu
¨ller glia, microglia, astrocytes), bipolar, horizontal, amacrine, and
ganglion cells (Livesey and Cepko, 2001;Vecino et al., 2016). Understanding the
molecular signals that regulate retinal cell fate is a common goal for neuroscientists
and ophthalmologists. To understand whether all the retinal cell types originate from
the same precursor, in 1990, the Cepko lab conducted an ex utero surgery and per-
formed subretinal viral injections into embryos at embryonic days 13e14 (E13-14)
to label retinal precursor cells (Turner et al., 1990). Among the retinal cells, RGCs
are one of the earliest to begin differentiation. Here, we review the regulatory mech-
anisms of RGC differentiation in rodents and human stem cells.
2.1 Regulatory mechanisms of retinal ganglion cell differentiation
Previous studies have discovered that eye development goes through several stages.
First, the anterior neural plate is developed and then specified into telencephalon,
134 CHAPTER 6 Stem cell therapies for glaucoma and optic neuropathy
eye field, diencephalon, and hypothalamus (Pillai-Kastoori et al., 2015). Subse-
quently, the neural plate evaginates and then invaginates to give rise to the optic
vesicle, which will develop into retina. Several eye-field transcription factors are
essential for eye formation including Pax6, Rx1, Lhx2 (LIM homeobox protein
2), Otx2 (orthodenticle homolog 2), Six3, ET, and Six6 (Zuber et al., 2003).
RGCs are the earliest cell type born during retinogenesis, while Pax6, Math5, and
Notch signaling pathways are central to regulating RGC fate specification (Mu
and Klein, 2004;Mu et al., 2005).
RGCs are retinal neurons that transfer visual information from the eye to the
brain through their extending axons. In the mouse, RGC promarkers start to express
by E12 and peak at E14. Among intrinsic transcription factors (TFs), the bHLH TF
Math5 (Atoh7) positively regulates RGC specification as Math5-deletion mice fail to
form optic nerves (Brown et al., 2001;Brzezinski et al., 2005,2012). However, cells
with Math5 expression also differentiate into other retinal neurons, indicating that
Math5 is necessary but not sufficient to drive RGC fate. So what other TFs are
required for RGC fate? We and others have reported that two genes from the
Sry-related high mobility box C (SoxC) superfamily, Sox4 and Sox11, are critically
important TFs for RGC differentiation as genetic ablation of both Sox4 and Sox11
dramatically impairs RGC and optic nerve development (Chang and Hertz, 2017;
Chang et al., 2017;Jiang et al., 2013;Kuwajima et al., 2017). Expression of Sox4
is capable of promoting human stem cell differentiation into RGC-like cells, indi-
cating that Sox4 is sufficient to drive RGC cell fate in vitro and in vivo (Chang
et al., 2017). A previous study suggested that SoxC and Math5 TFs regulate RGC
fate in a parallel manner (Chang et al., 2017). In addition to RGC-promoting TFs,
differentiation-suppressing genes such as Notch (Nelson et al., 2006,2007) and
RE1-Silencing Transcription factor (REST) (Chang et al., 2017;Mao et al., 2011)
are known to impede RGC development. Given the importance of TFs in regulating
gene expression, transcriptomic studies may reveal mechanisms downstream of TFs.
Thus, transcriptome studies of retinal development will be an interesting direction in
the future.
In addition to intrinsic RGC signaling, extrinsic signaling also contributes to
retinal development. Many neurotrophic factors such as brain-derived neurotro-
phic factor (BDNF) and ciliary neurotrophic factor (CNTF) reportedly drive
neuronal differentiation, survival, and growth in the retina (Kimura et al., 2016).
Recently, we reported that growth and differentiation factor-11 (GDF-11) and
GDF-15,whichbothbelongtothetransforminggrowthfactor-beta(TGF-b)super-
family, oppositely regulate RGC differentiation in the developing retina in vivo in
rodent retinal progenitor cells (RPCs) and in human embryonic stem cells (hESCs).
The opposing effects of GDF-11 and GDF-15 are partially explained by their abil-
ity to suppress or activate Smad-2, respectively. Pharmacologic or genetic
blockade of Smad-2 in vivo also promotes RGC specification. Genetic knockout
of GDF-15 but not GDF-11 slightly reduces photoreceptor cell numbers as well,
indicating that GDF-15 is not specific to RGCs but also to the development of other
retinal cell types.
2. Retinal cell fate specification 135
2.2 Stem cell to retinal ganglion cell differentiation
In 2006, a two-dimensional (2D) differentiation process to derive RGCs from hESC
was first established (Lamba et al., 2006). ESCs were directed to anterior neural fate
with Dickkopf-1 (Dkk-1, a Wnt inhibitor), Noggin (a BMP inhibitor), and insulin
growth factor 1 (IGF-1). Subsequently, the presence of basic fibroblast growth factor
(bFGF) pushed the stem cells to an eye field fate, an essential structure during retino-
genesis (Mathers et al., 1997;Furukawa et al., 1997). Then, they differentiated into
RPCs (CHX10-/PAX6-positive) and RGC-like cells (HUC/D-positive) (Fig. 6.1).
Meanwhile, mouse ESCs could also be directed to an RGC-like fate in the presence
of Dkk-1, LeftyA (Nodal inhibitor), Activin, and DAPT (Notch inhibitor) (Osakada
et al., 2008). While this method yielded large numbers of photoreceptors, the propor-
tion of RGC-like cells was only 10.1 1.7%. In the next few years, various
approaches were developed to generate RGC-like cells from ESCs or induced
pluripotent stem cells (iPSCs), and they commonly relied on regulating Wnt,
BMP, Nodal, and Notch signaling pathways. In 2014, an optimized method was
developed by differentiating hESC into neural rosettes and subsequent culture of
the neural rosettes in suspension (Riazifar et al., 2014). It was found that only those
RGC-like cells generated from the suspension culture condition could develop with
long fibers. Moreover, in the presence of fetal bovine serum (FBS), hESCs yielded a
significantly higher percentage of BRN3a-positive RGC-like cells. Electrophysio-
logical testing showed the differentiated cells to have electroactivities similar to
cortical neurons. Moreover, RGC-like cells could also be derived from both mouse
ESCs and human iPSCs with chemically defined recapitulation of the
FIGURE 6.1
2D RGC differentiation from stem cell following four stages including stem, neural
precursor, retinal precursor, and RGC-like cells.
136 CHAPTER 6 Stem cell therapies for glaucoma and optic neuropathy
aforementioned developmental regulators including Notch, FGF, and Shh (Teotia
et al., 2017). RNA sequencing revealed that the mouse RGC-like cells derived in
this approach had similar patterns of expression with bona fide E14 mouse RGCs.
Meanwhile, the researchers also cocultured hiPSC-RGCs with mouse superior
colliculus (a target of visual neurons) or inferior colliculus (a target of auditory
neurons). Their work demonstrated that these cells could discriminate specific and
nonspecific targets, and their sodium current had twice the mean amplitude in the
coculture condition, compared to the noncoculture condition.
On the other hand, in addition to supplying several signaling pathway regulators
during neural induction, researchers also direct RGC fate by overexpressing several
essential transcription factors. The most commonly used factors were Atoh7 (also
known as Math5), as inspired by developmental evidence. Overexpressing Atoh7
allowed the generation of RGC-like cells from both mouse and human iPSCs
(including hiPSCs reprogrammed from human Tenon’s capsule fibroblasts) (Chen
et al., 2010;Deng et al., 2016;Xie et al., 2014). It was also found that Sox4 over-
expression could greatly potentiate the differentiation of RGCs from human iPSCs,
and it showed a synergistic effect with Atoh7 on potentiating RGC differentiation
(Chang et al., 2017).
2.3 Retinal organoid differentiation
In 2011, stem cell differentiation of retinal tissue experienced a breakthrough with
the advent of retinal organoid formation. Optimized from stratified cerebral cortical
tissue differentiation, self-organized optic-cup structures were generated by the
addition of basement-membrane matrix components in mouse ESC differentiation
(Eiraku et al., 2011). To initiate the differentiation, signaling pathways including
Wnt and TGF-bwere regulated to recapitulate neurogenesis in vivo. After neural
epithelium fate was directed, and while in the presence of matrix components, optic
vesicleelike structures expressing the retinal cell marker Rx protruded from stem-
cell aggregations. Subsequently, this structure invaginated spontaneously and strat-
ified as in a retina. A year later, such 3D retinal tissues (later called retinal organoids)
were also successfully developed from human ESCs (Nakano et al., 2012).
Meanwhile, another group provided matrix components by plating stem cell
aggregates (embryoid bodies, EBs) on precoated substrates, which generated optic
vesicleelike structures from both normal and patient-specific hiPSC (Meyer
et al., 2011). In 2014, the process of retinogenesis was described in more detail
(Zhong et al., 2014). After plating on matrix-coated substrates, the EBs gain their
anterior neuroepithelial fate, expressing SOX1 and PAX6 (Fig. 6.2). Then, eye
fieldelike domains are developed, expressing LHX2 and Rx, followed by
CHX10-positive neural retina generation. Retinoid acid (RA) was added in
long-term cultures to promote photoreceptor maturation. Moreover, functional pho-
toreceptors were observed for the first time in these retinal organoids, demonstrated
by perforated-patch electrophysiological recordings that detected flash-triggered
responses in photoreceptors.
2. Retinal cell fate specification 137
Soon after, another group reported a differentiation system that could generate
highly enriched populations of optic vesicleelike tissues (Ohlemacher et al.,
2015). This system is widely used in present downstream studies of retinal organo-
ids. However, as further studies developed, researchers found that some hiPSC and
ESC lines were not capable of generating retinal organoids in the differentiation sys-
tems mentioned earlier. Therefore, researchers compared several factors in the dif-
ferentiation of various cell lines, including transcription factors essential to
retinogenesis and supplementary growth factors in previous retinal cell differentia-
tion protocols. Thus, they reported that with additional DKK-1, the disability of
generating retinal organoid in some cell lines could be rescued (Luo et al., 2018b).
In studies of RGCs, the retinal organoid can be a promising platform for invesi-
gating RGC development and neurite outgrowth. Neurite outgrowth modulation
with various soluble factors and substrates has been compared using retinal
organoidederived RGCs (Fligor et al., 2018). Meanwhile, a Brn3-reporter ESC
line was developed to facilitate RGC detection (Sluch et al., 2017). Utilizing this
cell line, RGC loss during retinal organoid development was observed (Capowski
et al., 2019).
In recent years, as RNA-sequencing techniques have developed, further efforts
were made to deeply examine the development of retinal organoids. To understand
similarities and differences between retinal organoids and in vivo retinas during
development, the transcriptome profiles between mouse iPSC-derived retinal orga-
noids and mouse retinas were compared (Brooks et al., 2019). It was found that spe-
cific signaling pathways were temporally dysregulated in developing retinal
organoids and genes involved in photoreceptor functions and survival were delayed
FIGURE 6.2
3D RGC differentiation from stem cell following four stages including stem cell, embryonic
body, neuroepithelium, and retinal organoid.
138 CHAPTER 6 Stem cell therapies for glaucoma and optic neuropathy
or reduced. These may be contributing reasons to the lack of functional maturation
of distinct cell types, including photoreceptors, in retinal organoids. Moreover, cone
and rod cell clusters in 8-month retinal organoids were identified by single-cell RNA
sequencing (Kim et al., 2019). Notably, these photoreceptor cells had similar single-
cell transcriptomes to the human macula. Functional tests demonstrated that cones in
retinal organoids expressed functional HCN1 channels (potassium/sodium
hyperpolarization-activated cyclic nucleotide-gated channel 1), which is a character-
istic feature of human photoreceptors. Moreover, RGC diversity was found in retinal
organoids, but although several RGC subtypes were identified in retinal organoids
using molecular markers (Langer et al., 2018), the subtype distribution may differ
from those in fetal retina (Luo et al., 2019).
3.Retinal cell transplantation
Neuronal cell degeneration is irreversible because neurons will die once injured and
will not be replaced. In the eye, neuronal degeneration happens commonly in age-
related macular degeneration (AMD), retinitis pigmentosa (RP), and glaucoma pa-
tients. Replacing lost retinal cells to connect with the endogenous cell of the host
retina might restore vision. Transplantation of photoreceptor sheets (Mohand-Said
et al., 1997;Ghosh et al., 1999), full-thickness fetal (Ghosh et al., 1998) or adult ret-
inas (Ghosh et al., 1999;Schuschereba and Silverman, 1992;Wasselius and Ghosh,
2001) was conducted by using the Silverman method (Silverman and Hughes, 1989)
with modifications. In 1998, Seiler and Aramant reported that transplantation of
intact fetal retinal sheets together with their RPE restored damaged rat retinas (Seiler
and Aramant, 1998). To increase donor cell survival, exogenous addition of glial-
derived neurotropic factor (GDNF) and brain-derived neurotrophic factor (BDNF)
has been employed in retinal sheet transplant experiments in rodents and has shown
functional improvement (Yang et al., 2010). For photoreceptor transplantation, a
phototransduction assay was conducted in a mouse model, showing that transplanted
photoreceptors can perform the light-to-dark shift similar to endogenous ones (Seiler
et al., 1999). Although there are several successful cases in photoreceptor transplan-
tation, there is still the limitation of long-term survival of transplanted tissue. Less
than 0.2% of transplanted cells were observed to integrate into the host retina
3 weeks posttransplantation (Mansergh et al., 2010). Despite low cell viability,
cell transplant is still a potential therapeutic strategy for vision restoration.
3.1 Primary retinal ganglion cell transplantation
RGCs are important neuronal cells in the eye, which receive visual information from
photoreceptors via bipolar cells and transfer the signals all the way to the brain
through their extending axons. RGCs degenerate in patients with glaucoma or optic
neuropathy. However, there is no cell regeneration or endogenous cell replacement
after RGC injury, which causes permanent vision loss. Therefore, cell
3. Retinal cell transplantation 139
transplantation has been proposed as a potential therapy for RGC replacement in
such cases. Some studies successfully transplanted primary rodent RGCs to the
host eye and observed donor cell integration, synapse formation, light response,
and axon extension into the optic chiasm (Hertz et al., 2014;Venugopalan et al.,
2016). Although using primary cells for transplantation is a potential approach,
resources of donor RGCs are very limited. Thus, stem cellederived RGCs are an
attractive alternative.
3.2 Stem cellederived retinal ganglion cell transplantation
Although various methods of deriving RGC-like cells directly from stem cells have
been reported, studies on transplanting these cells are relatively rare. In 2012, RGC
precursors were derived from human Mu
¨ller stem cells and transplanted onto the
inner retinal surface of an RGC-depletion rat model (Singhal et al., 2012). Trans-
planted cells were observed extending processes into the host RGC layer and
partially restored RGC function. However, when these cells were applied into the
feline eye, they aggregated in the vitreous cavity and elicited a severe inflammatory
response. Fortunately, after applying donor RGC precursors derived from feline-
Muller stem cells, combined with a collagen scaffold, an improvement was observed
in RGC functional examination (Becker et al., 2016). Recently, another group
derived functional RGCs from spermatogonial stem cells by viral transfection or
chemical induction (Suen et al., 2019). Subsequently, those derived RGCs were
injected into a glaucoma mouse model and donor cells were observed to survive
10 days posttransplantation, but functional improvement was not reported. Mean-
while, Zhang et al. differentiated RGC-like cells from hESCs and found them to
have immature responses to a GABA
A
receptor agonist (Zhang et al., 2019). By
intravitreal injection, these RGC-like cells were transplanted into an adult rat eye
and demonstrated successful migration into the host ganglion cell layer
(Fig. 6.3A). Although some studies have shown electrophysiological improvement
after transplantation, most of them only achieved graft survival, without evidence
of donor cell integration or synaptic reconnection.
3.3 Retinal organoid transplantation
Given the advantage of retinal organoids to highly mimic retinogenesis and produce
fine layers, they may be a promising source of donor cells for retinal transplantation.
To date, retinal organoids were mainly transplanted as dissociated RPCs. As early as
2008, Aoki et al. derived eye-like structures from ESCs, an early form of the retinal
organoid, and injected the dissociated cells into the mouse vitreous cavity (Aoki
et al., 2008). Cells were observed to spread along the GCL and further differentiated,
evidenced by RGC-specific markers, especially in RGC-degeneration models
(Fig. 6.3B). Recently, RPCs harvested from retinal organoids were injected into
the subretinal space of nonhuman primates (Chao et al., 2017). Cells survived and
migrated to the RGC layer. Although functional integration was not achieved,
140 CHAPTER 6 Stem cell therapies for glaucoma and optic neuropathy
GFP-labeled neurite growth had also been observed, targeting to the host optic nerve
head. To further purify the RPCs from retinal organoids, cells were sorted by c-Kit
(Zou et al., 2019), which is a type III receptor tyrosine kinase that binds to stem cell
factor (SCF). Then, c-Kit positive cells were injected into subretinal space of the rd1
model of mice and rats, and electrophysiological improvements were observed post-
transplantation. Although most of the “integrated” cells were then proven to be host
cells that received material transfer from donor cells, c-Kit þcells did modulate
microglia and may thereby provide a healthier microenvironment to delay retinal
degeneration.
Besides dissociated RPCs transplantation, several attempts had been made to
transplant the retinal organoid as a tissue sheet. Mandai et al. transplanted mouse
iPSC-derived retinal organoid sheets into an end-stage retinal degeneration model
(rd1) (Mandai et al., 2017). Direct contact between host bipolar cell terminals and
the presynaptic terminal of donor photoreceptors was visualized in this study. The
transplanted rd1 mice showed light-responsive behaviors and light responses were
recorded from host RGCs. Meanwhile, hESC-derived retinal organoid sheets were
also transplanted subretinally into a mouse model of photoreceptor degeneration
(Iraha et al., 2018). No rejection was observed, although it was a heterotransplanta-
tion. The tissue sheet achieved long-term survival and further maturation of photo-
receptors, and light responses were also recorded from host RGCs. As early as 2015,
there were attempts to transplant retinal organoid sheets into nonhuman primate
FIGURE 6.3
Transplantation of RGC-like cells/retinal progenitors from 2D (A) or 3D (B) differentiation
culture into rodent system. GFP or tdTomato fluorescence would be introduced in 2D or
3D culture, respectively, for facilitating visualization after transplant.
3. Retinal cell transplantation 141
models of photoreceptor degeneration (Shirai et al., 2016). Grafted sheets survived
in primate eyes and achieved further differentiation into a range of retinal cell types.
Immunostaining suggested the formation of synaptic connections between donor
photoreceptor and host bipolar cells. Unfortunately, electrophysiological examina-
tion was not performed in this study. Recently, it was reported that hiPSC-derived
retinal organoid sheets survived well in the host retina of primates over 2 years,
which is the longest follow-up report in a primate model to date (Tu et al., 2019).
In the graft, a substantial number of mature photoreceptors were observed, and visu-
ally guided saccades also suggested a mild recovery of light perception 1.5 years
after transplantation.
As mentioned earlier, most of these studies, especially those performed on larger
animal models, focused on photoreceptor degeneration models. One of the problems
that hinders retinal organoid transplantation into the inner eye of retinal degenera-
tion model animals was the variation of inner retinal anatomic structures among
species. In rodent eyes, the vitreous cavity is relatively much smaller, facilitating
the contact and migration of donor cells to the ganglion cell layer of the host retina.
However, in larger animal models, vitreous cavities make up most of the volume of
the eyeball, causing donor cell suspensions to reaggregate and fail to contact with
the inner layer of the host retina (Becker et al., 2016). Therefore, tissue engineering
of a donor sheet is thought to be a promising solution (Fig. 6.4A). RGC-like cells
FIGURE 6.4
RGC-like cells in a dissociated form (A) or combined with an engineered tissue scaffold
(B) are transplanted into a nonhuman primate system.
142 CHAPTER 6 Stem cell therapies for glaucoma and optic neuropathy
were collected from retinal organoids and seeded on a PLGA-scaffold. This RGC-
scaffold biomaterial (retinal sheet) supported neurite outgrowth for donor cells
and showed satisfying biocompatibility in rabbit and primate eyes (Li et al.,
2017). Further, the same group of researchers established the surgical procedure
for retinal sheet delivery and epiretinal fixation in primate eyes, which may facilitate
inner retinal transplantation in large animal models (Luo et al., 2018a)(Fig. 6.4B).
3.4 iPSCs for cell replacement therapy
The iPSC is a type of pluripotent stem cell that is reprogrammed from somatic cells,
which was pioneered by Takahashi and Yamanaka in 2006. The manipulation of cell
fate has led to new approaches in studies of personal cell replacement therapy.
Briefly, somatic cells including but not limited to fibroblast cell from the skin,
peripheral blood mononuclear cell (PBMC), and urine cells were collected from
host patients. By introducing four transcription factors (Myc, Oct3/4, Sox2, and
Klf4), somatic cells could regain pluripotency and then give rise to every cell
type in the body. Theoretically, cells derived from this patient-specific hiPSC had
the same genetic background as the host and the autologous transplantation could
with no risk of immune rejection. However, studies have shown that the hiPSC
had epigenetic variations, distinct DNA methylation patterns, and acquired copy
number variations (Mills et al., 2013;Nishizawa et al., 2016;Cahan and Daley,
2013), which may then induce an immune reaction to some extent. On the other
hand, although the multilineage differentiation potential of iPSCs has been proven
by teratoma formation, the differentiation capacity of various mature cells or tissues
can differ among cell lines and clones (Martinez et al., 2012). A recent study showed
that excluding OCT4 during reprogramming resulted in great improvement of iPSC
developmental potential (Velychko et al., 2019), since the overexpression of Oct4
led to off-target gene activation and epigenetic aberrations. Thus, researchers are
still further understanding the pluripotency of iPSCs, and before clinical application,
the safety of iPSC-derived donor cells including tumorigenesis and immunogenicity
need further evaluation.
When it comes to a cell replacement therapy for glaucoma and neuropathy,
despite the technical problem in transplantation mentioned earlier, whether the
donor neurons could reconnect to the host central neural system is another key
obstacle. In those limited studies of stem cellederived RGC replacement therapy
in primate model (Chao et al., 2017), no synapse formation was observed yet.
4.iPSCs for modeling of familial glaucoma
Glaucoma is a leading causes of blindness (Jonas et al., 2017) and, along with other
optic neuropathies, is characterized by the loss of RGCs (Stowell et al., 2017). IOP is
the most common risk factor associated with glaucoma and thought to be a primary
contributor to RGC cell death. IOP management is the current standard of care for
4. iPSCs for modeling of familial glaucoma 143
glaucoma patients; nevertheless, in some patients, this fails to stop the loss of RGCs
and progressive visual dysfunction. Unfortunately, vision loss due to RGC death is
irreversible, leading to bilateral blindness in as many as 14% of diagnosed patients
(Blomdahl et al., 1997).
The main types of primary glaucoma, including POAG, PACG, and NTG, were
distinguished by IOP and the mechanism of IOP increase. However, as they are all
forms of primary glaucoma, they share the same pathology of RGC loss. It had been
widely confirmed that primary glaucoma has a genetic etiology, especially in famil-
ial cases. Many genes are correlated with POAG such as caveolin-1 (CAV1)
(Thorleifsson et al., 2010), ABCA1, AFAP1, GMDS, PMM2, TGFBR3, FNDC3B,
ARHGEF12, GAS7, FOXC1, ATXN2, and TXNRD2 (Gharahkhani et al., 2014;
Chen et al., 2014;Li et al., 2015;Springelkamp et al., 2015;Bailey et al., 2016).
NTG (Anderson et al., 2001) and juvenile open angle glaucoma (JAOG) (Souzeau
et al., 2017) are two kinds of early-onset POAG. Genetic mutation of optineurin
(OPTN) (Aung et al., 2005) and myocilin (MYOC) (Souzeau et al., 2017;Turalba
and Chen, 2008) are contributors to NTG and PACG, respectively. There are eight
known genes involved in PACG including PLEKHA7, COL11A1, PCMTD1-
ST18, EPDR1, CHAT, GLIS3, FERMT2, and DPM2-FAM102 (Vithana et al.,
2012;Khor et al., 2016). Although there is no known candidate gene for exfoliation
syndrome glaucoma (XFG), two genomic regions LOXL1 (Thorleifsson et al., 2007)
and CACNA1A (Aung et al., 2015) are predicted for causing XFG.
In the last two decades, much efforts have been focused on the phenotypic
changes in the trabecular meshwork (TM) that are caused by genetic mutations,
such as MYOC mutation, which were thought to be the cause of dysfunction of
TM and leading to subsequent RGC death by increasing IOP (Joe et al., 2015). How-
ever, in recent years, the susceptibility and vulnerability of RGC to damage due to
genetic mutations were getting more and more attention. As a tool for investigation,
iPSCs are an “in-dish twin” of specific patients, carrying the genetic background,
and are a promising model for understanding the real pathological changes caused
by a given mutation. As primary glaucoma has proved to be a polygenic disease
(Qassim et al., 2020), the advancements in gene editing technology give us a chance
to identify the distinguishing functional changes caused by a specific mutation. With
such understanding, clinical suggestions can be made according to how much the
neuropathy in a specific patient is contributed by IOP increase and RGC
vulnerability.
The CRISPR/Cas9 system, a technology that can specifically modify, delete, or
correct precise regions of DNA, was first observed in the 1990s in prokaryotes (Moj-
ica et al., 1993). In 2012, the Doudna and Charpentier groups showed that a single
guide RNA (sgRNA) can effectively mimic the tracrRNA:crRNA complex, enabling
more efficient design (Jinek et al., 2012). For DNA editing, there are two DNA repair
mechanisms (Xu et al., 2018). One is homology directed repair (HDR), which occurs
during the S phase of dividing cells and uses a homologous DNA sequence as a tem-
plate for the reconstruction of broken DNA (Iyama and Wilson, 2013). Another one
144 CHAPTER 6 Stem cell therapies for glaucoma and optic neuropathy
is nonhomologous end joining (NHEJ), which occurs in dividing cells and also in
postmitotic nondividing cells (Iyama and Wilson, 2013). For treating RP,
CRISPR/Cas9-driven HDR has been used to mimic human RP in a mouse RP model.
One study observed the biallelic mutations of receptor expression enhancer protein 6
(REEP6) in several RP patients (Arno et al., 2016) and created a CRISPR/Cas9
knock-in of Reep6
L135P/L135P
in mice to discover loss of the outer nuclear layer
(ONL) (Arno et al., 2016), suggesting that this mutation of REEP6 may lead to
RP. Another study utilized CRISPR/Cas9 to knock-in the Rp9 gene in a murine
cone cell line (661 W cells) and showed a potential correlation between Rp9 and
RP (Lv et al., 2017). In the case of CRISPR/Cas9-driven NHEJ, disruption of
vascular endothelial growth factor (VEGF) secretion in the retinal epithelium
(RPE) (Kim et al., 2017) or mutation of VEFGR2 can reduce choroid neovasculari-
zation (CNV) (Huang et al., 2017), an early symptom of AMD.
In glaucoma, CRISPR/Cas9 was utilized in a TM cell line to generate a CAV1
knockout (KO) genotype (Wu et al., 2019). That study showed that the CAV1-KO
TM cells have reduced adhesion with higher extracellular matrix-degrading enzyme
expression, suggesting that CAV1 is important for the physiological regulation of
TM cells (Wu et al., 2019). A recent study applied CRISPR/Cas9 technology in
glaucoma patient-derived iPSCs to repair the genetic mutation for treating
X-linked retinitis pigmentosa (XLRP) (Bassuk et al., 2016). All the aforementioned
studies revealed that CRISPR/Cas9-driven DNA editing is a potential therapeutic
strategy for congenital and/or inherited eye diseases.
On the other hand, iPSCs have advantages in cell replacement therapy, especially
considering the homology of patient-specific iPSC-derived donor cells. Considering
the mutation carried by patient-specific iPSCs, gene editing could fix the mutation
before transplantation or differentiation.
Trabeculectomy, also known as filtering bleb, was first reported by Cairns in
1968 for treating glaucoma (Cairns, 1968). Trabeculectomy is a surgical procedure
of removing part of the trabecular meshwork, which can increase the outflow of fluid
from the eye and then release IOP (Cairns, 1968). To avoid the wound healing
response, antiscarring agents such as antimetabolites 5-fluorouracil (5-FU) (Linde-
mann et al., 2017) and mitomycin C (Fontana et al., 2006) are most commonly used
during or after surgery. Some cases show promising long-term effects of trabeculec-
tomy in POAG (Bendel and Patterson, 2018).
Moreover, it was found that some familial glaucoma patients had unexpected
outcomes after filtration surgery. A family carrying a Pro370Leu MYOC gene
mutation was followed for 10 years and demonstrated that with the passage of
each generation, the postoperation IOP declined further, and none of them were
prone to filtering bleb scarring (Zhuo et al., 2008). This phenomenon may also indi-
cate the genetic factor affecting the postsurgery outcome of glaucoma. In this
situation, hiPSCs from such families could be differentiated into fibroblasts to iden-
tify the mechanism of filtering bleb scarring in order to develop better medication to
maintain functional bleb.
4. iPSCs for modeling of familial glaucoma 145
5.Summary and future directions
In summary, understanding the regulatory mechanisms of RGC development will
help us to differentiate RGC from progenitor cells or stem cells. In the eye, to treat
diseases caused by genetic deficiency, human iPSC-derived retinal cells have
become an important tool for investigation via CRISPR/Cas9 editing. In glaucoma
or optic neuropathy, stem cellederived cell replacement provides a potential treat-
ment for retinal degenerative diseases. Although RGCs survive after transplant, pro-
motion of proper cell integration, synapse formation, and physiologic function will
be a challenge for restoring visual circuit.
With the fast advances in sequencing technology and bioinformatic analysis, re-
searchers will be able to further understand organoid differentiation. In the following
studies, an organoid that more closely resembles the in vivo development will be
derived, not only morphologically, but also at the levels of the transcriptome, epige-
nome, protostome, etc. As a highly simulating model, organoids can provide further
insights to retinal development. Moreover, in the assistance of gene-editing technol-
ogy, organoids carrying various gene mutations can be generated as a model of iden-
tifying the functions of specific genes and for drug screening.
Although the nonhuman primate has been used for studying stem cellederived
RGC replacement therapy, many challenges still limit progression. First, there is
the challenge to efficiently produce enough material for transplantation. Considering
the nonproliferation and fragility of neurons, donor cells must be harvested in large
quantities. Simultaneously, we need to optimize the time point of cells for transplan-
tation, in order to find a balance between the neuronal maturity and the ability to
migrate and integrate. Because of the larger vitreous cavity in primate models,
providing sufficient contact between donor cells and the host retina will be a prob-
lem that can benefit from tissue engineering advancements. With the proper tissue
engineering scaffold, we can not only delivery donor cells to a targeted position
in the retina, but also direct axon regeneration and control the release of growth fac-
tors and immunosuppressors, if necessary. Subsequently, studies will also focus on
axon reconnection and the following physiological evaluation, including visual elec-
troactivities and visualebehavior reaction.
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References 153
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Induced Pluripotent Stem
Cells (iPSC) in Age-related
Macular Degeneration
(AMD) 7
Graham Anderson, PhD
1,*
, Pierre Bagnaninchi, PhD
1
,
Baljean Dhillon, BMedSci(Hons), BMBS FRCS(Glasg), FRCOphth, FRCS(Ed),
FRCPE
2,3
1
Centre for Regenerative Medicine, Institute for Regeneration and Repair, The University of
Edinburgh, Edinburgh BioQuarter, Edinburgh, United Kingdom;
2
Centre for Clinical Brain
Sciences, University of Edinburgh, Edinburgh Bioquarter, Edinburgh, United Kingdom;
3
NHS
Lothian, Clinical Ophthalmology, Princess Alexandria Eye Pavilion, Edinburgh, United Kingdom
*Corresponding author. e-mail address: 1139761@ed.ac.uk.
Chapter outline
1. Background .......................................................................................................156
1.1 AMD .................................................................................................. 156
1.2 The RPE’s Role in the Subretinal Niche ................................................156
1.2.1 Support ..................................................................................... 156
1.2.2 Barrier Function ......................................................................... 159
2. Cellular Therapies for AMD ................................................................................. 159
2.1 Induced Pluripotent Stem Cell (iPSC)-derived RPE ................................ 161
2.2 Clinical Trials Utilizing iPSC-RPE in AMD ............................................. 162
2.3 Genomic Instability in iPSC-RPE, is Direct Reprogramming the Answer? .. 164
3. Future Directions ................................................................................................166
References .............................................................................................................167
Abstract
Age-related macular degeneration (AMD) is a leading cause of legal blindness in
many OECD nations, yet still has limited treatment options. Recent advances in
hPSC technologies promise to significantly improve the prognosis of those with
AMD through the use of iPSC-derived cell replacement therapies. Moreover, the
use of iPSC-derived tissues in vitro makes it possible to produce mature
patientederived tissues phenotypically similar to those found in vivo without the
scarcity or batch variability of embryonic stem cell (ESC)-derived tissue or primary
sources. In this chapter, we highlight the recent advances in the clinical applications
of iPSCs in the treatment of AMD and discuss the challenges this technology must
CHAPTER
155
Recent Advances in iPSCs for Therapy, Volume 3. https://doi.org/10.1016/B978-0-12-822229-4.00006-1
Copyright ©2021 Elsevier Inc. All rights reserved.
mediate in order to enter routine practice alongside novel techniques, which can be
deployed to accelerate this process.
Keywords:
Bioenergetics; Clinical trial; Dry AMD; iPSC; iPSC-RPE; Lipofuscin; Ophthalmology;
Photoreceptors; Pluripotency; Reprogramming; RPE; Synthetic-transcription factors; Tight junctions;
VEGF; Wet AMD.
1.Background
1.1 AMD
Age-related macular degeneration (AMD) is a leading cause of blindness among
industrialized nations and a significant source of morbidity globally (Wong et al.,
2014). The pathogenesis of AMD is broadly divided into two pathways, “wet”
and “dry.” Wet AMD (also known as exudative or neovascular AMD) is an aggres-
sive form of retinal degeneration wherein the underlying vasculature erupts into the
retinal space leading to upheaval of the retinal pigmented epithelium (RPE) and
scarring. In Europe, wet AMD accounts for approximately 10% of AMD diagnoses
but accounts for 90% of blindness diagnoses. However, wet AMD can be controlled
through the continued use of vascular endothelial growth factor (VEGF) blockers
(such as bevacizumab [Avastin] and ranibizumab [Lucentis]) and photodynamic
therapy (Detty et al., 2004;Sheu et al., 2015). Dry AMD (or nonexudative AMD)
is hallmarked by the presence of lipid and protein deposits, Drusen, visible upon
fundus exam, and progressive central vision loss without vascular involvement.
This is the result of geographic atrophy of the photoreceptor layer following the
degeneration of the RPE layer on which the viability of the photoreceptors is wholly
dependent (Schmitz-Valckenberg et al., 2016;Fleckenstein et al., 2018). Dry AMD
makes up the majority of AMD diagnoses in Europe; however, despite being less
aggressive than wet AMD, currently there are no effective treatments to slow or
reverse its progression (Mitchell et al., 2018).
The lack of treatment options, coupled with the relative immune privilege of the
eye and the ease with which the retina can be monitored, has made AMD an ideal use
case for the development of treatments with advanced cellular therapies based on
induced pluripotent stem cells (iPSCs).
1.2 The RPE’s Role in the Subretinal Niche
The RPE is a monolayer of polarized, hexagonal epithelial cells laden with eumela-
nin. RPE function can broadly be divided into two categories: support of the retinal
microenvironment and barrier function.
1.2.1 Support
The process of phototransduction in vertebrates relies wholly upon the isomerization
of 11-cis retinal into all-trans retinal as the first step of the visual cascade; however,
this process leaves the opsin bleached and unfit for use. Therefore, in order for long-
term vision to be achieved, the replenishment of 11-cis-retinal is critical.
156 CHAPTER 7 Induced Pluripotent Stem Cells
While dietary vitamin A can serve as a substrate for 11-cis-retinal production, the
majority of 11-cis-retinal is provided via a recycling process facilitated by the RPE
(Baehr et al., 2003). Genome association studies have implicated one of the genes
involved in this process, ABCR, in the pathogenesis of AMD and shown its mutation
to be the causative agent in Stargardt’s disease (Allikmets, 2000).
A central event in the visual cycle of the vertebrate retina is the phagocytosis of
bleached outer segments of overlying photoreceptors by the RPE. The opsin-rich
outer segments of the photoreceptors exist in a state of constant turnover instructed
both by lightedark cycles and endogenous circadian rhythms (LaVail, 1976;Grace
et al., 1999). This turnover minimizes the influence of photooxidative lipid peroxi-
dation upon the photoreceptors and provides a renewable substrate for the produc-
tion of 11-cis-retinal by the RPE (Besharse and Hollyfield, 1979). Photoreceptor
phagocytosis by the RPE is orchestrated via the binding surface integrin a
v
b
5
and
internalization protein Mer tyrosine kinase (MerTK) (Finnemann et al., 1997;Rog-
gia and Ueta, 2015). Mutations in the MerTK gene have been implicated in retinitis
pigmentosa (RP) but have been effectively ruled out as a cause of AMD (Al-khersan,
Kwong and Grassi, 2019). Furthermore, the RPE shuttles cholesterol-rich high-
density lipoproteins (HDL) derived from photoreceptor outer segments (POS)
apically and basally to avoid toxicity and provide a substrate for photoreceptor
regeneration (Storti et al., 2017).
One of the most obvious features of the RPE is their melanin-rich pigment. RPE
pigment is primarily made up of antioxidant eumelanin with only a small fraction
made up of the prooxidant pheomelanin, although this ratio does falter with age
(Ito et al., 2013). The primary function of the melanin within RPE is to capture scat-
tered light to reduce its interference with the photoreceptors and thereby improve vi-
sual acuity. This has the added benefit of limiting the degree of photooxidation that
takes place within the retina. Additional antioxidant protection is provided by antiox-
idant chromophores such as b-carotene, lutein, and zeaxanthin, which together bolster
the RPE’s ability to absorb light over a broad range of wavelengths. Dietary supple-
mentation of these antioxidant chromophores was previously shown to slow the pro-
gression of AMD in the landmark AREDS (Age-Related Eye Disease Study) clinical
trials (Kassoff et al., 2001;Chew et al., 2013) and rollout of these supplements to
patients is currently being explored (Lee et al., 2018). Lipofuscin, although usually
associated with aging pathologies, is also thought to have a protective role in the
retina, via the absorption of harmful light in the blue end of the spectrum.
However, its accumulation within the RPE with age is now believed to facilitate
blue-light phototoxicity, which may have a role in retinal degeneration (Sparrow
et al., 2002;Ozkaya et al., 2019).
Additionally, the RPE supports the outer retinal niche through the secretion of
multiple growth factors Fig. 7.1). Apically, the RPE secretes pigment epithelium-
derived growth factor (PEDF), a neurotrophic factor required for the normal devel-
opment of the neurosensory retina, including the photoreceptors (Tombran-Tink
et al., 1991), and a powerful antiangiogenic (Dawson et al., 1999). Basally, the
RPE secretes proangiogenic factors such as soluble vascular endothelial-derived
growth factor (VEGF) to support the development and maintenance of the chorioca-
pillaris (Saint-Geniez et al., 2009).
1. Background 157
The bioenergetics of the retina are primarily taken care of through the glucose
transport (GLUT-1) class of membrane channels (Sugasawa et al., 1994;Swarup
et al., 2019) alongside lactate uptake facilitated through the MCT1 and MCT3 trans-
porters (Philp et al., 1998).
Recently Kanow et al. (2017) demonstrated that the RPE sits in the center of a
bioenergetic ecosystem wherein the lactate secreted from the photoreceptor layer
during respiration inhibits glycolysis within the RPE, sparing the glucose and allow-
ing it to be utilized by the photoreceptors. Moreover, Chao et al. (2017) used targeted
liquid chromatographyetandem mass spectrometry (LC/MS-MS) to show that RPE
FIGURE 7.1
Illustration of the outer blooderetina barrier highlighting the molecules of interest in RPE-
driven homeostasis.
158 CHAPTER 7 Induced Pluripotent Stem Cells
shows a preference to proline as an energetic substrate, converting it to glutamate for
insertion into the Kreb’s cycle. Together, these studies illustrate how the RPE is able
to effectively support the bioenergetic requirements of the inner retina without itself
being a drain on resources.
1.2.2 Barrier Function
The blooderetina barrier (BRB) exists in two parts, the highly dynamic inner BRB
is maintained by the endothelial cells and pericytes of the retinal vasculature (Sun
and Smith, 2018) while the outer BRB is a product of the RPE. The structure of
the outer BRB is hallmarked by the presence of apical tight junctions between adja-
cent RPE wherein their extracellular membranes are entwined with one another via
tight-junction proteins such as those of the claudin and occludin families as well as
associated proteins such as ZO-1 (Caceres and Rodriguez-Boulan, 2020).
The RPE tight junctions are critical in maintaining the immune privilege of the
inner retina and preventing the invasion of immunomodulatory cells into the retinal
space. Consequently, the breakdown of the RPE-RPE tight junction is considered a
key tipping point between early and advanced AMD (Naylor et al., 2019).
Recent work by Benedicto et al. (2017) highlighted the synergistic relationship
between RPE tight junction integrity and the endothelial cells (EC) of the chorioca-
pillaris. Using contacting and noncontacting coculture methods, the researchers
were able to demonstrate that the endothelial cells of the retinal vasculature secrete
molecules that encourage improvement in tight-junction integrity and basement
membrane deposition in cultured fetal RPE.
Further experiments illustrated that this process is mediated through lysyl oxi-
dase expression (Smith-Mungo and Kagan, 1998), seemingly unique to ECs, and
adds credence to the assertion that dysregulation within the vasculature may often
precede retinal pathology (Benedicto et al., 2017;Paterson et al., 2020).
Moreover, the RPE is responsible for handling ionic and metabolic exchange
across the BRB. This process is primarily handled by apically located Na
þ
-K
þ
-
ATPase-driven pumps, which maintain ionic and pH homeostasis in the subretinal
space (Wetzel et al., 1999;Ropelewski and Imanishi, 2019). Interestingly, despite
its central role in retinal fluid balance and ionic control, the RPE exhibits relatively
low expression of the water transport channel aquaporin (AQP) (Verkman et al.,
2008;Sisto et al., 2019). This would suggest that the intraocular pressure (IOP) of
the eye, in concert with the AQPs of the neurosensory retina, is sufficient to drive
fluid from the subretinal space (Verkman et al., 2008); this in turn confers the adhe-
sion the RPE needs to form a tight barrier.
2.Cellular Therapies for AMD
Due to their central role in the function, and dysfunction, of the retina, the RPE has
been the target of numerous therapies for retinal degeneration, such as AMD, trialled
over the years. Pharmaceutical interventions targeting molecules associated with
2. Cellular Therapies for AMD 159
AMD, such as amyloid-b(Ding et al., 2011;Rong et al., 2019) and complement fac-
tors (Yehoshua et al., 2014), have shown limited success in treating dry AMD. The
dearth of pharmaceutical options available to patients has lead researchers to explore
the use of autologous or allogenic RPE transplants with or without the surrounding
photoreceptors and Bruch’s membrane (BrM)echoroid complex.
Early cell replacement approaches relied upon moving cells from a healthy area
of the retina and relocating them to the sight of geographic atrophy. One such tech-
nique, called ‘Retinal Translocation’ commonly involves injecting saline solution,
subretinally, to ‘bleb’ a small portion of the retina from its anchorage uponBrM.
This portion is then dissected free from the surrounding tissue and rotated 25e50
degrees before being replanted upon the choroid (Fig. 7.2)(Eckardt et al., 1999).
While this approach and related full thickness translocation interventions (Va n
Meurs and Van Den Biesen, 2003;Karasu and Erdo
gan, 2019) have shown some ef-
ficacy in improving the vision of patients with AMD, they place a considerable
health burden on the patients and carry risk of surgical complications related to peri-
operative bleeding and retinal detachment (MacLaren et al., 2005).
In order to deliver on the promise shown by the RPEechoroid translocation in-
terventions, it was clear that a more convenient source of cells, coupled with a more
straightforward deposition procedure, was sorely needed. The practice of intro-
ducing RPE allografts into the subretinal space began in the late 1980s, where Gou-
ras, Algvere, et al. used a combination of cultured fetal RPE patches and cell
suspensions to repair the retinas of individuals with wet and dry AMD (Gouras
and Algvere, 1996). These initial trials into the delivery of RPE within the subretinal
space highlighted the need for an intact BrM and correct RPE orientation to facilitate
stable engraftment, thus paving the way for patch-base cell replacement techniques
(Tezel et al., 1999).
One of the primary limitations of cell sources at the time was the limited avail-
ability of suitable fetal tissue and, conversely, the potential tumorigenicity of more
abundant sources such as the immortalized cell-line aRPE-19 (Wang et al., 2005).
FIGURE 7.2
Schematic of retinal rotation to relocate atrophic RPE away from the macula.
160 CHAPTER 7 Induced Pluripotent Stem Cells
This need remained largely unfilled before the discovery and scale-up of human em-
bryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs).
While several cellular therapies for AMD utilizing hESCs have been developed,
for this chapter, we will be focusing upon those that relied upon hiPSCs.
2.1 Induced Pluripotent Stem Cell (iPSC)-derived RPE
Since their development in 2006 (Takahashi and Yamanaka, 2006), iPSCs have been
used to derive tissue from every germ layer of the body, facilitating the production of
cardiomyocytes (Funakoshi et al., 2016), neurons (Pa¸sca et al., 2011) and hepato-
cytes (Yi et al., 2012), as well as RPE (Buchholz et al., 2009) and photoreceptors
(Lamba et al., 2010).
Compared to other tissues, the production of iPSC-derived RPE (iPS-RPE) is
relatively straightforward. This is due in large part to the fact that the RPE is part
of the “default pathway” of tissue differentiation. The default model of tissue devel-
opment suggests that ectodermal cells, in the absence of embryonic morphogens
such as bone morphogenic proteins (BMPs) and transforming growth factor-b
(TGF-b), will differentiate toward a neuronal fate (Stern, 2006;Suzuki and Vander-
haeghen, 2015). For iPSC cultures this means simply maintaining the undifferenti-
ated colonies in culture without specialized factors is sufficient to produce neurons
alongside retinal ganglion cells and a small proportion (w1%) of RPE (Maruotti
et al., 2013). Typically, differentiation can be improved by first including basic fibro-
blast growth factor (bFGF) in the iPSC growth medium until the cells are established
and then removing it (Buchholz et al., 2009).
RPE produced in this fashion has been shown to produce robust tight junctions
along with many of the visual cycle proteins found in primary RPE such as RPE-
specific protein 65 kDa (RPE65), Bestrophin (BEST), and microphthalmia-
associated transcription factor (MITF) while also being capable of phagocytosing
photoreceptor outer segments (POS). This highlights that even RPE derived using
this simple protocol retains the molecular machinery required to fulfill their roles
within the retina.
While simple and effective, contemporary methods for deriving RPE from iPSC
have started to move away from the use of transcription factors such as bFGF and
TGF-band now rely more upon small molecules to drive differentiation. This is
due in part to the potential unreliability of growth factorebased differentiation pro-
tocols within clinical applications as they can suffer from batch-to-batch variation.
Initially, working on hESCs, Idelson and colleagues demonstrated the effectiveness
of using the B vitamin nicotinamide (NIC) and the TGF derivative Activin A in
differentiating RPE (Idelson et al., 2009). It was later shown that NIC was able to
temper markers of AMD in iPSC-RPE derived from individuals with AMD (Saini
et al., 2017). The cytoprotective and differentiating action of NIC is thought to be
driven by its role as a substrate for nicotinamide adenine dinucleotide (NAD
þ
) pro-
duction. NAD
þ
, alongside its reduced form NADH, is a central player in cellular
respiration, thus adding evidence to the case that AMD is, in part, a product of
2. Cellular Therapies for AMD 161
sustained perturbation of metabolic homeostasis (Cano et al., 2014) and affirms the
role of bioenergetics in RPE development.
Alongside NIC, the addition of the dimeric epidithiodiketopiperazine (ETP)
fungal metabolite chetomin (CTM) has been shown to improve iPS-RPE differenti-
ation by four to fivefold over using NIC alone (Maruotti et al., 2015). However, it is
worth bearing in mind that differentiation efficiency can vary dramatically between
iPSC lines depending on which tissue they were original derived from due to epige-
netic memory.
Encouragingly, the NIC-CTM protocol has successfully been applied to iPSCs
derived from fetal lung fibroblast (IMR09-4; WiCell), CD34
þ
bone marrow samples
(BC1; Johns Hopkins), and skin fibroblasts (IPSCORE; WiCell (Smith et al., 2019),)
suggesting this protocol can be widely applied to iPSC from different sources.
2.2 Clinical Trials Utilizing iPSC-RPE in AMD
The reduced need for manipulations, such as colony dissection or selective trypsini-
zation, as well as the possibility of small moleculeedriven differentiation and rela-
tively short culture period, makes iPS-RPE an attractive cell therapy for good
manufacturing practice (GMP) scale-up and clinical translation. Additionally, the
autogenic nature of patient-derived iPSC-RPE means that immunosuppression is
not required, thus removing a common cause of adverse events in hPSC-RPE
replacement trials (Schwartz et al., 2015;McGill et al., 2018). It is for these reasons
that iPSC-RPE cells were the first iPSC tissues to make it to clinical trial, doing so in
less than 10 years after iPS cells were first described (Takahashi et al., 2007;Mandai
et al., 2017).
In their seminal paper, Mandai et al. were able to successfully transplant a sheet
of iPSC-RPE onto the atrophic retina of a patient with exudative AMD. As this was a
first-in-man study, the primary endpoint was not to cure the patient of AMD but to
prove that the iPSC-RPE cell product, alongside the deposition method, was reason-
ably safe and feasible. Two patients were recruited, patient 1 was a 77-year-old
woman of Japanese descent with exudative AMD in both eyes and patient 2 was
a 68-year-old Japanese man with exudative AMD in one eye. Both patients had
received anti-VEGF treatment prior to recruitment, with patient 2 also having
received PDT (Mandai et al., 2017).
In the study, iPSC-RPE was derived from dermal fibroblasts harvested via skin
biopsy of the patients’ inner arm. The patients’ fibroblast served both as a target
for reprogramming to iPSC and as a source of xeno-free feeder cells (following
mitomycin C inhibition) on which to culture the iPSC colonies.
To deliver the transcription factors necessary for transformation, the investiga-
tors chose to use two episomal vectors based on the EpsteineBarr virus nuclear
antigen-1 (oriP/EBNA-1). Episomal transfection is less efficient, and therefore
more costly, than conventional vectors such as lentivirus but has the distinct advan-
tage of being nonintegrating and biologically inactive, thus conferring it a more
favorable clinical risk profile (Bang et al., 2018).
162 CHAPTER 7 Induced Pluripotent Stem Cells
For this study, the investigators developed a highly robust quality assurance (QA)
framework in order to ensure the safety and efficacy of the cell product. This process
began at the iPSC derivation stage, the patient iPSC had to display good (>70%)
viability alongside characteristic iPSC morphology while also being free of any viral
contaminants such as mycoplasma. Karyotypic analysis was performed by way of
G-banding of 20 cells and Giemsa staining of 50 cells of each iPSC clone (32 clones
for patient 1, 40 clones for patient 2).
This genomic analysis was then backed up by RNA-seq, DNA methylation anal-
ysis, and single-cell expression analysis (Mandai et al., 2017). The inclusion of high-
ly sensitive genomic analyses was found to be extremely prescient in this trial as it
was revealed that patient 2’s iPSCs, and iPSC-RPE, displayed copy-number aberra-
tions on chromosome 12 as well in the STS gene on the X-chromosome. X-
chromosomal mutations are of particular concern in male patients due to the lack
of homozygous redundancy found in females (Baek and Aypar, 2017). All other
quality assessments, including the tumorigenicity assay, had come back clear; how-
ever, the investigators made the decision to not perform the iPS-RPE replacement
procedure on this patient.
The remaining patient received iPSC-RPE as planned, following the removal of
the neovascular membrane, which had established itself over the macula. Graft
thickness stabilized to w200 mm at 24 weeks postsurgery while graft area remained
around 5e6mm
2
after 2 years with only minor expansion after 1 year; this is attrib-
uted to the “flattening” of the RPE alongside an unfolding of the membrane it was
cultured upon. Visual acuity showed some signs of improvement (visual functioning
questionnaire score increased from 48.8 to 58.3) following the procedure; however,
this could be attributed to the removal of the neovascular membrane from the retina
(Mandai et al., 2017).
After 4 years, the iPSC-RPE graft seems to have remained largely intact showing
good cell spacing and stable choroidal geometry (Takagi et al., 2019). This trial has
laid the foundation for future iPS-RPE trials and highlighted the need for robust
genomic analysis to ensure the safety of the implanted iPS product.
Following the early cessation of the trial (Garber, 2015), there was a global re-
view of the position iPSC will have in regenerative medicine with most groups
agreeing that iPSC tissue banking would provide the most robust provision of tissue
in a clinical setting (Andrews et al., 2015). The use of banked iPSC relies implicitly
on HLA-matching patients with donors; therefore, going forward, the use of autol-
ogous iPSC will likely be restricted to individuals for whom HLA matching is not
possible or where the use of immunosuppression is contraindicated. Moreover, using
tissue banks also allows for more in-depth QA analysis to define which cells are suit-
able for transplantation.
To this end, recent advances in machine learning techniques now provide new
tools for assessing RPE-iPSC tissue viability using currently available imaging tech-
nologies. Schaub and colleagues have demonstrated the use of calibrated quantita-
tive bright-field absorbance microscopy (QBAM) to capture multiparametric
image information coupled with deep convolutional neural networks (DNN) and
2. Cellular Therapies for AMD 163
traditional machine learning (TML) algorithms can predict iPSC-RPE functionality
and viability (Schaub et al., 2020). The researchers trained a DNN, based upon Goo-
gLeNet (Szegedy et al., 2015), and TML algorithms using QBAM images of healthy
iPSC-RPE cultured with differentiation promoters (Aphidicolin) and inhibitors
(HPI4) to provide test sets for healthy and pathology differentiation.
This was then correlated with transepithelial electrical resistance (TEER) and
VEGF expression as measured by ELISA. The researchers were able to show
good accuracy for both the DNN and TML (85.4% and 76.4% respectively) in the
detection of AMD pathology in cultured iPSC-RPE and strong correlation between
predicted and measured TEER (Schaub et al., 2020). Since this approach relies only
upon a standard upright or inverted bright-field microscope equipped with commer-
cially available bandpass filters, it is highly amenable to incorporation into current
cell therapy workflows. The researchers hope this approach can noninvasively
provide release criteria metrics for iPSC-RPE and highlight batch outliers to be
excluded from cell banks/treatment regimens. Machine learning techniques and sim-
ple AI systems have the potential to significantly improve the robustness of current
QA frameworks around advanced cell therapy but must be adequately trained on a
wide set of donors and metrics in order to avoid bias (Tucker et al., 2020).
2.3 Genomic Instability in iPSC-RPE, is Direct Reprogramming the
Answer?
The canonical route to produce iPSCs, developed by Takahashi and Yamanaka, is
through the transfection of fibroblast cells with the transcription factors Oct-4,
Sox-2, klf-4, and c-Myc (OSKM) (Takahashi and Yamanaka, 2006).
In their original paper, they demonstrated that the integration of these factors into
the genome of the fibroblast was essential for effective reprogramming. However,
somatic integration of exogenous transcription factors presented a problem in
defining the risk landscape of iPSC therapies as integration begets genomic insta-
bility, which can lead to dedifferentiation and teratoma formation (Ben-David and
Benvenisty, 2011).
Moreover, following the beginning of the RIKEN iPS-RPE-AMD trial (Mandai
et al., 2017) the reliability of karyotyping has been called into question. Baker and
colleagues have shown that characterizing hPSC mosaicism via routine karyotyping
techniques is only reliable if genetic variants are present in at least 10% of the cell
population (Baker et al., 2016).
These findings cast a shadow over the development of regenerative medicine
applications reliant upon iPSCs and present a significant challenge, which must
be addressed.
In the clinical trial detailed in the previous section, the investigators made every
effort to reduce the risk of pluripotency: keeping iPSC culture time short (3e
4 weeks from transformation to RPE colony picking); using the less carcinogenic
164 CHAPTER 7 Induced Pluripotent Stem Cells
L-Myc over the conventional, but more carcinogenic, c-Myc; and using nonintegrat-
ing episomal vectors instead of integrating vectors such as lentivirus. Nonetheless,
the generated iPSC product still harbored genomic instabilities that could have jeop-
ardized the patients’ treatment.
Perhaps the most straightforward means of addressing the issue of pluripotency
is to avoid the pluripotent stage of the process all together and instead utilize direct
lineage reprogramming to create RPE. To date, direct lineage reprogramming has
been used to make a wide variety of tissues such as myoblasts (Davis et al.,
1987), motor neurons (Son et al., 2011), and RPE (Zhang et al., 2014) [for full re-
view see (Xu et al., 2015)] from fibroblast cultures (Fig. 7.3).
Focusing on the RPE, Zhang and colleagues demonstrated the possibility of
creating BEST-positive RPE from human foreskin fibroblasts (HFF) without
needing to induce pluripotency (Zhang et al., 2014). Drawing on the knowledge
of the molecular pathways of RPE development, the researchers identified a pool
of candidate transcription factors (TF): Rax, Crx, Pax6, Mitf, Otx2, and Nrl. Initial
experiments utilizing these six TFs elicited no production of Best1
þ
RPE cultures.
The researcher believed this was due to a lack of epigenetic plasticity within the
HFFs. The solution lay in the finding that two of the classic iPSC reprograming
TFs, c-Myc and Klf4, are highly expressed in adult RPE (Salero et al., 2012).
The researchers tested the hypothesis that adding these factors to their TF pool
would increase transduction efficiency and found it to be true.
FIGURE 7.3
The classical route for producing RPE from iPSC as well as the direct lineage conversion
techniques currently being explored.
2. Cellular Therapies for AMD 165
With their updated protocol, Zhang and colleagues were able to take HFFs to an
RPE-like state within 35 days with Best1
þ
cells appearing after 12 days. The inves-
tigators next demonstrated which of the TFs were sufficient to induce RPE reprog-
raming by removing them one at a time.
This revealed that Klf4, Nrl, and Pax6 are unnecessary in the HFF-RPE reprog-
ramming protocol bringing the TF pool back down to five factors (Zhang et al.,
2014).Currently, it is difficult to directly compare directly reprogrammed HFF-
RPE with those produced from iPSC or hESC as there are no studies explicitly
comparing them on an expression or functional basis.
The most crucial metric that needs to be elucidated is how genomically stable
these cells are compared to iPSC-RPE as this will be a significant bottleneck to clin-
ical implementation.
3.Future Directions
The future of iPSC use in clinical applications will depend heavily on the ability of
researchers to control their genomic stability in vivo. One possible solution being
explored in nonretinal tissues is the use of synthetic transcription factors to directly
reprogram cells. Synthetic transcription factors (syn-TFs) are engineered modular
genomic elements usually composed of a DNA-binding domain alongside a catalytic
or scaffold element. Through the use of syn-TFs it is possible to multiplex relevant
genes downstream of tunable enhancers, thus allowing for precise control over ge-
netic, and epigenetic, expression (for review see (Keung et al., 2015). Moreover,
the advent of Crispr/Cas9 genome editing has a greatly improved the throughput of
syn-TF production and accelerated their use in cellular reprogramming. Recent studies
have shown that syn-TFs can be used to reprogram fibroblasts into skeletal muscle
cells (Liu et al., 2016) through synthetic upregulation of the endogenous MyoD gene.
More recently, Matjusaitis et al. (2019) demonstrated the use of syn-TFs to
perform lineage conversion of fibroblasts to oligodendrocyte progenitor-like cells,
thus cementing the feasibility of using syn-TFs to reprogram fibroblasts into
neuronal lineages while also demonstrating that these cells are able to engraft
in vivo. Critical in the selection of appropriate TFs for lineage conversion, and there-
fore for syn-TF production, is the identification of master controllers and master reg-
ulators, which inhabit central positions in the transcription networks required for
successful conversion. For lineage conversion to be fully exploited in the production
of RPE, these regulators must first be identified through the use of unbiased tran-
scriptomics (Vierbuchen and Wernig, 2011;Black et al., 2016).
Going forward, we await the completion of further iPSC-RPE clinical trials using
RPE produced from tissue banked iPSC, such as those in the United Kingdom (Da
Cruz et al., 2018) and Japan, which will help inform the risk landscape of iPSC-RPE
transplantation and identify which patients can be best served using iPSC-based
intervention.
166 CHAPTER 7 Induced Pluripotent Stem Cells
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Considerations in using
human pluripotent stem
cellederived pancreatic
beta cells to treat type 1
diabetes
8
Wei Xuan Tan
1,2,*
, Hwee Hui Lau
1,3,*
, Nguan Soon Tan
3,4
, Chin Meng Khoo
2,5
,
Adrian Kee Keong Teo
1,2
1
Stem Cells and Diabetes Laboratory, Institute of Molecular and Cell Biology, A*STAR, Singapore,
Singapore;
2
Yong Loo Lin School of Medicine, National University of Singapore, Singapore,
Singapore;
3
School of Biological Sciences, Nanyang Technological University, Singapore,
Singapore;
4
Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore,
Singapore;
5
Division of Endocrinology, National University Hospital, Singapore, Singapore
Chapter outline
1. Introduction ................................................................................................ .......174
2. hPSC-based clinical trials .................................................................................. 175
3. Considerations for hPSCs for clinical trials ..........................................................176
3.1 hESCs versus hiPSCs........................................................................... 181
3.2 Genotype of donors .............................................................................. 182
3.3 Allogeneic versus autologous transplantation ......................................... 183
4. hiPSC biobanking efforts .................................................................................... 183
5. Generation of hPSCs ...........................................................................................185
5.1 Regulations on the generation of hPSCs for clinical use .......................... 185
5.2 Tissue acquisition and somatic cell isolation ......................................... 186
5.3 Reprogramming into hiPSCs ................................................................. 187
5.4 hiPSC culture and expansion ................................................................ 189
6. Manufacturing hPSC-derived pancreatic beta cells for cell therapy .......................189
6.1 Challenges in generating cGMP-grade hPSC-derived pancreatic beta
cells for therapy .................................................................................. 190
6.2 Strategies to prevent tumor formation ................................................... 192
7. hPSC-based therapy for type 1 diabetes ..............................................................193
7.1 Manufacturing of PEC-01 cells ............................................................. 194
CHAPTER
*
These authors contributed equally.
173
Recent Advances in iPSCs for Therapy, Volume 3. https://doi.org/10.1016/B978-0-12-822229-4.00012-7
Copyright ©2021 Elsevier Inc. All rights reserved.
7.2 VC-01 clinical trial .............................................................................. 194
7.3 VC-02 clinical trials ............................................................................. 195
7.4 Preclinical study by Semma therapeutics .............................................. 196
8. Future outlook and concluding thoughts .............................................................. 196
Acknowledgments ...................................................................................................197
References .............................................................................................................197
Abstract
Type 1 diabetes (T1D) is characterized by the autoimmune destruction of insulin-
producing beta cells in the pancreas. Consequently, T1D patients produce little to
no insulin, resulting in absolute insulin deficiency and an inability to regulate blood
glucose levels effectively. Insulin replacement is the mainstream treatment for
T1D, but it comes with a major drawbackdthe risk of hypoglycemia unawareness.
As such, replenishment of glucose-sensing and insulin-producing beta cells via cell
replacement therapy is believed to have great therapeutic value for T1D patients.
Human pluripotent stem cells (hPSCs) are considered an attractive source for the
derivation of insulin-producing beta-like cells for cell replacement therapy. How-
ever, several challenges need to be addressed before hPSC-derived beta-like cells
can be used for T1D treatment. In this chapter, we provide a snapshot of the current
progress of hPSC therapy with an emphasis on T1D, discuss some of the clinical
considerations and technical strategies in the generation of current good
manufacturing practice (cGMP)-compliant hPSC-derived beta-like cells and the
future outlook for T1D hPSC therapy.
Keywords:
Allogeneic; Beta cells; Biobank; Cell therapy; cGMP; cGTP; Clinical trials; Insulin;
Islets; Pluripotent; Regenerative medicine; Reprogramming; Stem cells; Transplantation; Type 1
diabetes.
1.Introduction
Type 1 diabetes (T1D) is classically an autoimmune disease whereby the body’s im-
mune system destroys its insulin-producing beta cells in the pancreatic islets of
Langerhans. Consequently, T1D patients produce little to no insulin, resulting in ab-
solute insulin deficiency and an inability to regulate blood glucose levels effectively.
Thus, hyperglycemia occurs. The underlying cause of T1D includes a combination
of genetic and environmental factors (Van Belle et al., 2011;Todd, 2010;Bluestone
et al., 2010). T1D accounts for about 10% of all diabetes cases globally, and approx-
imately 1.1 million children and adolescents had T1D in 2019 (International Dia-
betes Federation (IDF), 2019). Although T1D typically develops in children or
adolescents, it can develop in individuals of any age (International Diabetes Feder-
ation (IDF), 2019). T1D patients exhibit chronic hyperglycemia, which can lead to
several complications, including but are not limited to stroke, cardiomyopathy, cor-
onary heart disease, nephropathy, and blindness (Lotfy et al., 2017). In addition,
T1D patients are at very high risk of developing severe hyperglycemia crisis known
as diabetic ketoacidosis.
174 CHAPTER 8 Considerations in using human pluripotent stem cell
The therapy for T1D patients is total insulin replacement, which has to be
delivered via an insulin pump or multiple times a day typically via injectable
means, although noninjectable insulin delivery methods exist with lower efficiencies
(Heinemann et al., 2017). The major drawback of insulin therapy is the risk of
hypoglycemia, especially in patients with hypoglycemia unawareness, as insulin
replacement does not mimic the finely tuned insulin secretion by beta cells in
response to glucose stimulation (Control and Group, 1993;McCall and Farhy,
2013). As such, replenishment of glucose-sensing and insulin-producing beta cells
via cell replacement therapy is believed to have the greatest therapeutic value for
T1D patients.
Allogeneic islets from human cadaveric donors represent one of the most prom-
ising sources to obtain functional beta cells for transplantation. Indeed, the whole
pancreas (Kelly et al., 1967;Gruessner and Sutherland, 2005) and islet transplanta-
tion (Shapiro et al., 2000,2006;Hering et al., 2005;Berney et al., 2009) in T1D
patients has been shown to be effective in normalizing hyperglycemia and relieving
the patients’ dependence on exogenous insulin. However, allogeneic transplantation
of the pancreas or islets requires lifelong immunosuppression in the recipients
(Robertson et al., 2006) and is greatly limited by the number of cadaveric donors
(Shapiro et al., 2017). Moreover, global access to transplantable human islets is
restricted as the islet isolation procedure requires a specialized facility, and long-
distance transportation of islets for transplantation has its challenges (Ng et al.,
2019). With the rapid advancement of human pluripotent stem cell (hPSC) research
and a greater understanding of its clinical potential in the past decade, the use of
hPSCs has become a promising approach to generate functional beta cells for cell
therapy in T1D patients. Here, we provide a snapshot of the current progress of
hPSC therapy with an emphasis on T1D, discuss some of the clinical considerations
and technical strategies in the manufacturing of hPSC-derived beta-like cells and the
future outlook for T1D hPSC therapy.
2.hPSC-based clinical trials
It has been more than two decades since the discovery that human embryonic stem
cells (hESCs) could be isolated from preimplantation human embryos and expanded
indefinitely in vitro (Thomson et al., 1998). hESCs are defined as a population of
pluripotent cells derived from the inner cell mass of a preimplantation human blas-
tocyst. The intrinsic ability of hESCs to self-renew and differentiate into the three
germ layers and most of the somatic cells in the body makes them attractive as a
candidate for cell-based therapy. Since then, several protocols have been developed
to differentiate hPSCs into various functional cell types to potentially replace
diseased cell types in chronic and degenerative diseases. In the case of T1D therapy,
several groups have managed to generate differentiation protocols that can produce a
decent amount and purity of insulin
þ
beta-like cells (Rezania et al., 2014;Russ et al.,
2. hPSC-based clinical trials 175
2015;Schulz et al., 2012;Pagliuca et al., 2014). However, several hurdles, including
ethical considerations and posttransplantation immune rejections, pose challenges
for hESCs in reaching the clinics. The discovery of induced pluripotent stem cells
(iPSCs) by Takahashi and Yamanaka (2006) opened up the possibility of avoiding
these issues. This revolutionary discovery demonstrated that fibroblasts could be
reprogrammed into iPSCs by introducing the four Yamanaka factorsdOct4,
Sox2, c-Myc, and Klf4dthrough retroviral means. Using the same method, Yama-
naka’s group subsequently reported the generation of the first human iPSCs (hiPSCs)
from human fibroblasts in 2007 (Takahashi et al., 2007). Since then, a colossal
amount of effort has been invested by many research groups to optimize the gener-
ation of hiPSCs for clinical applications.
The discovery of hiPSC has led to an exponential increase in the number of pa-
pers reporting the various reprogramming methods (Maherali et al., 2008;Okita
et al., 2008;Sommer et al., 2009;Stadtfeld et al., 2008;Warren et al., 2010;Kim
et al., 2009), including the use of different cocktails of transcription factors (Yu
et al., 2007;Nakagawa et al., 2008;Zhao et al., 2008), small molecules (Huangfu
et al., 2008;Mikkelsen et al., 2008;Shi et al., 2008), and different starting cell types
(Loh et al., 2009;Aasen et al., 2008;Hanna et al., 2008;Ye et al., 2009;Loh et al.,
2010). With some successes in the optimization of reprogramming, a number of clin-
ical trials have begun using cell products derived from hiPSCs. We summarize the
ongoing clinical trials involving the transplantation of either hESC- or hiPSC-
derived cells to treat various diseases in Table 8.1, to allow for an appreciation of
the high potential of hPSCs in regenerative medicine. Despite the high number of
ongoing trials, only one company, ViaCyte, Inc., is targeting to treat T1D (Table 8.1).
In contrast, other major economy-burdening diseases such as heart and neurological
diseases have at least two institutes/organizations conducting clinical trials using
hPSC-derived cells (Table 8.1). Discussing the deeper considerations of using
hPSCs in diabetes, clinical trials may help to understand the technical barriers of
this approach.
3.Considerations for hPSCs for clinical trials
Among the hPSC-based clinical trials listed in Table 8.1, 24 of them use hESCs while
eight of them use hiPSCs. Among the eight clinical trials that use hiPSCs, six of them
use allogeneic hiPSCs while two of them use autologous hiPSCs (Table 8.1). Before
the approval of a cell-based clinical trial, thorough validation studies are required to
be done on the cell type(s) to be used. The choice of cell type(s) is extremely impor-
tant before embarking on the arduous journey from preclinical studies to the actual
clinical trial itself. In this section, we provide an in-depth discussion of three consid-
erations for the choice of hPSCs for clinical trials: the use of hESCs versus hiPSCs,
the genotype of the donors, and allogeneic versus autologous transplantation.
176 CHAPTER 8 Considerations in using human pluripotent stem cell
Table 8.1 Current cell therapy clinical trials involving the use of hPSCs (list is correct as of February 2020).
Disease
Year of
commencement
Type of
hPSCs
Intended cell type to be
transplanted Company/Institute
Status of
clinical
trial
Clinical trial
identifier
Blood disorders
Beta-
thalassemia
2015 Autologous
hiPSCs
hiPSC-derived
hematopoietic stem cells
The Third Affiliated
Hospital of
Guangzhou Medical
University
eNCT03222453
Cancer
Advanced solid
tumors
2019 Allogeneic
hiPSCs
hiPSC-derived natural
killer (NK) cells
Fate Therapeutics Phase 1 NCT03841110
Heart diseases
Advanced
ischemic heart
disease
2013 hESCs I6 hESC-derived cardiac
progenitors
Assistance
PubliquedHo
ˆpitaux
de Paris
Phase 1
completed
in 2018
NCT02057900
Cardiomyopathy 2018 Allogeneic
hiPSCs
hiPSC-derived
cardiomyocyte sheet
Osaka University
Graduate School of
Medicine
Phase 1/2 UMIN000032989
Heart failure 2019 Allogeneic
hiPSCs
hiPSC-derived
cardiomyocyte
Help Therapeutics eNCT03763136
Immunological diseases
Steroid-resistant
acute graft
versus host
disease
2017 Allogeneic
hiPSCs
Mesenchymoangioblast
(from hiPSCs)-derived
mesenchymal stem cells
Cynata Therapeutics Phase 1 NCT02923375
Continued
3. Considerations for hPSCs for clinical trials 177
Table 8.1 Current cell therapy clinical trials involving the use of hPSCs (list is correct as of February 2020).dcont’d
Disease
Year of
commencement
Type of
hPSCs
Intended cell type to be
transplanted Company/Institute
Status of
clinical
trial
Clinical trial
identifier
Metabolic diseases
Type 1 diabetes 2014 hESCs
CyT49
hESC-derived pancreatic
endoderm cells loaded in
an encapsulated device
VC-01
ViaCyte, Inc. Phase 1/2 NCT02239354
2017 hESCs
CyT49
hESC-derived pancreatic
endoderm cells loaded in
a delivery device VC-02
ViaCyte, Inc. Phase 1/2 NCT03163511
2017 hESCs
CyT49
hESC-derived pancreatic
endoderm cells loaded in
a delivery device VC-02
ViaCyte, Inc. Phase 1
completed
in 2018
NCT03162926
2019 hESCs
CyT49
hESC-derived pancreatic
endoderm cells loaded in
a delivery device VC-02
Center for Beta Cell
Therapy in Diabetes
and ViaCyte, Inc.
Phase 1 Center for Beta
Cell Therapy in
Diabetes (2018)
Neurological diseases
Spinal cord
injury
2010 hESCs H1 Oligodendrocyte
progenitor cell (OPC)
GRN-OPC1
Geron Corporation Canceled Lebkowski (2011),
Thies and Murry
(2015)
2015 hESCs H1 Oligodendrocyte
progenitor cell (OPC) AST-
OPC1
Asterias
Biotherapeutics
Phase 1/2a
completed
in 2018
NCT02302157
Parkinson’s
disease
2017 hESCs Q-
CTS-hESC-
1
hESC-derived neural
precursor cells
Chinese Academy of
Sciences
Phase 1/2 NCT03119636
2018 Allogeneic
hiPSCs
hiPSC-derived
dopaminergic progenitors
Kyoto University
Hospital
Phase 1/2 UMIN000033564
178 CHAPTER 8 Considerations in using human pluripotent stem cell
Retinal diseases
Dry age-related
macular
degeneration
(AMD)
2011 hESCs
MA09
hESC-derived retinal
pigment epithelium (RPE)
MA09-hRPE
Astellas Institute of
Regenerative
Medicine (AIRM)
Phase 1/2
completed
in 2015
NCT01344993
2012 hESCs
MA09
hESC-derived retinal
pigment epithelium (RPE)
MA09-hRPE
Cha Biotech Phase1/2a NCT01674829
2015 hESCs HAD-
C 102
hESC-derived RPE
OpRegen
CellCure
Neurosciences
Phase1/2a NCT02286089
2015 hESCs hESC-derived RPE Chinese Academy of
Sciences, Institute of
Zoology
eChiCTR-OCB-
15007054
2015 hESCs hESC-derived RPE Regenerative Patch
Technologies
Phase 1/2 NCT02590692
2017 hESCs hESC-derived RPE Chinese Academy of
Sciences
Phase 1/2 NCT03046407
2018 hESCs hESC-derived RPE Chinese Academy of
Sciences
Phase 1/2 NCT02755428
Myopic macular
degeneration
2013 hESCs
MA09
hESC-derived retinal
pigment epithelium (RPE)
MA09-hRPE
University of
California, Los
Angeles
Phase 1/2.
Canceled.
NCT02122159
Neovascular
AMD
2017 Allogeneic
hiPSCs
hiPSC-derived RPE Kobe City Medical
Center General
Hospital
eUMIN000026003
Retinitis
pigmentosa
disease
2015 hESCs hESC-derived RPE Chinese Academy of
Sciences, Institute of
Zoology
eChiCTR-OCB-
15007055
Severe ocular
surface disease
ehESCs hESC-derived RPE Eye Institute of
Xiamen University
eChiCTR-OCB-
15005968
Continued
3. Considerations for hPSCs for clinical trials 179
Table 8.1 Current cell therapy clinical trials involving the use of hPSCs (list is correct as of February 2020).dcont’d
Disease
Year of
commencement
Type of
hPSCs
Intended cell type to be
transplanted Company/Institute
Status of
clinical
trial
Clinical trial
identifier
Stargardt’s
macular
dystrophy (SMD)
2011 hESCs
MA09
hESC-derived retinal
pigment epithelium (RPE)
MA09-hRPE
AIRM Phase1/2
completed
in 2015
NCT01345006
2011 hESCs
MA09
hESC-derived retinal
pigment epithelium (RPE)
MA09-hRPE
AIRM Phase 1/2
completed
in 2015
NCT01469832
2012 hESCs
MA09
hESC-derived retinal
pigment epithelium (RPE)
MA09-hRPE
Cha Biotech Phase 1
completed
in 2015
NCT01625559
Wet AMD 2013 Autologous
hiPSCs
hiPSC-derived RPE RIKEN Bio-Resource
Center (BRC)
Phase 1 UMIN000011929
2015 hESCs
SHEF-1
hESC-derived RPE Pfizer Phase 1 NCT01691261
2017 Allogeneic
hiPSCs
hiPSC-derived RPE RIKEN BRC Cyranoski (2017)
SMD and AMD 2015 hESCs hESC-derived RPE Federal University of
Sa
˜o Paulo
Phase 1/2 NCT02903576
2015 hESCs hESC-derived RPE Southwest Hospital,
China
Phase 1/2 NCT02749734
This list only includes clinical studies that involve the transplantation of hPSC-derived cell products into patients. Follow-up studies are not included.
180 CHAPTER 8 Considerations in using human pluripotent stem cell
3.1 hESCs versus hiPSCs
When iPSC was first discovered, it was reported to be similar to ESC in terms of cell
morphology, expression of pluripotency markers, transcriptome profile, telomerase
activity, and differentiation ability (Takahashi and Yamanaka, 2006;Takahashi et al.,
2007). However, there has been a huge debate on whether iPSCs are truly identical to
ESCs. Subsequent reports have suggested that iPSCs are different from ESCs in
terms of gene expression profile (Chin et al., 2009;Ghosh et al., 2010;Marchetto
et al., 2009), epigenetic profile (Deng et al., 2009;Doi et al., 2009), neuronal differ-
entiation ability (Hu et al., 2010), and the ability to complement tetraploid embryos
(Stadtfeld et al., 2010) (which is the most stringent test of pluripotency that mouse
ESCs [mESCs] can achieve). Notably, it has been shown that some of the differences
between ESCs and iPSCs can be attributed to the retention of gene expression
(Ghosh et al., 2010;Marchetto et al., 2009) or epigenetic memory (Kim et al.,
2011;Ohi et al., 2011) from the somatic cells that the iPSCs were derived from.
On the other hand, other studies have shown that iPSCs are indistinguishable
from ESCs in terms of gene expression profile (Guenther et al., 2010;Newman
and Cooper, 2010;Bock et al., 2011), DNA methylation status (Bock et al.,
2011), and neuronal differentiation competency (Boulting et al., 2011). Interest-
ingly, Carey et al. (2011) showed that most of their iPSC clones achieved tetraploid
complementation (Carey et al., 2011) as opposed to the observations from Stadtfeld
and et al. (2010), despite both groups having used similar reprogramming methods.
One of the key differences between the methods used by both groups is the order of
the reprogramming factors in the expression vector, which might alter the expression
level of each factor (Yamanaka, 2012).
The differences between iPSCs and ESCs were reviewed in a paper by Yamanaka
in 2012 (Yamanaka, 2012). It was highlighted that the studies that favored similar-
ities between iPSCs and ESCs were performed with a relatively large number of
iPSC and ESC clones (>10). In contrast, studies that showed distinct differences be-
tween the two were conducted with a relatively lower number of clones (<10)
(Yamanaka, 2012). As variations between different ESC clones have been well-
documented, iPSC clones could have a larger variation due to “incomplete” reprog-
ramming. The reprogramming efficiency can be affected by many technical vari-
ables such as reprogramming factor combination, order of the factors in the
expression vector, delivery method, and culture conditions during the reprogram-
ming process (Yamanaka, 2012). Consequently, the iPSC clones that were not
reprogrammed “perfectly” could be significantly different from ESCs while there
would also be some iPSC clones that are indistinguishable from ESCs. Yamanaka
suggested that it is therefore crucial for an evaluation criterion to be established
to select for iPSC clones, especially for clinical purposes.
Despite being the current gold standard for hPSCs and not having quality varia-
tions that stem from reprogramming, hESCs have two major limitations that restrict
its potential for cell therapy. The first limitation is the ethical concerns with the
3. Considerations for hPSCs for clinical trials 181
destruction of a human embryo to derive a hESC line. This concept stands against
the religious and personal beliefs of many and thus limits the appeal of hESC-
based cell therapy. Moreover, ethical concerns of the public can influence the deci-
sion of policymakers. The current policy in the United States is that new hESC lines
can only be created using leftover and unwanted frozen embryos from in vitro fertil-
ization (IVF) procedures. Therefore, the number of available clinical-grade hESC
lines is certainly insufficient to cover the majority of the human leukocyte antigen
(HLA) haplotypes in the population. It has been established that the matching of
HLA loci, specifically the HLA-A, HLA-B, and HLA-DR genes, significantly re-
duces the risk of graft rejection (Opelz and Do
¨hler, 2007;Johnson et al., 2010)
and the need for immunosuppression (Aydingoz et al., 2007). The limited number
of hESC lines contributes to a second major limitation of hESCs as compared to
the use of hiPSCsdimmune rejections. Like most organ or tissue transplantations,
hESC-derived cell products face the risk of graft failure due to immune rejection by
the host. Therefore, immunosuppression therapy is required, bringing about poten-
tial adverse effects such as bacterial infection.
The one distinct advantage of using hiPSCs for cell therapy is the possibility of
finding an HLA-matching donor or even using patient-derived autologous cells to
reduce the risk of immune rejection or the need for immunosuppression. The use
of encapsulation devices, as seen in ViaCyte’s clinical trial, will then act as an addi-
tional safety feature to prevent immune rejection of grafts. Another advantage
hiPSCs may have over hESCs is the ability to determine the “phenotypic health”
of the donor cells before the generation of hiPSCs. For hESCs, besides sequencing
the genome, there is currently no effective means to predict whether that particular
genotype is at risk of developing any age-related chronic disease in the future.
Despite these advantages of hiPSCs, they could be inferior to hESCs in that they
may have accumulated somatic mutations over time before reprogramming. The var-
iable efficiency of “complete” reprogramming, as opposed to the default pluripotent
state of hESCs, also implies that some hiPSC lines may not be “as pluripotent as”
hESCs. For companies such as ViaCyte, the use of one extremely well-
characterized hESC line with an optimized pipeline is still the preferred strategy
for cell therapyebased clinical trials, as opposed to dealing with the clonal vari-
ability of hiPSCs.
3.2 Genotype of donors
Many quantitative analyses have identified diabetes-associated gene variants that
could contribute to an increased risk of disease manifestation (Fuchsberger et al.,
2016;Flannick et al., 2019;Mahajan et al., 2018;European Consortium for
IDDM Genome Studies, 2001;Cox et al., 2001). For example, the transcription fac-
tor 7-like 2 (TCF7L2) gene is one of the most established and robust candidates for
T2D association. Several single nucleotide polymorphisms (SNPs) in the TCF7L2
gene have been independently linked with T2D risk and T2D-related traits
182 CHAPTER 8 Considerations in using human pluripotent stem cell
(Fuchsberger et al., 2016;Flannick et al., 2019;Gjesing et al., 2011;Florez et al.,
2006;Grant et al., 2006;Palmer et al., 2011). As most of the known causal T2D-
associated variants affect beta cell function (McCarthy, 2010), the donors must be
devoid of these causal variants as much as possible. Certain gene variants have
also been reported to display ethnic-specific susceptibility to diabetes risk (Fuchs-
berger et al., 2016;Flannick et al., 2019). This can be one of the factors for consid-
eration in recipient matching. With these considerations in mind, the whole genome
profile of the donor should be screened against a carefully determined gene variant
panel covering a range of chronic diseases including diabetes mellitus.
3.3 Allogeneic versus autologous transplantation
By using HLA-matched donors in allogeneic transplantation, the risk of immune
rejection is significantly lowered (Opelz and Do
¨hler, 2007;Johnson et al., 2010).
Once a hiPSC bank covering diverse HLA types has been established (Taylor
et al., 2012), it may then be possible to use a limited and defined number of hiPSC
lines to treat a large number of individuals. These hiPSCs can be subjected to thor-
ough quality checks to ensure “complete” reprogramming and then be used in a
long-term manner. However, for patient-specific autologous transplantation, there
is a need to subject newly made hiPSCs to rigorous quality checks, which can be
prohibitively expensive. Hence, for practical reasons, an allogeneic donor system
is likely to be more financially sustainable. Importantly, the ready-made bank of
HLA-typed hiPSCs circumvents excessive waiting time for cell differentiation and
production. However, a caveat is that even HLA-matched tissue transplant faces a
risk of immune rejection and may still require immunosuppression (Aydingoz
et al., 2007;Patterson et al., 1986). It may be possible to circumvent this problem
by using genome editing technology to knock out HLA genes in hiPSCs (Jang
et al., 2019). However, this approach is expected to be an extremely cumbersome
process that will be subjected to tight scrutiny in terms of genome stability and
safety of the cells.
A counterargument supporting autologous hiPSC-based cell transplantation
arises from the assumption that there will not be any immune reaction. Surprisingly,
a study suggests that this is not always the case (Zhao et al., 2015). Nonetheless,
autologous hiPSCs are not likely to be suitable for T1D therapy as the transplanted
beta-like cells might succumb to the dysregulated autoimmunity present in T1D
patients.
4.hiPSC biobanking efforts
Given that personalized autologous hiPSC-based transplantation is currently
projected to be too costly, a current viableoptionistocreateahiPSCbiobank
that has a series of hiPSCs that encompass most of the HLA types. According to
the World Health Organization (WHO) HLA nomenclature, there are 21, 44, and
4. hiPSC biobanking efforts 183
15 different alleles of HLA-A,HLA-B,andHLA-DR genes, respectively (Marsh
et al., 2010). This would mean that there are possibly 13,860 different HLA hap-
lotypes, requiring 13,860 different hiPSC lines, which is near impossible for any
biobank to have. However, the combination of HLA genes for a haplotype is not
completely random due to gene linkage, and as such, some HLA haplotypes are
more common than others. Hence, careful selection of homozygous HLA haplo-
types will allow better coverage of the haplotypes using fewer numbers of clones.
It has been estimated that 50 HLA homozygous lines screened from a pool of
24,000 individuals can provide HLA-matched cells for 90.7% of the Japanese pop-
ulation (Nakatsuji et al., 2008). That said, the number of required clones will be
higher if the estimation was done in highly multiethnic countries (Gourraud
et al., 2012), thereby increasing the complexity and cost.
Multiple efforts to create hiPSC biobanks for research and clinical purposes
are currently underway in Europe, Asia, and North America (Kim et al., 2017;
The Scientist, 2014;Morrison, 2017;Huang et al., 2019). In North America,
several government-owned and private companies are actively expanding their
hiPSC banks. These include the California Institute for Regenerative Medicine
(CIRM), Coriell Institute, WiCell Research Institute, New York Stem Cell Founda-
tion Research Institute, Rutgers University Cell and DNA Repository (RUCDR)
and Fujifilm Cellular Dynamics International (The Scientist, 2014;Huang et al.,
2019). Notably, CIRM aims to create the largest hiPSC bank with 9000 lines
derived from 3000 individuals (The Scientist, 2014) and currently possesses the
most number of hiPSC lines of at least 1500 (Huang et al., 2019). In Europe, the
two main efforts in hiPSC banking are led by the European Stem cells for Bio-
logical Assays of Novel drugs and prediCtive toxiCology (StemBANCC) and
the European Bank for induced pluripotent Stem Cells (EBiSC) (Morrison,
2017;Huang et al., 2019). Apart from the Europe-wide networks, individual
countries also have their hiPSC banking efforts, such as I-Stem in France, the Hu-
man Induced Pluripotent Stem Cell Initiative (HipSci) in the United Kingdom,
hPSCreg in Germany, and the Spanish National Stem Cell Bank in Spain (Kim
et al., 2017;The Scientist, 2014). Based on a recent review, EBiSC and HipSci
are reported to have at least 800 hiPSC lines in their depository (Huang et al.,
2019). In Asia, many countries, such as India, Japan, Korea, Singapore, Taiwan,
and Thailand, have initiated efforts in hiPSC banking as well (Kim et al., 2017;
Huang et al., 2019). Being the leader in hiPSC clinical trials, Japan also established
the first hiPSC bank in the world, initiated by the Center for iPSC Research
and Application (CiRA) that is mainly for regenerative medicine (Saito et al.,
2014). The RIKEN BRC has also established a hiPSC bank that holds 480
hiPSC lines that can be used for cell therapy, drug discovery, or research. These
hiPSCs can be distributed both locally and internationally (RIKEN Bio-
Resource Center, 2018).
184 CHAPTER 8 Considerations in using human pluripotent stem cell
5.Generation of hPSCs
5.1 Regulations on the generation of hPSCs for clinical use
The United States Food and Drug Administration (FDA) plays an important role in
regulating stem cellebased products for clinical applications. In general, all human
tissue/cell products intended for clinical use possibly need to comply with the CFR
Title 21 (21CFR1271, under FDA regulation). Safety remains the major concern in
stem cellebased therapy; hence all cellular products intended for clinical applica-
tions are to be generated in accordance to the current good manufacturing practice
(cGMP). The cGMP provides guidelines to assure the proper design, monitoring,
and control of manufacturing processes and facilities. This is aimed at ensuring
the safety, purity, and hopefully efficacy of the product. The design and operation
of a cGMP-compliant cell-engineering laboratory are essential for the production
of cellular products intended for clinical application.
To supplement cGMP, Subparts C and D of 21CFR1271 define the current good
tissue practices (cGTPs) that describe the procedures to prevent the introduction,
transmission, and spread of communicable diseases by the human cellebased pro-
ducts (https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?
CFRPart¼1271&showFR¼1). The cGMP and cGTP guidelines comprise documen-
tation including (but not limited to) personnel training, standard procedures, envi-
ronment control, storage, and distribution of hPSCs (21CFR1271) (Fig. 8.1). On
top of the procedures involved in the production pipeline, cGTP guidelines extend
to the regulation of tissue acquisition, which also includes addressing ethical consid-
erations (Fig. 8.1).
Under cGTP guidelines, it is required to define tissue requirements before donor
screening to determine eligibility (21CFR1271.50) based on test results for the rele-
vant communicable disease agents or diseases (RCDADs). Informed consent is also
required to ensure donors are adequately informed about the specific aspects of the
program they participate in. Under the guidelines for Stem Cell Research and Clin-
ical Translation (by the International Society for Stem Cell Research, ISSCR), the
Defining tissue requirements
Donor eligibility determination
Donor recruitment
Establish SOP for tissue/cell acquisition
cGMP
(Ensure proper design, monitoring and
control of manufacturing processes)
cGTP
(Prevention of the introduction,
transmission, or spread of communicable
diseases)
cGMP production personnel training
cGMP facility preparation
Equipment documentation
Production and process control records
Defining distribution procedures
Testing and release for distribution
Records and reports for all procedures above
Review and approval - IRB
Informed consent from donor
Human tissue or somatic cell acquisition
Ethical considerations
CFR Title 21
FIGURE 8.1
Simplified flowchart of processes required for cGMP-grade hPSCs.
5. Generation of hPSCs 185
derivation of hiPSCs from acquired tissues or somatic cells is to be subjected to the
local human subjects review committee for approval. An additional specialized hu-
man embryo research oversight (EMRO) review process will be required if the
research involves the generation of human embryos or entails the sensitive aspects
of the use of totipotent hPSCs (https://www.isscr.org/docs/default-source/all-isscr-
guidelines/guidelines-2016/isscr-guidelines-for-stem-cell-research-and-clinical-trans
lationd67119731dff6ddbb37cff0000940c19.pdf). Therefore, besides being compliant
with FDA regulation, the use of human biomaterial will need to be approved by the
Institutional Review Board (IRB) before donor recruitment can take place.
As part of the cGTP guidelines, all cell products meant for therapy must be
cultured in fully defined conditions to achieve high reproducibility and in
xenogeneic-free conditions to prevent possible xenogeneic infections and immune
rejections (European Medicines Agency, 2011). Many of the hPSC lines that were
developed in the early days would have been exposed to xenogeneic agents and/
or feeder cells during the differentiation process as they were originally developed
for research purposes. However, some of these lines can be converted to cGMP-
compliant products after going through strict validation tests and subsequently
adhering to cGMP guidelines (Devito et al., 2014). It is indeed very challenging
to develop fully cGMP-cGTP-compliant hPSC lines in which the entire
manufacturing procedure, from tissue acquisition, reprogramming, hiPSC clonal
expansion to the creation of a master and working cell banks (Fig. 8.2), adheres fully
to the guidelines. Ideally, cGTP and cGMP conditions should be adhered to right
from the first handling of the cells or instilled as early as possible in the pipeline.
In this section, we discuss some of the important clinical and technical consider-
ations for generating hiPSCs.
5.2 Tissue acquisition and somatic cell isolation
A variety of human somatic cells can be used to generate hiPSCs. Examples include
fibroblasts (Takahashi et al., 2007), keratinocytes (Aasen et al., 2008), hematopoietic
stem cells from the bone marrow (Ye et al., 2009), cord blood (Ye et al., 2009), and
peripheral blood (Loh et al., 2009) and peripheral blood mononuclear cells (PBMCs)
Human somatic cells
Fibroblasts
PBMCs
Cell isolation &
expansion
hiPSC isolation &
expansion
Cell expansion & banking
Reprogramming
Non-viral-based
Viral-based
Non-integrative
Integrative
Translational
application
Feeder or feeder-free
FIGURE 8.2
The workflow for generating hiPSCs.
186 CHAPTER 8 Considerations in using human pluripotent stem cell
such as lymphocytes (Hanna et al., 2008;Loh et al., 2010). Among these, skin fibro-
blasts and PBMCs have emerged to be the two most common tissue sources for
hiPSC generation given their ease of access and amenability to be reprogrammed
into hiPSCs. To obtain skin fibroblasts, a skin punch biopsy is taken from a donor.
The skin biopsy will be cultured for a few weeks to obtain sufficient fibroblasts. The
site at which the skin biopsy is performed is critical as exposure to the sun could
result in the accumulation of UV-induced mutations that would compromise the
quality and safety of hiPSC-derived cells generated for translational applications.
Alternatively, the acquisition of PBMCs through routine venipuncture is less inva-
sive than getting a skin punch biopsy. Hence, this approach has gained traction
over the years despite the higher cost in the initial purification and maintenance
of blood cells. The other advantage of reprogramming PBMCs over skin fibroblasts
is the ease of cell isolation and a faster rate of expansion. The ease of obtaining a
sufficient amount of PBMCs for reprogramming circumvents the poorer reprogram-
ming efficiency of the cells (Loh et al., 2010). However, the major caveat in using T
lymphocytes for reprogramming is the V(D)J rearrangement in the T cell receptor
(TCR) genes, which can lead to the formation of T cell lymphomas (Serwold
et al., 2007).
There have been long-standing discussions on whether the type of tissue/cells
initially used to generate hiPSCs could eventually impact their differentiation poten-
tial. Several studies have shown that iPSCs retain the DNA methylation profiles of
their somatic tissue origins. The residual distinct transcriptional and epigenetic pat-
terns in hiPSCs could favor differentiation along lineages relevant to the parental or-
igins while being restrictive to alternative cell lineages (Doi et al., 2009;Polo et al.,
2010;Kim et al., 2010). Interestingly, iPSCs reprogrammed from human pancreatic
islet cells uniquely retained open chromatin structures at key beta cell genes and
differentiate more readily into insulin
þ
cells than nonebeta cellederived iPSCs
(Bar-Nur et al., 2011;Thurner et al., 2017). The high efficiency of beta cell differ-
entiation achieved using this approach is highly desirable for regenerative medicine.
However, this strategy has not been widely adopted due to the limited amount of beta
cells available from human cadaveric donors, which poses a substantial technical
challenge given the low reprogramming efficiency (Bar-Nur et al., 2011).
5.3 Reprogramming into hiPSCs
hiPSCs can be generated via viral or nonviral reprogramming methods. Each method
differs in their reprogramming efficiencies for selected cell types (Fig. 8.2).
Integration-free reprogramming methods should be adopted to generate hiPSCs suit-
able for clinical applications.
Some non-viral-based reprogramming methods include the use of minicircle
DNA vectors, episomal vectors, RNAs, and proteins. The minicircle DNA vector
reprogramming method involves the delivery of supercoiled DNA free of bacterial
plasmid backbone elements (such as ORI and antibiotic resistance gene cassette)
via lipid-mediated transfection or nucleofection. When expressed in human adipose
5. Generation of hPSCs 187
stromal cells, a plasmid/minicircle vector containing a single cassette of four reprog-
ramming factors LIN28,NANOG,SOX2,OCT4, and reporter GFP gene can deliver a
reprogramming efficiency of 0.005% (Jia et al., 2010;Narsinh et al., 2011). The
introduced minicircle plasmid backbone is then excised using PhiC31-based intra-
molecular recombination system to obtain a transgene-free hiPSC (Jia et al.,
2010;Narsinh et al., 2011). The elimination of the use of viruses in this method
makes it attractive for clinical application. However, this method is not commonly
used mainly due to its poor reprogramming efficiency.
Episomal vectors are promising for transient expression of reprogramming fac-
tors to achieve integration-free iPSCs. A commonly used episomal vector for reprog-
ramming is the Epstein Barr virus-derived vector due to its ability to result in stable
and extended expression of reprogramming factors in somatic cells. The episomal
vectors can be introduced into somatic cells via electroporation or nucleofection.
However, it is recognized that the episomal vectors will be diluted out of cells
with each division; and the hiPSCs generated will lose the episomes within
10e15 passages (Okita et al., 2011;Schlaeger et al., 2015). Although the episomes
replicate extrachromosomally, occasional integration into the genome can still occur
(Schott et al., 2014). Therefore, individual hiPSC clones will need to be thoroughly
screened for exogenous gene integration to confirm normal karyotype.
To eliminate the risk of plasmid integration, direct delivery of synthetic RNA
encoding the four Yamanaka factors and LIN28 into human somatic cells was first
introduced in 2010 by Warren and colleagues. This system involves modifications
of in vitro transcribed RNAs to evade the host antiviral defense response to ssRNA
by the somatic cells. The reprogramming efficiency of this approach is the highest
among all other nonintegrative systems, with 2% of neonatal fibroblasts successfully
reprogrammed into iPSCs within 17 days (Warren et al., 2010). By adding LIN28 on
top of the four Yamanaka factors, culturing at 5% oxygen, and supplementing with
valproic acid in the culture medium, the reprogramming efficiency was further
enhanced to 4.4% (Warren et al., 2010). The synthetic RNA reprogramming method
requires consecutive, daily transfections and hence makes it technically cumber-
some for larger-scale application. Although the synthetic RNA reprogramming
approach is appealing for its high efficiency, the high dosage of MYC mRNA could
also represent a high oncogenic risk (Gonzalez et al., 2011). Further exploration to
exclude the use of the potentially oncogenic MYC in reprogramming could be useful
in deriving clinically compliant hiPSCs.
A method that would guarantee the absence of unwarranted effects of exogenous
genetic materials in the iPSCs generated is the direct delivery of the reprogramming
factors as proteins. Kim and colleagues reported fusing the carboxy termini of
OSKM with a Myc-tag and a tract of poly-arginine transduction domain. The engi-
neered fusion reprogramming factors were stably expressed in HEK293 cells, and
the cellular extracts were applied to human neonatal fibroblasts multiple times
over 6 weeks before the cells were dissociated and plated onto feeders for the iPSCs
to emerge (Kim et al., 2009). However, this protein-based system is slow and de-
livers a low reprogramming efficiency.
188 CHAPTER 8 Considerations in using human pluripotent stem cell
Compared to non-viral-based methods, viral-based reprogramming systems
generally achieve higher reprogramming efficiencies and are applicable to a wider
range of cell types. However, there is a risk of genome integration. As lentiviral
and retroviral transductions are known to lead to random genome integration,
hiPSCs that are meant for clinical applications should not be generated using either
virus (Gonzalez et al., 2011). Adenovirus could serve as an option for integration-
free expression vehicle to derive hiPSCs. However, the reprogramming efficiency
is extremely low, at 0.0002% for human fibroblasts (Zhou and Freed, 2009). Alter-
natively, Sendai virus is a cytoplasmic RNA vector that has been shown to be able to
generate integration-free hiPSCs from a variety of somatic cells at higher efficiency
of 0.1%e1% (Ban et al., 2011;Seki et al., 2010;Fusaki et al., 2009). Sendai viral
extracts for Yamanaka factors are commercially available. The downside of the Sen-
dai system is that the virus will take approximately 10 passages after transduction to
be diluted out of the cells, and thorough screening has to be performed to ascertain
that the hiPSCs are virus-free.
5.4 hiPSC culture and expansion
Defined hPSC culture media and reagents are critical to achieve the stringency
required to use hiPSC-derived cells in the clinic. Traditionally, a layer of mitotically
inactivated mouse embryonic fibroblasts (MEFs) is required to support the mainte-
nance of hPSCs (Leonardo et al., 2012). However, there are concerns that undesired
foreign pathogens and antigens from the xenogeneic culture could trigger an im-
mune response in patients. For safety reasons, the use of murine-derived feeder cells,
animal-derived components found in matrices, culture medium, and passaging re-
agents should be fully avoided. Chemically defined medium and reagents would,
therefore, provide a standardized and reproducible culture condition suitable for
hPSC-derived cells to be used in humans. Besides conforming to cGMP, safety
testing and traceable documentations (such as Drug Master Files, https://www.fda.
gov/drugs/guidances-drugs/drug-master-files-guidelines) are required for submis-
sion to FDA in order for the media to be deemed suitable for clinical trials. hPSC
culture media have evolved over the years and cGMP-grade media suitable for clin-
ical translation are now commercially available.
6.Manufacturing hPSC-derived pancreatic beta cells for
cell therapy
hPSCs have the potential to generate an unlimited pool of pancreatic beta cells for
treating diabetes via cell replacement therapy. Numerous differentiation protocols
have been developed in the past two decades to create human pancreatic beta-like
cells in a dish (Loo et al., 2018). These protocols were built on the knowledge gained
from rodent models (Santosa et al., 2015). hPSCs are directed to differentiate into
beta-like cells using a cocktail of growth factors and small molecules, modeling
6. Manufacturing hPSC-derived pancreatic beta cells for cell therapy 189
the key stages of pancreatic beta cell development, such as definitive endoderm,
primitive gut tube, pancreatic progenitor, endocrine progenitor, and finally beta
cells. These protocols have also been applied to various hESC and hiPSC lines
(Table 8.2). Typically, the percentage of insulin
þ
cells obtained varies among the
protocols (Rezania et al., 2014;Russ et al., 2015;Pagliuca et al., 2014;Agulnick
et al., 2015;Yabe et al., 2017;Zhang et al., 2009). To manufacture pancreatic
beta cells for clinical application, the existing differentiation processes still require
fine-tunings to be robust, reproducible, and efficient in generating a large pool of
beta cells of optimal quality.
6.1 Challenges in generating cGMP-grade hPSC-derived
pancreatic beta cells for therapy
Given that different hPSC lines may exhibit differing differentiation efficiencies
(Osafune et al., 2008;Rouhani et al., 2014), it is important to decide on the choice
of hPSC line and specific differentiation protocol to be used as early as possible in
the manufacturing process. Kelly et al. (2011) reported that the NKX6.1
þ
cell pop-
ulation derived from three hESC lines (CyT49, CyT209, and MEL1) ranged from
6% to 45%, even though the same differentiation protocol was used (Kelly et al.,
2011). In a recent study by Wesolowska-Andersen et al. (2019) and, several
notable differences in the “omics” profiles of pancreatic progenitors were
observed between three differentiation protocols. These early differences likely
contributed to the varied differentiation efficiencies as these cells progressed
further toward the endocrine lineage (Wesolowska-Andersen et al., 2019).
While it is now possible to obtain a decent population (w20%e60%) of insulin
þ
cells using various differentiation protocols, these in vitro derived beta-like cells do
not necessarily always secrete insulin upon glucose challenge. At times, in vivo
maturation by transplanting hPSC-derived beta-like cells into an animal is required
to obtain functional pancreatic beta cells (Table 8.2). The specific molecular factors
and signaling pathways active in the in vivo microenvironment that trigger the func-
tional maturation of beta-like cells are still not understood. In general, this inconsis-
tency in generating functional hPSC-derived beta-like cells is a biological variable
that remains an obstacle for translation.
The past two decades have witnessed the progression of differentiation protocol
from planar 2D to 3D/spheroid suspension culture that has achieved a higher per-
centage and maturity of hPSC-derived beta-like cells (Loo et al., 2018). However,
it is important to note that individual cells in the hPSC spheroid could be subjected
to different concentrations of growth factors or small molecules due to their spatial
orientation. As a result, it may be inevitable that the pancreatic differentiation gives
rise to a mixed population of pancreatic endocrine cells as well as some residual un-
differentiated cells. The presence of undifferentiated hPSCs poses an important
safety concern when utilizing hPSC-derived cells for treating T1D patients. In
one early rodent study, the histopathological analysis showed that there were
some OCT4
þ
and SSEA-1
þ
cells scattered throughout the grafted tissue
190 CHAPTER 8 Considerations in using human pluripotent stem cell
Table 8.2 hPSC-derived INS
þ
cells in the past decade (2009e19).
Ref/Cell line Differentiation condition % INSDcells GSIS
Recipients/
transplant site
Zhang et al., 2009
hESC (H1, H9)
hiPSC (Ca, C2, C5)
2D Matrigel culture 25% Yes, in vitro e
Rezania et al., 2013
hESC (H1)
2D culture on Matrigel, spinner flask suspension
culture
w60% Yes, in vivo SCID subcutaneous
with encapsulation,
kidney capsule
Rezania et al., 2014
hESC (H1)
hiPSC (episomal
iPSC)
2D culture on Matrigel, aireliquid interface,
suspension culture on ULA plate
w50% Yes, in vitro and
in vivo
NSG, SCID, NOD-
SCID mice kidney
capsule
Pagliuca et al., 2014
hESC (HUES8)
hiPSC (hiPSC-1,
hiPSC-2)
Spinner flask suspension culture with stirring >30% Yes, in vitro and
in vivo
SCID, NRG-Akita
mice kidney capsule
Russ et al., 2015
hESC (MEL1,
INS
GFP/W
)
Suspension culture on low-adherent plate with
shaking
w60% Yes, in vitro and
in vivo
NSG kidney capsule
Agulnick et al., 2015
hESC (CyT49)
Suspension culture on nontreated plate/bottle with
rotation agitation Reaggregation
50% Yes, in vivo SCID subcutaneous
with encapsulation
(Encaptra-20)
Nostro et al., 2015
hESC (H1, H9,
MEL1, INS
GFP/W
,
NKX6.1
GFP/w
)
hiPSC (38-2, MSC-
iPSC1, BJ-iPSC1)
Early-stage embryoid bodies on low-cluster plate,
later-stage dissociation to single cells and culture on
gelatin-coated plate
w64% Yes, in vivo NSG mammary fat
pad, kidney capsule
Millman et al., 2016
hiPSC (non-diabetic:
ND-1, ND-2. T1D
hiPSC)
Spinner flask suspension culture with stirring 24%e27% Yes, in vitro and
in vivo
SCID kidney capsule
Manzar et al., 2017
hiPSC (T1D hiPSC
line)
Culture on feeder, 3D culture embedded in Matrigel 56% Low, in vivo B6 mice
Yabe et al., 2017
hiPSC (TkDN4-M,
253G1, 454E2)
2D culture on Matrigel, aggregates culture on ULA
plate
w30% Yes, in vitro NOD-SCID kidney
capsule
Selected protocols that are able to generate at least 20% of INS
þ
cells are shown.
6. Manufacturing hPSC-derived pancreatic beta cells for cell therapy 191
posttransplantation (Fujikawa et al., 2005). Teratoma formation occurred in six out
of 10 transplanted mice and failed to treat T1D in the rodent model (Fujikawa et al.,
2005). Teratoma formation is also subjected to some degree of experimental vari-
ability. Within the same laboratory setup and utilizing the same protocol, Kroon
et al. (2008) and Kelly et al. (2011) reported a 15% and 46% occurrence of teratoma
formation, respectively (Kelly et al., 2011;Kroon et al., 2008). Overall, this tumor-
igenic property of hPSC-derived cells, in this case in hPSC-derived beta cell “orga-
noids/spheroids,” remains a concern for T1D cell replacement therapy.
6.2 Strategies to prevent tumor formation
One way to resolve tumor formation in hPSC-derived beta-like cells is to perform
cell sorting to purify the cell population of interest before transplantation. Kelly
et al. (2011) identified CD142 as a cell surface marker for pancreatic endoderm
(PE) cells (not beta cells). They showed that enriched PE cells significantly reduced
the formation of teratomas in grafts when transplanted into rodents (Kelly et al.,
2011). In a recent publication by Veres et al. (2019), the authors identified CD49a
as a beta cellespecific surface marker (Veres et al., 2019). By dissociating stem
cellederived beta-like cells and magnetically sorting them based on CD49a expres-
sion before reaggregation, the authors demonstrated that the purity of the beta-like
cell population was enriched to 80% (Veres et al., 2019). However, it has also been
reported that the expression of CD49a is not specific to beta cells in adult human
islets (Baron et al., 2016). The specificity of the surface markers should thus be scru-
tinized, and the use of multiple surface markers to increase specificity and enrich-
ment should be explored. On the other hand, human islets consist of a mixed
population of pancreatic endocrine cells that function synergistically to achieve
glucose homeostasis. The eventual cell sorting to enrich a specific pool of pancreatic
beta cells may thus not be an ideal approach for diabetes therapy. It is currently
debatable whether the transplantation of hPSC-derived beta cells or pseudo-islets
that resemble the bona fide human islets would be a more appropriate approach
for cell replacement therapy in T1D patients.
An alternative strategy to purifying desired human islet cell types would be to
eliminate the residual undifferentiated hPSCs. One approach is to use suicide con-
structs to eliminate cells with tumorigenic potential. Suicide constructs include toxic
genes such as the herpes simplex virus thymidine kinase (HSV-TK) and nitroreduc-
tase (NTR). These genes are lethal to the host cell when expressed and only in the
presence of a specific substrate (Fareed and Moolten, 2002). These toxic genes
can be controlled by a promoter that is active only in dividing cells, such as the hu-
man telomerase reverse transcriptase (hTERT) promoter to select against tumori-
genic cells (Albanell et al., 1999). In hPSC-based therapy, the tumorigenic cells
are usually pluripotent cells that have escaped the differentiation process (Qadir
et al., 2019). The suicide genes can be designed to be controlled by pluripotent
gene promoters as well, such as the OCT4 gene promoter. Importantly, the suicide
constructs have to be introduced into the hPSCs using genome editing tools such
192 CHAPTER 8 Considerations in using human pluripotent stem cell
as CRISPR. Suicide constructs are effective in preventing teratoma formation in ro-
dent models (Qadir et al., 2019). Qadir et al. (2019) used a double fail-safe approach,
whereby the human TERT promoter controlled the HSV-TK gene, and the constitu-
tively active NTR gene was floxed with the CRE gene controlled by the insulin pro-
moter, thus specifically selecting only for INS
þ
cells that are nondividing.
Another potential approach is to use monoclonal antibodies (mABs) to specif-
ically kill hPSCs by recognizing antigens that are found on hPSCs but not on their
differentiated progenies (Tan et al., 2009). In 2009, Tan et al. discovered a mAB,
named mAB 48, that specifically recognizes the podocalyxin-like protein-1
(PODXL) protein on hESCs and causes oncosis to them but not to the differentiated
cells (Tan et al., 2009;Schriebl et al., 2012). This approach is advantageous
compared to the use of suicide constructs because it does not involve genome-
edited cells, which is associated with an additional layer of regulation. Although
both strategies are still immature for clinical application, they are promising in func-
tioning as a safety net in preventing teratoma formation. The occurrence of teratoma
in hPSC-based clinical trials may disrupt confidence and impede the progress of this
technology.
Besides repeatedly differentiating hPSCs into beta-like cells, another approach to
minimize undifferentiated cells in the end product will be to maintain self-renewing
endodermal progenitors. Some studies have reported on long-term cultures of human
endodermal derivatives (Cheng et al., 2012) or self-renewing pancreatic progenitors
that can be serially passaged, expanded in vitro on feeders as well as cryopreserved
(Hannan et al., 2013;Sneddon et al., 2012;Trott et al., 2017). The current assump-
tion is that long-term cultures of these endodermal differentiated progenitor cells no
longer contain tumorigenic hPSCs. Therefore, the risk of teratoma formation is
greatly reduced when these starting materials are used to generate human beta-
like cells. However, current protocols rely on feeder layers to maintain the self-
renewing capacity of these endodermal/pancreatic progenitors. To meet clinical re-
quirements, the protocols will need to be further modified to achieve xenogeneic-
free culture conditions. Another consideration is the long-term expandability of
the endodermal progenitors (Trott et al., 2017) since large quantities of beta cells
(w9500 islets per kg of body weight) are required for diabetes therapy (Gerber
et al., 2018). However, despite the numerous obstacles that make the translation
of in vitro derived pancreatic beta-like cells to clinical use challenging, recent years
have seen the first leap into a hPSC-based therapy for T1D patients.
7.hPSC-based therapy for type 1 diabetes
In 2008, ViaCyte, Inc. (previously Novocell, Inc. (ViaCyte Incoporated, 2010)) pub-
lished a stepwise protocol that could differentiate hESCs into pancreatic endoderm
cells (PEC-01) in an adherent culture system. The cells were able to secrete insulin
in response to glucose after in vivo maturation and could reverse diabetes in mice
(Kroon et al., 2008). Subsequent cell sorting assessments identified that only the
7. hPSC-based therapy for type 1 diabetes 193
pancreatic progenitor cells (marked by CHGA
/NKX6.1
þ
/PDX1
þ
) formed the
functional component of PEC-01, while the immature multihormonal cells as part
of the heterogeneous cell population did not (Kelly et al., 2011). To progress into
the clinics, it is pertinent that this differentiation system is cGMP-compliant,
xenogeneic-free, robust, efficient, and scalable. In fact, in 2012, ViaCyte reported
that their CyT49 hESC line could be cultured and expanded in a feeder-free system
and differentiated into pancreatic progenitors in a scalable fashion using a rotatory
suspension system (Schulz et al., 2012). They also reported that their protocol is
highly reproducible and is relatively efficient in generating about 30%e40% of
pancreatic progenitor cells, which could consistently function after in vivo matura-
tion (Schulz et al., 2012). ViaCyte has begun the manufacturing of cGMP-grade
PEC-01 cells and has begun testing them in clinical trials, as described in the next
two sections.
7.1 Manufacturing of PEC-01 cells
The production of PEC-01 cells begins with their clinical-grade hESC line CyT49.
ViaCyte has set up a tiered cryopreservation system for the CyT49 hESC line that
is cGMP-compliant (Schulz, 2015). They established a robust and scalable feeder-
free culture system for the CyT49 hESC line by using xenogeneic-free serum
replacement, KnockOut Serum with GMP-grade Heregulin b-1 and Activin A for
self-renewal (Schulz, 2015). hESCs are passaged as single cells using Accutase
for precise control over seeding density for consistent and predictable expansion.
They reported that a frozen vial of 100 million CyT49 cells could consistently be
thawed and expanded to a sufficient amount of cells for differentiation in four
passages throughout 15 days (Schulz, 2015). For differentiation into PEC-01, hESCs
are dissociated into single cells and allowed to reaggregate using a roller bottle sys-
tem for 1 day to form uniform spheroids of pluripotent cells. These cells are then
subjected to a four-stage differentiation protocol that comprises various specification
cues to direct them to form pancreatic progenitors. The PEC-01 cells are cryopre-
served and various release tests, such as sterility, mycoplasma, endotoxin, thaw
yield, pancreatic progenitor composition, gene expression, and percentage of plurip-
otent cells, will be conducted on thawed cells (Schulz, 2015).
7.2 VC-01 clinical trial
In 2014, ViaCyte began its first phase 1/2 clinical trial, which implanted the VC-01
combination product (PEC-Encap), consisting of PEC-01 cells encapsulated in an
Encaptra device, in an open-label, dose-escalating study of safety, tolerability, and
efficacy (Clinical Trial Identifier: NCT02239354). After clearing the release tests
on thawed PEC-01 cells, the VC-01 combination product was prepared over
3 days, from the thawing of PEC-01 cells to formulating and loading into the Encap-
tra device (Schulz, 2015). Two different sizes of capsules (VC-01-250 and VC-01-
20) of the VC-01 combination product were implanted into patients by injecting into
194 CHAPTER 8 Considerations in using human pluripotent stem cell
the subcutaneous layer. The larger unit VC-01-250 was the intended size to be
tested, while the smaller unit VC-01-20 was to be extracted at various time points
for histological and other analyses. The Encaptra device was designed to house
all the PEC-01 cells and allow the host blood vessels to grow on the exterior. This
allows nutrients such as glucose, proteins, and oxygen to reach the cells and for in-
sulin to diffuse out into the circulatory system. As blood vessels are unable to pene-
trate the Encaptra device, immune cells are unable to reach the transplanted PEC-01
cells and thus, are able to avoid immune rejection. Therefore, patients receiving VC-
01 do not require immunosuppression. Besides, the Encaptra device will prevent the
outgrowth of any potential tumor, providing an additional layer of safety for the pa-
tients. With the implantation at the subcutaneous layer, the graft can also be easily
retrieved for safety purposes.
The clinical trial was split into two cohorts. Patients in cohort 1 were implanted
with a subtherapeutic dose of VC-01 to evaluate safety and tolerability while pa-
tients in cohort 2 were implanted with the intended therapeutic dose, with the
goal of evaluating the efficacy of VC-01. In June 2018, ViaCyte reported the 2-
year data of their cohort 1 study at the American Diabetes Association (ADA) con-
ference (ViaCyte Incoporated, 2018). Their VC-01 combination product was shown
to be safe and well tolerated with no abnormal growth observed. The major side ef-
fects reported were due to surgical procedures and/or postoperative care. However, it
appears that most of the cells did not survive longer than 12 weeks, likely due to hyp-
oxia as a result of the host launching a foreign giant body response to the graft (Pul-
len, 2018). The positive news is that the Encaptra device seemed to be effective in
preventing immune rejection as no donor-specific antibodies were detected in pa-
tients. Although there were low levels of engraftment, in vivo maturation of pancre-
atic progenitors to endocrine islet cells after transplantation was observed in regions
with good host tissue integration and vascularization, after both 12 weeks and
2 years. Analysis of the explants showed that some of these matured islet cells
were able to produce insulin (ViaCyte Incoporated, 2018). These data were mostly
positive in terms of proving the safety and tolerability of the VC-01 product and that
the implanted PEC-01 cells can undergo in vivo maturation in patients to form
insulin-producing cells, in the case of successful engraftment. Subsequent strategies,
such as improving the design of the Encaptra capsule, are currently being developed
to enhance engraftment success rate (ViaCyte Incoporated, 2018).
7.3 VC-02 clinical trials
In 2017, ViaCyte launched two more clinical trials using a similar product, VC-02
(PEC-Direct). VC-02 is similar to VC-01, except that blood vessels can enter the de-
vice and interact more closely with the PEC-01 cells. The direct vascularization will
improve engraftment success rate but will also require immunosuppression therapy,
thereby making VC-02 more suitable for patients with a more severe form of T1D
(ViaCyte Incoporated, N.D.). ViaCyte launched a small-scale phase 1 trial to eval-
uate if VC-02 can be maintained safely for up to 4 months in a small group of
7. hPSC-based therapy for type 1 diabetes 195
patients with T1D (Clinical Trial Identifier: NCT03162926). The phase 1 trial was
completed in 2018. In parallel, they also launched a phase 1/2 trial that is currently
ongoing. Fifty-five patients with T1D are being recruited for an open-label study and
will be split into two cohorts, similar to the VC-01 clinical trial. The purpose of
cohort 1 is to evaluate if the implants can be maintained in patients for up to 2 years,
whereas that of cohort 2 is to evaluate the efficacy of VC-02.
7.4 Preclinical study by Semma therapeutics
As one of the problems faced by ViaCyte in the VC-01 phase 1/2 clinical trial was
the large patient-to-patient variability (Pullen, 2018), a potential approach to
circumvent this issue is to transplant functional beta-like cells that do not require
in vivo maturation. It has been reported that functional beta-like cells can be gener-
ated from hPSCs purely in vitro, without the need for in vivo maturation (Pagliuca
et al., 2014;Veres et al., 2019). Two of the authors in both papers subsequently
founded Semma Therapeutics (later acquired by Vertex Pharmaceuticals (Vertex
Pharmaceuticals Incorporated, 2019)), a company that is committed to treating
T1D using hPSC-derived islet cells. In the original protocol, the differentiation of
endocrine progenitors into functional beta-like cells required the use of fetal bovine
serum (Pagliuca et al., 2014), which renders the cell product unsuitable for clinical
use. After a few years of optimization, Semma Therapeutics announced that they had
managed to generate functional islet cells from hPSCs in vitro using a protocol that
is serum-free, cGMP-compliant, and highly reproducible (Pullen, 2018). They are
currently optimizing the delivery system in order to offer immune protection to
the cells before proceeding to clinical trials.
8.Future outlook and concluding thoughts
The success rate in achieving long-term insulin independence (>5 years) postallo-
geneic islet transplantation is approaching 50% (Barton et al., 2012;McCall and
Shapiro, 2012). The major downside of allogeneic islet implant is the requirement
for lifelong administration of immunosuppressant drugs to prevent graft rejection.
Moreover, to achieve insulin independence, infusions from multiple donors are often
required. The ability to generate unlimited quantities of hPSC-derived pancreatic
beta-like cells could theoretically resolve the issue of scarcity of cadaveric islet do-
nors. The future of T1D hPSC-based cell therapy is likely to move toward trans-
planting allogeneic functional beta-like cells to achieve insulin independence and
restore normoglycemia. Currently, some of the challenges for routine hPSC-based
cell therapy are (1) demonstration of the functionality of these beta-like cells
in vivo in humans, (2) host response to the transplanted cells, and (3) potential tumor
formation. Multiple concurrent strategies such as cell transplant in encapsulation de-
vices, knockout of immune genes in transplanted cells, and building a comprehen-
sive cGMP-compliant hiPSC biobank hold great promises for diabetes patients
196 CHAPTER 8 Considerations in using human pluripotent stem cell
awaiting islet replacement therapy. Further refinements to these technical aspects
will bring us closer to realizing the use of hPSC-derived islets or beta cells for
the treatment of T1D patients.
Acknowledgments
The authors thank the members of the Teo laboratory for the critical reading of this
manuscript.
Funding
W.X.T. is supported by the NUS Research Scholarship. H.H.L. is supported by the Institute of
Molecular and Cell Biology (IMCB) Scientific Staff Development Award (SSDA) for her
part-time Ph.D. A.K.K.T. is supported by IMCB, A*STAR, NMRC OFYIRG16may014,
A*STAR ETPL Gap Funding ETPL/18-GAP005-R20H, NMRC OF-LCG/DYNAMO, Lee
Foundation Grant SHTX/LFG/002/2018, Skin Innovation Grant SIG18011, FY2019 Sing-
Health Duke-NUS Surgery Academic Clinical Programme Research Support Programme
Grant, Precision Medicine and Personalised Therapeutics Joint Research Grant 2019, Industry
Alignment FunddIndustry Collaboration Project (IAF-ICP) I1901E0049, and the 2nd
A*STAR-AMED Joint Grant Call 192B9002.
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References 203
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Induced pluripotent stem
cells for treatment of heart
failure 9
Shigeru Miyagawa, Yoshiki Sawa
Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Suita,
Osaka, Japan
Chapter outline
1. Introduction ................................................................................................ .......206
2. Trend of cell therapy .......................................................................................... 207
3. Production of iPSCs and establishment of a cardiogenic differentiation method .....208
3.1 Cytokine-based CM differentiation of iPSCs ........................................... 208
3.2 Small moleculeebased myocardial differentiation of iPSCs..................... 209
3.3 Direct reprogramming for cardiomyocyte generation ............................... 209
4. Large-scale cell culture for cell transplantation therapy .......................................209
4.1 Bioreactor system for generating CMs for cell transplantation therapy ......210
5. Proof of concept in iPSC-CM sheet for heart failure and immunologic study
of iPSCs ............................................................................................................ 211
6. Removal of undifferentiated iPSCs to confirm safety for clinical applications .........213
7. Development of new drug-based heart failure therapy using disease-specific
iPSCs ................................................................................................................214
7.1 Disease-specific iPSCs and drug discovery screening in cardiovascular
disease............................................................................................... 214
7.2 Drug discovery screening using iPSC-CM tissue ..................................... 215
7.3 Future plans and perspectives in drug screening using iPSCs .................. 217
References .............................................................................................................218
Abstract
The prognosis of severe heart failure that is mainly due to cardiomyopathy is
remarkably poor. Although the outcomes of maximal pharmacological therapy,
surgical treatments such as left ventricular assist device, or heart transplantation
have improved significantly in recent years, drawbacks include lack of donors,
necessity of permanent immunosuppressive treatment, serious complications
including cerebral hemorrhage, and the financial burden of treatment.
Regenerative medicine is expected worldwide to overcome these shortcomings. The basic
technology required for regenerative medicine using stem cells such as iPS cells and ES
cells has made remarkable progress in the last 10 years. For severe heart failure, treatment
CHAPTER
205
Recent Advances in iPSCs for Therapy, Volume 3. https://doi.org/10.1016/B978-0-12-822229-4.00004-8
Copyright ©2021 Elsevier Inc. All rights reserved.
development using regenerative medicine techniques such as cell transplantation and
tissue transplantation has recently progressed, and clinical application has begun.
It is expected that iPS cells and tissues using them will not only improve cardiac
function by cell supplementation but also contribute to elucidation of pathological
conditions. In recent years, the pace of new drug development has decreased, partly
due to the inefficiency of screening technology. A drug discovery screening system
using iPS cellederived from healthy subjects or diseased patients may create an
efficient and innovative means of drug discovery technology.
This article summarizes the current state of research on heart failure treatment
using autologous or allogeneic cells, especially studies utilizing iPS cells, and drug
screening using disease-specific iPS cells in cardiomyopathy.
Keywords:
Allogeneic; Arrhythmia; Cardiomyocyte; Cell sheet; Cell therapy; Cytokine therapy;
Differentiation; Direct reprogramming; Drug screening; Heart failure; Immune rejection; Induced
pluripotent stem cells; Large-scale culture; Tissue engineering; Tumorigenicity.
1.Introduction
Severe heart failure is a highly fatal disease. Despite the remarkable progress in
medical technology, the mortality and morbidity rates remain high worldwide and
the quality of life following heart failure is significantly reduced (Cohn, 2014). Med-
ications are a priority in the treatment for heart failure patients at a reversible stage
of the disease (Fig. 9.1A). When heart failure is irreversible, treatment can involve
the left ventricular assist device (LVAD) or heart transplant. Heart transplantation is
the most effective treatment for severe heart failure. A new heart can prolong life and
alleviate heart failure symptoms. However, there is a shortage of donors worldwide,
which will likely continue (Longnus et al., 2014). Alternatively, destination therapy
using LVAD has been clinically performed. Major concerns of this approach are
complications that include infection and cerebral thrombosis (Abraham and Smith,
2013). In addition, bridge to recovery therapy that improves left ventricular function
by unloading the severely dilated left ventricle after LVAD implantation is a
FIGURE 9.1
Progression of pathology in heart failure and treatment options.
(A) Standard treatment for chronic heart failure. (B) Innovations in treatments for heart
failure.
206 CHAPTER 9 Induced pluripotent stem cells for treatment of heart failure
promising therapeutic option. However, the approach may be difficult to transition
into a universal treatment because it depends on the viability of the damaged
myocardial tissue (Miyagawa et al., 2016b). The development of new therapies
that can prevent heart failure from worsening before an irreversible stage or are
alternatives to LVAD and heart transplantation in an irreversible stage may open a
new era of treatment for heart failure.
As an option to overcome this situation, there is an increasing expectation for
regenerative medicine worldwide (Fig. 9.1B). However, regenerative medicine is
not capable of completely repairing severe heart failure. Thus, there is an urgent
need to develop a new treatment to replace heart transplantation and LVAD. Basic
research and clinical applications of cell and tissue transplantation are progressing
in severe heart failure. In this article, we outline the conventional cell therapy and
summarize nonclinical studies on the treatment of heart failure using iPSCs.
2.Trend of cell therapy
Numerous basic studies have been conducted on cell therapy for heart failure, and
many clinical studies have been performed (Miyagawa et al., 2011;Strauer et al.,
2002;Menasche et al., 2008;Meyer et al., 2006;Behfar et al., 2014;Hashimoto
et al., 2018). Cells can be administered via intramuscular injection using a syringe,
cardiac tissue can be transplanted under open thoracotomy, and transcatheter trans-
plantation can be done via the coronary artery (Strauer et al., 2002;Menasche et al.,
2008;Hare et al., 2012).
A clinical study performed in France was the first reported cell transplantation
treatment for heart failure (Menasche et al., 2008). In this clinical study, autologous
myoblasts were collected from the lower leg and transplanted into myocardial tissue
with a syringe during the thoracotomy. Unfortunately, the approach was not effective
in patients with heart failure, and a fatal arrhythmia was reported. It was speculated
that the arrhythmia occurred due to the electrical characteristics of myoblasts. How-
ever, considering the fact that autologous myoblast cell sheet transplantation per-
formed in Japan did not cause a lethal arrhythmia (Miyagawa et al., 2017), the
fatal arrhythmia may have been due to the transplantation method. In addition,
many clinical reports have described transplantation of bone marrow mesenchymal
stem cells, myocardial stem cells, and embryonic stem cell (ESC)-derived cardio-
myocytes (CMs) into heart failure patients (Miyagawa et al., 2011;Hashimoto
et al., 2018). These various cells have been clinically applied. Their therapeutic
effects largely depend on angiogenesis, antifibrosis, and antiinflammatory activities
of cytokines secreted from the cells. Basic research has revealed that c-kit positive
cells have a poor ability to induce differentiation into CMs, similar to other somatic
stem cells (Murry et al., 2006;van Berlo et al., 2014;Wang et al., 2018). As a conse-
quence, clinical effects mainly depend on the angiogenic effects of cytokines. In
fact, c-kit positive cells have already been clinically applied (Bolli et al., 2011).
However, the report of the clinical trial was retracted because of the lack of
2. Trend of cell therapy 207
reliability regarding the laboratory work for cell preparation (The Lancet, 2019).
ESCs have the potential to differentiate to CMs. Cardiac patches containing differ-
entiated CMs have been transplanted into patients with ischemic cardiomyopathy
(Menasche et al., 2015a,b;2018). The approach still needs to be sufficiently verified
concerning safety and efficacy. iPSCs can also differentiate to cardiac myocytes
(Yoshida et al., 2018;Yu et al., 2013). Basic research demonstrated that iPSCs
can be induced to differentiate to CMs and that these transplanted CMs may provide
contractile force directly to failing hearts (Higuchi et al., 2015). Unlike somatic stem
cells, differentiated CMs have the ability to secrete cytokines in addition to their
potential to contribute directly to the contractile force in a failing heart. Nonclinical
research aimed at clinical applications has begun (Hashimoto et al., 2018;Chow
et al., 2017;Laflamme et al., 2007).
3.Production of iPSCs and establishment of a cardiogenic
differentiation method
iPSCs are generated from multiple cell sources, including fibroblasts, T cells, adi-
pose tissue, and umbilical cord blood, using Oct-3/4, Sox2, c-Myc, and Klf4 reprog-
ramming factors (Bayart and Cohen-Haguenauer, 2013). Sendai virus vectors and
plasmid vectors are used for gene transfer of these reprogramming factors into
somatic cells. Differentiation of iPSCs to CMs is induced by several cell culture pro-
tocols, described further, regardless of the cell source or reprogramming factors. In
addition, the process of direct reprogramming, in which CMs are directly induced
from somatic cells by reprogramming specific factors, has been also reported (Gour-
die et al., 2016;Ieda et al., 2010;Song et al., 2012;Xin et al., 2013). This section
describes how to induce CM differentiation from reprogrammed somatic cells and
pathophysiological characters of differentiated CMs.
3.1 Cytokine-based CM differentiation of iPSCs
A standard in vitro protocol for differentiation of iPSCs to CMs was first reported in
2008 (Yang et al., 2008). The authors introduced multiple factors into ESCs to
generate CMs. As a first step, activin A and bone morphogenetic protein 4 (BMP4)
are added to express the WNT signaling protein that induces mesoderm
differentiation. The WNT inhibitor DKK1 is added to identify cardiac mesoderm
with vascular endothelial growth factor or basic fibroblast growth factor (bFGF)
that promotes cell growth and maturation (Yang et al., 2008). This differentiation
method has made it possible to reproduce self-pulsating embryoid bodies with an
expression of heart-specific markers, such as cardiac troponin T, cardiac myosin
light/heavy chain, and connexin 43 (Miki et al., 2012). However, in spite of the repro-
ducibility of the cardiogenic differentiation method, the rate of differentiation into
CMs or maturation of CMs depends on the cell line or culture conditions (Yu et al.,
2013;Kawamura et al., 2012). Mechanical stretching, addition of various humoral
208 CHAPTER 9 Induced pluripotent stem cells for treatment of heart failure
factors, and microRNA modification can each enhance the maturation of immature
CMs differentiated from iPSCs or the production of ventricular CMs (Yu et al.,
2013;Ren et al., 2011). In addition, some iPSC lines expressing the CXCL4/PF4
gene when undifferentiated can easily differentiate to form CMs (Ohashi et al.,
2019). These differentiated CMs have almost the same microstructures and physiolog-
ical behaviors as native CMs. However, there are some differences, such as the poor
expression of myosin heavy chain, which is a marker of cardiomyogenic maturation,
and the poorly polarized expression of connexin43 on the cell membrane of CMs.
3.2 Small moleculeebased myocardial differentiation of iPSCs
Regenerative treatment using cytokine-based iPSC-derived CMs (iPSC-CMs) may
be an expensive treatment for heart disease, since it requires a large number of func-
tional CMs for transplantation. Cytokines are made from biological materials. The
regulatory approval for their use could be a hurdle for clinical application. Synthetic
small molecules might potentially solve this problem. Some small molecules that
modulate WNT signaling can induce differentiation of iPSCs to CMs (Minami
et al., 2012;Sharma et al., 2015) or can enhance cytokine-based cardiomyogenic dif-
ferentiation of iPSCs to CMs (Ren et al., 2011). Highly pure ventricular myocytes
can be obtained using synthetic small molecules.
3.3 Direct reprogramming for cardiomyocyte generation
Direct reprogramming of somatic cells using specific transcription factors or micro-
RNAs in the absence of iPSCs can induce the differentiation multiple cell sources,
such as dermal fibroblasts, cardiac fibroblasts, and skeletal myoblasts, to CMs
in vitro (Guo et al., 2015). This direct reprogramming technique may be a potential
candidate for cardiac regenerative therapy because it shows less tumorigenicity
compared to iPSC-CMs (Gourdie et al., 2016;Ieda et al., 2010;Song et al., 2012;
Xin et al., 2013). However, it is necessary to verify the efficiency in myocardial dif-
ferentiation and the compatibility of the generated CMs with native CMs concerning
their microstructures and physiological behavior (Chen et al., 2012).
4.Large-scale cell culture for cell transplantation therapy
iPSC-CMs are a promising treatment to compensate for lost organ function. Howev-
er, the number of cells required to repair damaged organs depends on the organ and
disease severity. In particular, cell therapy for heart failure requires many cells
compared to other organs, on the order of approximately 100 million to 1 billion
iPSC-CMs per patient (Jing et al., 2008;Lock and Tzanakakis, 2009). It is important
to develop a technology for the large-scale culture of iPSC-CMs that can yield large
quantities of highly purified cells. Various large-scale culture systems are currently
being developed.
4. Large-scale cell culture for cell transplantation therapy 209
It is difficult to prepare a sufficient number of iPSC-CMs for heart failure
because the commonly used method for culturing ESCs and iPSCs involves flat
cell culture dishes (Kawamura et al., 2012). Cell preparation for clinical use must
be done in highly regulated areas, such as cell processing centers and cell isolators,
which may make cultivation difficult due to space and labor limitations. In addition,
handling a large number of dishes increases the risk of contamination and fewer cul-
ture vessels may reduce the variation in cell properties from the viewpoint of quality
control. This section provides the latest information on large-scale cell culture sys-
tems for iPSCs and CMs.
4.1 Bioreactor system for generating CMs for cell transplantation
therapy
In a bioreactor system, cells are cultured in bottles and are protected from the outside
air, potentially reducing the chance of contamination (Fig. 9.2). In the culture me-
dium, parameters that include pH, oxygen, and temperature are monitored and rigor-
ously adjusted to produce high-quality CMs. Bioreactors occupy less space, require
less labor, and are less expensive concerning reagents and media. Bioreactor culture
of ESCs and iPSCs has been demonstrated to be an effective way to expand these
cells. For induction of differentiation, iPSCs are usually cultured in three-
dimensional cell clusters called embryoid bodies. The aggregated cells differentiate
FIGURE 9.2
Production process of hiPSC-derived cardiomyocyte sheet.
iPSCs undergo undifferentiated maintenance and expansion by a two-dimensional
culture method, then form embryoid bodies, and are subjected to suspension rotation
culture in a bioreactor system. Cardiomyocyte differentiation is induced via the sequential
addition of differentiation-inducing factors to cardiomyocytes at an appropriate time.
These cells are stored frozen, thawed a few days before use, and then cultured for sheet
formation in a temperature-responsive culture dish for 2 days.
210 CHAPTER 9 Induced pluripotent stem cells for treatment of heart failure
into CMs in the reactor and are collected and dispersed as single cells on culture
dishes. Immature cells are eliminated. CMs grown in the bioreactor can form
sheet-like grafts with synchronized beats in vitro using temperature-responsive
dishes (Matsuura et al., 2012). Therefore, the bioreactor system may be an ideal
application for clinical preparation of iPSC properties in terms of stem cell mainte-
nance, expansion, and differentiation. Conversely, although it is necessary to
eliminate undifferentiated iPSCs in a large number of CMs to confirm the safety
of iPSC therapy, bioreactor systems have some difficulties in elimination of imma-
ture cells compared with planar culture because the bioreactor system increases the
number of CMs by culturing embryoid bodies.
5.Proof of concept in iPSC-CM sheet for heart failure and
immunologic study of iPSCs
For severe heart failure featuring remarkable fibrosis and substantial loss of CMs,
the resupply of healthy CMs is crucial. CM transplantation may replenish CMs in
infarct lesions that have been depleted of these cells. In recent years, it has been
reported that iPSCs can differentiate into various cells including CMs that are phys-
iologically and anatomically homologous to native CMs (Yu et al., 2013).
A CM sheet can be prepared using human iPSC-CMs. A proof of concept for the
same cardiac tissue using a large animal heart failure model has been described
(Kawamura et al., 2012;Ishida et al., 2019). In addition, X-ray diffraction analysis
using high-power synchrotron radiation demonstrated that the transplanted iPSC-
CM sheet repeatedly contracts and relaxes in the recipient heart and may function
as a working myocardium. The synchronized beat of the cardiac tissue may have
a direct effect on the contraction of the recipient’s heart (Higuchi et al., 2015).
The iPSC-CM sheet not only functions as a working tissue, but also secretes hepa-
tocyte growth factor and other cytokines from the tissue, producing angiogenesis in
the transplanted organ and improving blood flow (Kawamura et al., 2012).
In addition, expression patterns of complements, such as N-glycan expressed in
iPSCs, are similar to the pattern in mature CMs in the process of differentiation,
which may be important in verifying the immunogenicity of cell-derived CMs
(Kawamura et al., 2015). Furthermore, HLA homo iPSC-CMs were reported to sup-
press immunogenicity in cynomolgus monkey allograft experiments (Kawamura
et al., 2016). For clinical applications, HLA homo iPSC constructed by Center for
iPS Cell Research and Application, Kyoto University (CiRA), will be immunolog-
ically effective in transplantation into matched patients. After transplantation of
HLAehomogeneous cynomolgus monkey iPSC-CM sheets into HLA-matched indi-
viduals who were immunosuppressed, the transplanted cells had disappeared by
4 months after transplantation with preservation in functional recovery, showing
cell engraftment and functional efficacy. Therefore, the role of the iPSC-CM sheet
in improved function in heart failure does not depend on its synchronization with the
recipient heart, but rather on the angiogenic effect due to secreted cytokines.
5. Proof of concept in iPSC-CM sheet for heart failure 211
Alternatively, iPSC-CM sheets have proven superior to other somatic stem cell
sheets in functional recovery. The functional significance of iPSC-CM sheets may
be due to the mature blood vessels formed by both endothelial cells and smooth
muscle cells. In order to accentuate the characteristics of iPSC-CM sheets, it seems
necessary to improve the CM engraftment rate after transplantation.
In the future, efficacy can be further improved by promoting the engraftment ef-
ficiency of transplanted iPSC-CMs. To improve the engraftment efficiency in vivo, it
is necessary to suppress the immunogenicity of iPSCs and construct a developed
blood vessel to supply oxygen and nutrition to the transplanted tissue (Miyagawa
et al., 2016a,b). Regarding the suppression of immunogenicity, basic research is
needed to elucidate the mechanism of the immune reaction of iPSC-CMs.
Autologous iPSC transplants have the same origin as immunocompetent cells.
Thus, they can adapt to the immune response and are expected to be recognized as
“self” via acquired and natural immunity, to establish self-immune tolerance.
However, syngeneic iPSC-CMs that have weakly expressed MHC class 1 are
attacked by natural killer (NK) cells and are rejected after transplantation (Naka-
mura et al., 2019;Bix et al., 1991;Karre, 2008). The enhanced expression of MHC
class 1 or use of a methodology to block DNAM-1 and other molecules, which are
activating receptors of NK cells, may reduce immune rejection caused by innate
immunity (Nakamura et al., 2019). Recently, iPSCs with modified MHC class 1
have been produced (Xu et al., 2019). These cells may further reduce immunoge-
nicity after transplantation. Control of acquired immunity is also indispensable for
maintaining engraftment of transplanted cells. Simultaneous transplantation of an
allogeneic iPSC-CM sheet and bone marrow mesenchymal stem cells suppresses
killer T cells and improves CM engraftment after transplantation (Yoshida et al.,
2020).
Concerning the construction of nutritional blood vessels that can maintain trans-
planted tissues, maturated blood vessels with smooth muscle cells and vascular
endothelial cells are derived from the omentum. They have abundant vascular net-
works after simultaneous transplantation of the iPSC-CM sheet and omentum
(Kawamura et al., 2013). Nonclinical studies have shown that coimplantation of a
CM sheet and omentum maintains CM engraftment with a developed vascular
network and excellent functional recovery (Kawamura et al., 2013).
When transplanting iPSC-CMs to a failing heart, a major concern is an induc-
tion of arrhythmia. Fatal arrhythmias may occur when iPS cellederived CMs are
transplanted into myocardium with a needle, particularly early after transplanta-
tion (Shiba et al., 2016). When the artificial myocardial tissue is damaged with a
needle, an entry wave occurs around the damaged site and that site is a source
of arrhythmia. The arrhythmia is completely cured by transplantation of a CM
sheet derived from iPSCs onto damaged myocardial tissue (Li et al., 2020).
Conversely, when the iPSC-CM sheet was transplanted into a porcine myocardial
infarction model, no fatal arrhythmia was detected on the Holter electrocardiogram
(Kawamura et al., 2012).
212 CHAPTER 9 Induced pluripotent stem cells for treatment of heart failure
6.Removal of undifferentiated iPSCs to confirm safety for
clinical applications
Clinical trials using human pluripotent stem cells (hPSCs) such as human ESCs
(hESCs) (Schwartz et al., 2012,2015;Song et al., 2015) and human iPSCs (hiPSCs)
(Reardon and Cyranoski, 2014) have been conducted as regenerative medicine-
related treatments for heart failure. One of the most important issues to address is
safety, including the prevention of tumor formation in hiPSCs after transplant
(Masuda et al., 2014). For safer clinical application of hiPSCs, it is essential to
efficiently remove residual undifferentiated cells that may be capable of tumorige-
nicity. In this section, recent advances in the removal of hiPSCs are discussed.
Survivin is highly expressed in various cancers and aids in survival of tumor
cells. Survivin inhibitors are being developed as targeted cancer drugs. Survivin
and BCL10 are preferentially expressed in hiPSCs and survivin inhibitors are
completely cytotoxic to hPSCs in vitro (Lee et al., 2013). Only one administration
of a surviving inhibitor to hPSCs has completely prevented the formation of tera-
tomas after transplantation into immunodeficient mice.
Ben-David et al. (2013a) have shown that hPSC survival depends on oleic acid
metabolism, suggesting a unique role for lipid metabolism in hPSCs. Through high-
throughput screening of 52,000 small molecules, the authors reported that the oleic
acid synthesis inhibitor PluriSIn #1 was effective in selectively removing hPSCs.
Claudin-6 is a tight junction protein that has been identified as a specific surface
marker for hPSCs (Ben-David et al., 2013a,b). The authors demonstrated the specific
expression of claudin-6 in hPSCs and its value in selectively removing undifferen-
tiated cells.
Metformin is widely used as a treatment for diabetes. The drug is an AMP-
activated protein kinase agonist with diverse functions that include antitumor
effects. Vasquez Martin et al. (Vazquez-Martin et al., 2012) demonstrated that
metformin limits the tumorigenic potential of mouse iPSCs.
Other strategies to collect cardiomyocytes rely specifically on either metabolic
characteristics or surface markers. Tohyama et al. (2013) reported that only CMs
could survive culturing in a nonglucose (lactic aciderich) medium and reported
that depleting glucose removed undifferentiated iPS cells.
The CD30 antigen is highly expressed as a surface antigen of undifferentiated
iPSCs, and a method using an antibody against CD30 antigen has been developed
(Sougawa et al., 2018). A CD30 antibody-binding drug that bound to the CD30 an-
tigen of undifferentiated iPSCs reportedly induced apoptosis of the target cells and
showed no tumorigenicity in myocardial tissue transplants treated with the drug in
immunodeficient mice. Moreover, undifferentiated human iPSCs showed enhanced
expression and activation of transient receptor potential cation channel subfamily V
member 1 (TRPV-1) channels through transient culture at 42C, resulting in selec-
tive apoptosis of these cells without adverse impact on the viability of iPSC-CMs
(Matsuura et al., 2016). Cultivation at 42C sufficiently eliminated undifferentiated
6. Removal of undifferentiated iPSCs to confirm safety 213
human iPSC in fibroblast sheets and prevented tumor formation after transplantation
of the sheet onto subcutaneous tissue of nude rats (Matsuura et al., 2018).
For clinical applications of iPSCs, it is important to evaluate the contamination of
undifferentiated cells that are tumorigenic cells. Undifferentiated iPSCs specifically
express Lin28 and quantification of undifferentiated iPSCs has been successful by
measuring gene expression by RT-PCR. The degree of Lin28 expression correlated
with tumorigenicity in tumorigenicity tests using NOG mice (Ito et al., 2019).
7.Development of new drug-based heart failure therapy
using disease-specific iPSCs
The prognosis of early-onset severe heart failure due to cardiomyopathy is extremely
poor. Clarifying the pathology of the disease and development of new treatments are
urgently needed. Some types of dilated cardiomyopathy and hypertrophic cardiomy-
opathy are caused by mutations in genes, such as cardiac sarcomere constituent
genes and structural protein genes (Reichart et al., 2019). The development and
use of high-speed sequencing revealed a higher frequency of genetic abnormalities
that appeared more often than expected. Although drug treatments mainly aim at
correcting neurohumoral factors for heart failure, these treatments target the down-
stream pathologies, such as fibrosis. A new approach should consider more upstream
disease.
In the development of drug treatments for idiopathic cardiomyopathy, it is neces-
sary to stratify the pathophysiology in cardiomyopathy according to their causative
genes and develop effective drugs capable of curing upstream disease. In this regard,
basic research using disease-specific iPSCs could be useful.
Alternatively, in order to accelerate the treatment with new drugs, it is necessary
to develop a new drug discovery screening system that is different from the conven-
tional drug discovery methods. A drug screening system using iPSCs is expected be
an efficient drug discovery technology and bring about a revolution concerning
drug-based heart failure therapy.
In this section, we review the current status of research on disease-specific iPSCs
in cardiomyopathy and drug screening using iPSCs.
7.1 Disease-specific iPSCs and drug discovery screening in
cardiovascular disease
Cells differentiated from disease-specific iPSCs are likely to reproduce the original
pathology of the patient on the culture dish. As an example of drug discovery using
iPSCs, disease-specific iPSCs were prepared from patients with fatal osteodysplasia
and achondroplasia and were differentiated to pathological chondrocytes to illustrate
the pathophysiology in vitro. Tsumaki et al. discovered that statins are effective on
fatal osteodysplasia and achondroplasia by use of drug discovery screening using
214 CHAPTER 9 Induced pluripotent stem cells for treatment of heart failure
pathological chondrocytes and achieved the first drug repositioning using iPSCs
(Yamashita et al., 2014). Unlike drug discovery, drug repositioning already has
information on drug toxicity, so it is possible to deliver drugs to patients without
delay. It is expected that drug repositioning using iPSCs will be performed to treat
various diseases.
Concerning cardiovascular diseases, pathological analysis using iPSCs and iPSC
differentiated myocardium has been carried out mainly for cardiomyopathy caused
by genetic abnormalities. In particular, studies have been done on hereditary
arrhythmia disease, such as QT prolongation syndrome, and on the prolongation
of action potential duration and abnormal channel currents in iPSC differentiated
myocardium established from patients with KCNQ1 (Wang et al., 2012), KCNH2
(Matsa et al., 2011;Itzhaki et al., 2011), and SCN5A gene abnormalities have
been reproduced in vitro.
Concerning cardiomyopathy, Myh7 gene abnormality (Lan et al., 2013) and
MYBPC3 gene abnormality (Tanaka et al., 2014) have been reported as models of
hypertrophic cardiomyopathy. The disease phenotypes, including cellular hypertro-
phy, abnormality in sarcomeres, and potassium metabolism in cardiomyopathy, have
been reproduced. There are a wide variety of genes causing dilated cardiomyopathy;
each disease-specific iPSC has been established and their pathological conditions in
diseased myocytes have been analyzed. iPSC differentiated myocardium with
LMNA gene abnormality shows cell senescence and cell death, and iPSC differen-
tiated myocardium with TNNT2 gene abnormality demonstrates decreased contrac-
tility, abnormal intracellular calcium kinetics, and abnormal distribution of
sarcomere constituent proteins (Ebert et al., 2012). In addition, a study of TTN
molecular disruption mutation reported abnormal sarcomeric structure and de-
creases in mechanical stretch and responsiveness to drug stimulation in iPSC differ-
entiated myocardium (Hinson et al., 2015).
Arrhythmogenic right ventricular cardiomyopathy is a rare disease that causes
right ventricular-specific enlargement and fatal arrhythmia. Disease iPSCs from
patients with PKP2 gene abnormalities have been established and reduced expres-
sion of proteins involved in electrical conduction, electrophysiological abnormal-
ities, and intracellular lipid deposition have been reproduced (Caspi et al., 2013;
Ma et al., 2013).
Other authors established disease-specific iPSCs in patients with dilated cardio-
myopathy and found that this abnormality was a point mutation (R173W) of
Troponin T and highly expressed Serca2a. The introduction of beta blockers could
improve the pathology in dilated cardiomyopathy (Wu et al., 2015). It has also
been reported as a method for evaluating cardiotoxicity of drugs using iPSC-CMs
(Liang et al., 2013).
7.2 Drug discovery screening using iPSC-CM tissue
In recent years, research on drug discovery using single cells of iPSC-CMs has been
carried out (van Mil et al., 2018). Screening of single cells has not been achieved by
7. Development of new drug-based heart failure therapy 215
screening for receptors expressed by CMs, cardiomyocyte contractile proteins, and
intracellular signals.
Conversely, the phenotype of heart failure is often due to not only CMs, but also
other factors, such as cardiac fibrosis and in particular the interaction between extra-
cellular matrix and CMs mainly composed of the integrin-laminin system. Consid-
ering the aforementioned characteristics of myocardium, drug screening based on
myocardial tissue consisting of CMs and other cells is necessary to verify an accu-
rate drug response (Fig. 9.3). A recent paper reported creating three-dimensional
myocardial tissue using CMs differentiated from iPSCs to elucidate drug efficacy
and drug toxicity (Zuppinger, 2019). To verify the antifibrotic response of the
cardiac tissue to drugs, experimental myocardial tissue amplified cardiac fibrosis,
showing pathological and functional similarity to dilated cardiomyopathy in vitro.
Three-dimensional myocardial tissues composed of CMs, fibroblasts, and
vascular endothelial cells, which are the main constituents of the myocardium,
have been established and examined to gauge the responsiveness of myocardial tis-
sues to various drugs (Zuppinger, 2019). CMs differentiated from human iPSCs and
fibroblasts were coated with fibronectin and a thin gelatin film on the cell surface,
FIGURE 9.3
Drug screening system using iPSC-derived cardiomyocyte tissue.
(A) Construction of three-dimensional cardiac tissue (3D-CT) using hiPSC-derived
cardiomyocyte (hiPSC-CM) and other types of cells. Abbreviations are: hiPSC, human
iPSC; hiPSC-CM, human iPSCederived cardiomyocyte: and ECM, extracellular matrix,
(B) In vitro pathological model of cardiac fibrosis.
216 CHAPTER 9 Induced pluripotent stem cells for treatment of heart failure
using the alternate layering method to create iPSC-derived cardiac tissue (Takeda
et al., 2018)(Fig. 9.3A). This cardiac tissue showed higher cell density and expres-
sion of extracellular matrix-related and gap junction-related genes compared to
cardiac tissue containing only CMs. In addition, in this artificial myocardium, a
synchronized beat was observed and showed a high contractile performance.
When the HERG K channel blocker E-4031 was administered to this cardiac tissue,
a concentration-dependent drug response was confirmed in these drug screening
models. However, in the cell-mixed model, the response was observed at a lower
concentration compared to the tissue of CMs alone. Three-dimensional myocardial
tissue composed of CMs, fibroblasts, and vascular endothelial cells has excellent
structural and functional properties and better sensitivity to drugs and has already
been demonstrated to be a new drug screening tool (Takeda et al., 2018).
We hypothesized that myocardial tissue using human iPSC-CMs would be useful
as an evaluation system for antifibrotic drugs and conducted the following studies.
Transforming growth factor-beta (TGF-b), a fibrosis stimulating factor, was added
to cardiac tissue comprising fibroblasts and smooth muscle cells. Analysis concern-
ing extracellular matrix (ECM), matrix metalloproteinase (MMP), and other
molecules revealed increased expression of collagen type (Col) I, fibronectin,
MMP-2, and an increased Col1/Col3 ratio (Fig. 9.3B). Furthermore, when the func-
tion of this fibrosis-amplified myocardial tissue was analyzed, the contraction/relax-
ation speed and force were decreased by TGF-bstimulation. Moreover, when
hepatocyte growth factor, which has an antifibrotic effect, was added to this
fibrosis-amplified myocardial tissue, the increase in ECM production and the
decrease in contraction/relaxation rate due to TGF-bwere suppressed (Iseoka
et al. manuscript in preparation). Cardiac tissue derived from human iPSCs can eval-
uate not only changes in ECM production, but also effects on CM function in
response to fibrosis induction or suppression stimulation. This could also provide
a useful evaluation system for the development of antifibrotic drugs as a model of
cardiac fibrosis. In the future, it will be necessary to verify drug efficacy using
cardiac tissue pathologically and functionally similar to dilated cardiomyopathy.
This will help achieve drug repositioning or establish newly developed drugs as
precision medicine.
7.3 Future plans and perspectives in drug screening using iPSCs
The movement away from the use of animals in drug development is occurring glob-
ally and nonclinical studies are being replaced by in vitro studies. Among them,
iPSC-related technology can be developed to international standards. Recent
advances in genetic analysis have revealed that the pathogenesis of a disease varies
greatly depending on the causative gene, and it is necessary to elucidate the patho-
physiology in diseases and develop therapeutic methods based on the causative gene.
Hindering this effort, there is no animal model that accurately verifies drug efficacy
and safety for humans. Drug discovery using human iPSCs may be an alternative to
animal-based drug development. In the future, a new compound screening system
7. Development of new drug-based heart failure therapy 217
that can accurately analyze drug efficacy and safety will be established based on sci-
entific innovations, such as regenerative tissue construction, iPSC technology
(Fig. 9.4), drug discovery technology, and next-generation gene analysis technology.
This system may accelerate drug repositioning by screening for approved drugs and
analyzing the basic pathology of the refractory diseases, creating the possibility to
screen candidate medicine in a huge drug library that fundamentally cures refractory
diseases. This approach may greatly contribute to the establishment of a new treat-
ment system for cardiovascular intractable diseases, mainly cardiomyopathy.
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218 CHAPTER 9 Induced pluripotent stem cells for treatment of heart failure
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Induced pluripotent stem
cells in liver disease 10
M. Teresa Donato
1,2
, Marı
´a Pelecha
´
1
, Laia Tolosa
1
1
Unidad de Hepatologı
´
a Experimental, Instituto de Investigacio
´n Sanitaria La Fe, Valencia, Spain;
2
Departamento de Bioquı
´
mica y Biologı
´
a Molecular, Facultad de Medicina, Universidad de
Valencia, Valencia, Spain
Chapter outline
1. Introduction ................................................................................................ .......226
2. Generation of iPSCs and differentiation into hepatic phenotype .............................227
2.1 Differentiation of iPSCs into hepatocyte-like cells .................................. 228
2.2 Correction of iPSCs ............................................................................. 228
2.3 Differentiation of iPSCs into other hepatic cells ..................................... 229
2.4 Cocultures and 3D cultures of iPSC-derived cells ................................... 230
3. Use of iPSCs in liver disease ..............................................................................231
3.1 Indications of cell therapy for liver diseases........................................... 231
3.2 Bioartificial liver systems to bridge patients to liver transplantation ......... 233
3.3 iPSCs in animal models of liver disease ................................................ 233
3.4 Limitations and challenges of the clinical use of iPSCs .......................... 235
4. iPSC for modeling liver disease ..........................................................................236
5. Hepatotoxicity studies ........................................................................................ 238
6. Conclusions.......................................................................................................241
Abbreviations..........................................................................................................241
Acknowledgments ...................................................................................................241
References .............................................................................................................242
Abstract
The development of suitable and reproducible liver cell models is fundamental
for regenerative medicine, drug screening, and disease modeling. Human pri-
mary hepatocytes are the gold standard not only for hepatic cell therapy but also
for preclinical toxicological screening or the study of liver disease; however,
their limited availability, variability, and phenotypic instability hamper their
use. Recent advances in stem cell technology have allowed the generation of
induced pluripotent stem cells (iPSCs) from different somatic cell types and
their differentiation into hepatocyte-like cells (HLCs), which can provide un-
limited cell source for their use in hepatology studies. We review in detail the
advances in the protocols for generating iPSCs and differentiate them into
CHAPTER
225
Recent Advances in iPSCs for Therapy, Volume 3. https://doi.org/10.1016/B978-0-12-822229-4.00011-5
Copyright ©2021 Elsevier Inc. All rights reserved.
HLCs and the application of these toolsindifferentfieldssuchaslivercell
transplantation, drug-induced liver injury, or to model liver disease.
Keywords:
3D models; Acute liver failure; Animal models; Cell transplantation; Clinical
translation; Differentiation; Disease modeling; End-stage liver disease; Hepatocyte-like cells;
Hepatocytes; Hepatotoxicity studies; iPSCs; Liver cells; Liver-based inborn metabolic errors; Safety
assessment.
1.Introduction
The liver is the largest organ in the body with many specific functions such as plasma
protein secretion, carbohydrate and lipid metabolism, bile acid and urea synthesis,
metabolic homeostasis or detoxification, and vitamin and carbohydrate storage
(Chen et al., 2018). Although liver has a considerable regenerative capacity, chronic
liver diseases caused by genetic disorders, viral infections, or drug-induced pertur-
bations are a worldwide public health threat that can lead to end-stage liver disease
(ESLD) (Kakinuma and Watanabe, 2019). The only effective and curative treatment
of ESLD is organ transplantation although it is limited by the availability of organs
among other limitations.
Primary human hepatocytes (PHHs) are the standard cells used for both liver cell
transplantation and in vitro studies (Soldatow et al., 2013). Cultured PHHs maintain
key hepatic-specific functions (Gomez-Lechon et al., 2014) and have been exten-
sively used as a tool for anticipating drug metabolism and for assessing the risk
of drug hepatotoxicity in human (Gomez-Lechon et al., 2008;Gomez-Lechon
et al., 2004;Hewitt et al., 2007;O’Brien et al., 2004,2006) as well as in disease
model (Caron et al., 2019) and for cell transplantation (Pareja et al., 2020;Tolosa
et al., 2016). However, the use of primary hepatocytes is hindered by the increasing
shortage of suitable donor livers for hepatocyte isolation as well as by the insuffi-
cient functional quality, drastic loss of hepatic functionality over time (particularly
drug-metabolizing capability) (Godoy et al., 2013;Gomez-Lechon et al., 2004,
2008;Hewitt et al., 2007;Soldatow et al., 2013), and great susceptibility to cryopres-
ervation and thawing of hepatocytes (Fox and Chowdhury, 2004;Hansel et al., 2016;
Iansante et al., 2018).
Research on induced pluripotent stem cells (iPSCs), which are adult cells that have
been genetically reprogrammed to an embryonic stem cellelike state, points to them
as an attractive alternative to the use of primary cells. iPSCs can differentiate into all
cell types of the body, including liver cells, and their ability of self-renewal and in vitro
expansion makes them a very promising cell source for generating suitable functional
hepatocyte-like cells (HLCs) (Forbes et al., 2015;Nicolas et al., 2017;Singh et al.,
2015;Yu et al., 2014). A huge number of HLCs could be made readily available to
any patient on an as-needed basis for hepatic cellebased therapies, both for pro-
grammed treatment for liver-based metabolic disorders and emergency use in patients
with acute liver failure (ALF), acute-on-chronic liver failure (ACLF), or ESLD
(Hannan et al., 2013;Nicolas et al., 2017;Singh et al., 2015;Tolosa et al., 2016;
226 CHAPTER 10 Induced pluripotent stem cells in liver disease
Yu et al., 2014), but also these HLCs could be a useful cell source for development of
models and drug discovery (Chen et al., 2018).
In this chapter, we will explain the different strategies recently describedtorepro-
gram somatic cells to the pluripotency, their differentiation to HLCs, and their potential
use to provide a real prospect of making unlimited numbers of HLCs available to:
(i) provide cell-based therapy for liver diseases (both hereditary liver diseases and in
other patients with liver disease as an alternative to organ transplantation); (ii) develop
new models for the study of liver disease; and (iii) use them for drug discovery and pre-
clinical studies of drug-induced liver injury (DILI) (Fig. 10.1).
2.Generation of iPSCs and differentiation into hepatic
phenotype
Since the first generation of iPSCs from somatic cells by Yamanaka (Takahashi
et al., 2007;Takahashi and Yamanaka, 2006), researchers have focused mainly on
two aspects: methods to induce somatic cell reprogramming and which somatic cells
FIGURE 10.1 Use of iPSC in hepatology.
iPSCs can be produced from different cell types obtained from patients and
reprogrammed into a pluripotent state. These cells can be genetically corrected and
differentiated into hepatocyte-like cells (HLCs) or directly differentiated. HLCs can be
used in drug screening, cell therapy, or disease modeling.
The figure was created with BioRender.
2. Generation of iPSCs and differentiation into hepatic phenotype 227
to reprogram. Currently, iPSCs can be obtained from different cell sources (fibro-
blasts, blood cells, urine cells, or amnion cells) for a review (Chhabra, 2017;
Gerbal-Chaloin et al., 2014) and through distinct approaches (integrative and non-
integrative methods) that have been previously reviewed in detail (Yu et al., 2014).
Gene editing of patient-derived iPSCs for the correction of underlying genetic
defects has been also proposed in the case of using these cells for autologous trans-
plantation, which would reduce problems of immune rejection and also for modeling
disease (Zhang et al., 2011). In this case, safety assessment should be more exhaus-
tive since gene editing tools can generate off-target effects.
2.1 Differentiation of iPSCs into hepatocyte-like cells
Several protocols that mimic liver development stages and that normally include
three basic steps have been described to produce HLCs from both embryonic
stem cells (ESCs) and iPSCs (Hannan et al., 2013;Tolosa et al., 2015). The first
step uses Activin A, BMP4, LY294002, and Wnt3a to induce endoderm; as a second
step the hepatic specification is achieved through the addition of FGF2, FGF4, and
BMP4 to get bipotential cells called hepatoblasts that can differentiate into both he-
patocytes and cholangiocytes. Finally, the hepatic differentiation and maturation us-
ing HGF and oncostatin M allow for obtaining HLCs. In the recent years, in addition
to the use of different combinations of growth factors, the addition of small mole-
cules (Asumda et al., 2018) or microRNAs (Deng et al., 2014), the coculture with
other cell types (Takebe et al., 2014), and the use of 3D configurations (Gieseck
et al., 2014) have been also proposed to improve the hepatic differentiation of iPSCs.
Numerous studies have demonstrated that both ESCs and iPSCs can be differen-
tiated into HLCs; however, the characterization of the HLCs sometimes is only
based on the analysis of the expression of few hepatic markers by means of immu-
nofluorescence or RT-PCR and does not include functional analysis. Hepatocytes
perform many essential functions, including ureogenesis, metabolic homeostasis,
or detoxification (Chen et al., 2018), so, depending on their subsequent use, the char-
acterization should focus on specific functions. For instance, in the case of DILI, the
cytochrome P450 (CYP) activities should be specifically assessed since many drugs
require bioactivation, whereas in the case of the clinical use of the cells, specific
functions depending on the disease of the selected patient should be covered.
2.2 Correction of iPSCs
For patients with monogenic inherited metabolic diseases, gene correction should be
applied if the derived HLCs are thought to be used in cell transplantation or for the
understanding of the disease. The most widely used tools for genome editing are
zinc-finger endonucleases, transcription activator-like effector nucleases, and clus-
tered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-
associated system (Cas9) (reviewed by Chang et al., 2018). For instance, in a study
of Omer et al. (2017), the CRISPR/Cas9 system was used to correct an LDLR
228 CHAPTER 10 Induced pluripotent stem cells in liver disease
mutation of iPSCs derived from a patient with hypercholesterolemia. In this case, the
genetic correction restored LDLR-mediated endocytosis (Omer et al., 2017).
Gene editing has shown to be a useful potential tool for the application of patient-
derived iPSCs for the correction of underlying genetic defects that could allow the
autologous transplantation and, thus, reduce problems of immune rejection.
2.3 Differentiation of iPSCs into other hepatic cells
Although hepatocytes are the major cell type of the liver and account for the approx-
imately 80% of the hepatic volume and quantity, the liver is composed by other
parenchymal (cholangiocytes) and nonparenchymal cells (Kupffer cells (KCs), liver
sinusoidal endothelial cells, and hepatic stellate cells (HSCs)) that also play impor-
tant functions in the liver (Fig. 10.2). Their important role in disease has also been
described.
On the one hand, cholangiocytes are biliary epithelial cells that constitute the
biliary tree. Their main function is the modification and transport of bile, which
is crucial for the removal of waste products of the body such as cholesterol, bili-
rubin, hormones, or toxins from the liver (Maepa and Ndlovu, 2020). As
described before, as hepatocytes, they are derived from hepatoblasts, so the first
FIGURE 10.2 Differentiation of induced pluripotent stem cells (iPSCs) into hepatic cells.
The in vitro differentiation of iPSCs into different parenchymal (hepatocytes,
cholangiocytes) and nonparenchymal (Kupffer cells, liver sinusoidal endothelial cells, and
hepatic stellate cells) cells, which play important specific roles in the liver, has been
described.
The figure was created with BioRender.
2. Generation of iPSCs and differentiation into hepatic phenotype 229
differentiation steps are common with the protocols of hepatocyte differentiation. From
this point, several protocols have been described for the differentiation of pluripotent
stem cells to cholangiocytes using factors such as EGF, IL-6 (Dianat et al., 2014), or
the modulation of Notch signaling (Sampaziotis et al., 2015). It has been shown that
cholangiocytes-like cells derived from patients can recapitulate disease (i.e., cystic
fibrosis) in vitro and validate drug efficacy (Ogawa et al., 2015;Sampaziotis et al.,
2015).
On the other hand, the differentiation into nonparenchymal cells has been also
described. KCs are liver resident macrophages with phagocytic and scavenger func-
tions that play an important role in diseases such as fibrosis, viral hepatitis, steato-
hepatitis, or DILI (Maepa and Ndlovu, 2020). The differentiation of iPSCs into KCs
by adding BMP4, VEGF, and IL-3 among other factors (Tasnim et al., 2019) has
been recently described. These KCs were able to perform phagocytosis and secrete
cytokines (Tasnim et al., 2019), so HLCs and KCs could be derived from the same
patient and used for modeling disease but also used for regenerative purposes.
Liver sinusoidal endothelial cells (LSECs) are functionally unique because of
the high activity of receptor-mediated endocytosis and their role in liver regen-
eration after injury (Chen et al., 2020). Different groups have differentiated
pluripotent cells into different endothelial cells or have used HUVECs, although
up to the moment only one group has described the differentiation of iPSCs into
LSCEs (Koui et al., 2017).
Finally, under physiological conditions, HSCs have as a main function the stor-
age and transport of vitamin A. However, when liver is damaged, HSCs activate and
change into an activated state, which promotes the secretion of the extracellular ma-
trix (ECM) involved in liver injury repair (Chen et al., 2020). Coll et al. (2018)
described the differentiation of iPSCs into HSCs by adding BMP4, FGF1, FGF3,
and palmitic acid and suggested them in combination with HLCs as a model for
the study of liver fibrosis (Coll et al., 2018).
2.4 Cocultures and 3D cultures of iPSC-derived cells
In previous decades, liver studies have focused on 2D cell cultures; however, actual
liver tissue is 3D, and therefore, to address the limitations of 2D cell cultures,
researchers have focused on creating 3D microenvironments that mimic native envi-
ronment (Chen et al., 2020). The use of a 3D system offers the advantage to better
preserve cell-to-cell interactions and self-aggregate to reproduce the cellular organi-
zation of the organ. The engineered scaffolds (decellularized organs, natural macro-
molecules, or synthetic biodegradable polymers) can provide a 3D environment for
cell attachment, proliferation, and maturation.
Functional and mature organoids are preferable for regenerative medicine pur-
poses (Kuse and Taniguchi, 2019). In this sense, Takebe et al. (2013) succeeded
in generating a three-dimensionally vascularized liver by coculturing human
iPSCsederived hepatic endoderm cells with human umbilical vein endothelial cells
(HUVECs) and mesenchymal stem cells (MSCs) (Takebe et al., 2013). Another
230 CHAPTER 10 Induced pluripotent stem cells in liver disease
interesting approach is organ-on-a-chip technology, which enables perfusion and
cell interactions in a microfluidic device and allows a better metabolic performance
of the cells (Schepers et al., 2016).
3.Use of iPSCs in liver disease
Cell-based liver therapies are foreseen as a valuable therapeutic option to replace or
complement whole organ transplantation by recovering and stabilizing the lost meta-
bolic function for acute and chronic liver diseases. However, the scarce availability
of liver tissue to isolate good-quality cells, the low engraftment capability of the
cells into the host liver mainly due to the rejection of transplanted cells as well as
the difficulties to monitor and predict rejection (Soltys et al., 2017) limit its wider
application. Human iPSCs differentiated into HLCs could be used as autologous
cell therapies that would avoid immune rejection but that in some cases could
require gene correction and/or help to create biobanks of readily available HLCs
for the emergency treatment of ALF.
3.1 Indications of cell therapy for liver diseases
ESLD and life-threatening metabolic defects are a major cause of mortality world-
wide and imply one of the highest healthcare burdens (Tolosa et al., 2016; Williams,
2006; Blachier et al., 2013). The only effective and curative treatment for these irre-
versible diseases is organ transplantation; however, the shortage of organs has led
transplant centers to extend their criteria and accept marginal donors. Nevertheless,
the demand for organs and the mortality in the waiting list increase, and there is an
obvious need for alternative treatments to support life until an organ is available or
endogenous liver regenerates, avoiding organ transplantation (Viswanathan and
Gupta, 2012). From a clinical point of view, transplantation of PHH or HLCs derived
from iPSCs could signify an alternative to whole organ transplantation for correcting
genetic disorders that result in metabolically deficient states, in ALF or ESLD, or for
maintaining liver function in patients who do not meet the clinical eligibility criteria
to be candidates for liver transplantation due to different reasons (i.e., advanced age,
other diseases, or cardiovascular factors). The wider application of cell transplanta-
tion would imply patient morbidity, improved quality of life, and better survival
rates (Tolosa et al., 2016). Table 10.1 summarizes the diseases in which hepatocyte
transplantation has been clinically applied in humans.
Liver-based inborn metabolic disorders are rare diseases that are caused by de-
fects in hepatic enzymes or proteins with metabolic functions, such as receptors
or transporters. Liver transplantation is not always the first therapeutic option in chil-
dren due to invasiveness, recipient’s age, or the need of lifelong immunosuppressive
therapy (Hansel et al., 2014;Jorns et al., 2012). For those patients, cell transplanta-
tion could be an appropriate treatment to provide the missing liver function without
replacing the whole organ and avoiding its side effects. In fact, hepatocyte
3. Use of iPSCs in liver disease 231
transplantation has been used in pediatric patients with distinct kinds of inborn he-
patic metabolic disorders (Hansel et al., 2014;Jorns et al., 2012) with encouraging
results (Table 10.1). In this case, the use of autologous cells would imply genetic
correction and more safety assessment than if no correction is required, but the
avoidance of immune rejection has led researchers to point them as an appealing op-
tion in regenerative medicine.
On the other hand, recently, the use of cell therapy to reverse the fibrosis and
inflammation in nonalcoholic fatty liver disease (NAFLD) has been proposed
Table 10.1 Indications of cell therapy for liver diseases.
Liver diseases with indication of
cell therapy References
Inborn metabolic disorders
❖CriglereNajjar syndrome type 1
❖Urea cycle defects
- Ornithine transcarbamylase
- Carbamoyl phosphate synthetase
type 1 deficiency
- Citrullinemia
- Argininosuccinate lyase deficiency
❖Glycogen storage disease type I
- Type Ia
- Type Ib
❖Refsum disease
❖Phenylketonuria
❖Tyrosinemia type 1
❖Factor VII deficiency
❖Primary hyperoxaluria
❖Familial hypercholesterolemia
❖Progressive familial intrahepatic
cholestasis
Ambrosino et al. (2005), Ribes-Koninckx et al. (2012)
Legido-Quigley et al. (2009),Meyburg et al. (2009),
Ribes-Koninckx et al. (2012),Stephenne et al.
(2005, 2006)
Defresne et al. (2014),Lee et al. (2007),Muraca
et al. (2002),Ribes-Koninckx et al. (2012),
Stephenne et al. (2012)
Sokal et al. (2003)
Soltys et al. (2017),Stephenne et al. (2012)
Ribes-Koninckx et al. (2012)
Dhawan et al. (2004),Quaglia et al. (2008)
Guha et al. (2005)
Barahman et al. (2019),Caron et al. (2019)
Brinkert et al. (2018),Quaglia et al. (2008)
Other diseases
❖Patients with indication of LT
- Acute liver failure
- Acute on chronic liver failure
- Nonalcoholic steatohepatitis
❖Patients with no indication of LT
- Patients with postoperative liver
failure after partial hepatic
resection
- Patients with chronic
decompensation of an end-stage
liver disease
- Other Patients with no indication
of LT (i.e., advanced age,
alcoholism)
Bilir et al. (2000),Fisher and Strom (2006),He
et al. (2019),Li et al. (2016),Pareja et al. (2013),
Starkey Lewis et al. (2019),Strom et al. (1999)
Habeeb et al. (2015),Liang et al. (2017),Mito
et al. (1992)
232 CHAPTER 10 Induced pluripotent stem cells in liver disease
(Pais et al., 2016). Another possible indication of liver cell transplantation would be
as a metabolic support in patients with ALF or ACLF, which could serve as a bridge
to liver transplantation (Hansel et al., 2014). Moreover, patients with ESLD and no
indication of LT could benefit from cell therapy, which could delay disease progres-
sion (Lai et al., 2015)(Table 10.1).
3.2 Bioartificial liver systems to bridge patients to liver
transplantation
As explained before, current efforts aim to recapitulate the tridimensional organiza-
tion of the liver by using multiple cell types and both synthetic compounds or extra-
cellular matrices from decellularized livers (Mazza et al., 2015) with the idea of
transferring them to the clinical practice.
On the one hand, the decellularization of whole organs and subsequent reseeding
with relevant cell types (Mazza et al., 2015) would allow the construction of
implantable organs. With this approach, the vascular framework is retained, which
is a major advantage, although the high number of cells needed is a key limitation.
Liver support systems are extracorporeal devices that support the regeneration of
a patient’s liver and buy time until a suitable donor organ is available (Lee et al.,
2015). There are nonbiological and bioartificial liver (BAL) support systems that
have been produced by several companies. For instance, a BAL device prevented ce-
rebral manifestations of ALF in an allogeneic model of drug-overdose ALF (Glori-
oso et al., 2015).
Finally, liver bioprinting implicates the accurate construction of complex
parenchymal organ structures, including the vascular network (Lee et al.,
2015). 3D bioprinting enables the fabrication of biomimetic tissues with the pre-
cise delivery and placement of living cells, biomaterials, and growth factors,
which can be printed either in a specific pattern for the development of the final
tissue structure or on an already existing 3D matrix (scaffold) (Kryou et al.,
2019). The most commonly used techniques for bioprinting are laser-based tech-
niques, inject printing, and microextrusion printing (Kryou et al., 2019). BAL is
considered one of the most promising tools as a therapeutic method for severe
liver diseases and in the field of regenerative medicine that will progress in the
future years.
3.3 iPSCs in animal models of liver disease
Despite the fact that up to the moment iPSC-HLCs have not been used in humans,
their efficacy has been proved in different animal models of liver disease, mainly
mice with induced acute or chronic liver failure although iPSC-HLCs have been
also used in models of metabolic deficiencies (Table 10.2).
One of the first studies in showing the in vivo efficacy of HLCs derived from
iPSCs used a liver failure model with dimethylnitrosamine (DMN) (Liu et al.,
2011). Liu et al. demonstrated the engraftment of cells comparable to PHH as
3. Use of iPSCs in liver disease 233
well as the increased survival of the animals treated with iPSC-HLCs, indicating
their suitability for their clinical translation.
In order to recapitulate early organogenesis, as said before, Takebe et al. (2013)
cultivated hepatic endoderm-iPSCs with HUVECs and MSCs that self-organized
Table 10.2 Use of human iPSCederived hepatocyte-like cells for the
treatment of liver disease in different animal models.
Cells Animal model Results Reference
Human iPSC-HLCs Mice with
chronic liver
failure induced
with DMN
Increased survival,
engraftment, and
repopulation
Liu et al.
(2011)
Human iPSC-HLCs Mice with
chronic liver
failure induced
with CCl4
Reduction of bilirubin
and LDH levels,
reduction of fibrosis,
increased survival rate
Asgari et al.
(2013)
Human organ buds
generated from iPSCs,
MSCs, and HUVECs
Mice with
ganciclovir-
induced liver
failure
Vascularization of organ
buds, increased survival
Takebe
et al. (2013)
Human iPSC-derived
hepatocyte sheet
Mice with CCl4-
induced acute
liver failure
Increased survival and
albumin levels in
transplanted animals
Nagamoto
et al. (2016)
Human iPSC-HLCs Mice with acute
and chronic liver
failure induced
with CCl4
Increased survival rate in
acute model and
reduction of fibrosis in
chronic
Takayama
et al. (2017)
Human liver organoids
generated from single
donor-derived iPSC
endoderm, endothelial
cells, and mesenchymal
cells
Mice with acute
liver failure
induced with
diphtheria toxin
Improved survival rate Nie et al.
(2018)
Human iPSC-HLCs Hemophilia B
model (factor IX
deficient mice)
Shorter bleeding in
transplanted animals
Okamoto
et al. (2018)
Human iPSC-HLCs
spheroids
Partial
hepatectomy
(50%) and Fah
/mice
Body weight recovery
and decrease of liver
damage markers
Rashidi
et al. (2018)
Mouse TALEN-corrected
iPSCs differentiated into
HLCs
Arginase-1
deficient mice
Extended survival and
recovery of the
expression of Arginase
expression in liver
Sin et al.
(2018)
Human iPSC-HLCs Gunn rat
(UGT1A1
deficient model)
Decrease of
hyperbilirubinemia,
bilirubin glucuronidation
Fourrier
et al. (2020)
234 CHAPTER 10 Induced pluripotent stem cells in liver disease
and showed a high metabolic capacity and performed specific liver functions
(Takebe et al., 2013). Additionally, the mesenteric transplantation of the liver
buds derived from iPSCs rescued the mice with drug-induced lethal liver failure
(Takebe et al., 2013).
From then, iPSC-HLCs have been used as single cells (Asgari et al., 2013;
Okamoto et al., 2018) or in 3D structures (sheets or organoids) (Nagamoto et al.,
2016;Rashidi et al., 2018) in animal models of acute or chronic liver failure and
in models of metabolic liver disease (Table 10.2), with heartening results, and since
different clinical trials are ongoing for other diseases, it is foreseen that this could be
applicable in the future years.
3.4 Limitations and challenges of the clinical use of iPSCs
Before the clinical application of iPSCs-derived HLCs, some important safety issues
such as long-term safety, tolerability, and immunogenicity should be addressed. The
first thing to consider is the fact that reprogramming itself can induce both genetic
and epigenetic defects in iPSCs, and it is possible that those defects can directly or
indirectly promote immunogenicity and tumorigenicity in vivo. In fact, it has been
recently reported that the genomic translocation detected in the iPSCs will create
fusion proteins and new immunogenic determinants (Yoshihara et al., 2017). iPSCs
are characterized by their self-renewal ability and pluripotency that make them a
promising tool in regenerative medicine, but make them also responsible for a
considerable tumorigenic potential (Laurent et al., 2011).
On the other side, cell therapy for hereditary liver diseases with patient-specific
iPSC-derived HLCs would require gene correction before or after reprogramming.
In this case, personalized cell therapy with patient’s iPSCs would avoid rejection,
and thus immunosuppression, although the immunogenicity of iPSCs and their de-
rivatives is still divisive (Araki et al., 2013;Zhao et al., 2011). In fact, differential
immune recognition between differentiated and undifferentiated pluripotent cells
has been demonstrated (Guha et al., 2013;Saljo et al., 2017;Tan et al., 2014). iPSCs
are epigenetically abnormal and inherited epigenetic signature of parental cells
could explain abnormal expression of immunogenic proteins expressed during the
iPSCs differentiation (Zhao et al., 2015). Moreover, undifferentiated iPSCs present
a lower expression of MHC class I and the complete absence of MHC class II anti-
gens compared to the differentiated cells (Chen et al., 2015).
In the case of the use of iPSCs-derived cells for the treatment or genetic meta-
bolic defects, genome editing is mandatory; however, due to the possible off-
target effects (edits in the wrong place) and mosaicism (the fact that some cells
are edited but others do not), safety is a major concern. In this sense, the enhance-
ment of efficiency and safety of genome editing will provide cell-based therapies
closer to the clinic for patients with inborn metabolic diseases (Haeussler et al.,
2016).
When talking about cell therapy, short- and long-term engraftment of the trans-
planted cells is major limitation, and many researchers have focused on the
3. Use of iPSCs in liver disease 235
improvement of this key issue through different strategies. iPSC-HLCs could have a
proliferative advantage over PHH, although other combined strategies such as the
partial reversible embolization of the portal vein (Dagher et al., 2009) or irradiation
of the native liver that increase homing should be also taken into account to improve
the clinical results.
Unlike other applications, the clinical use of HLCs derived from iPSCs requires
working under good manufacturing practices (GMP) guidelines (Asgari et al., 2010)
for both reprogramming of cells into a pluripotent state and differentiation into the
hepatic phenotype. Another challenge for the clinical transplantation of iPSC-HLCs
is to achieve enough material for transplantation and the evident remarkable cost of
getting enough tissue mass to maintain the hepatic functionality (Habka et al., 2015),
since a total of 2.0 10
8
viable cells/kg for each patient has been proposed as an
optimal and safe dose in humans (Soltys et al., 2017).
4.iPSC for modeling liver disease
The understanding of liver diseases is limited by the lack of appropriate disease
models that reveal the pathophysiology of chronic liver disease although genetically
modified animal models have helped to elucidate their mechanisms (Kakinuma and
Watanabe, 2019). New approaches are being developed in order to achieve more
patient-specific and ethical permissive human cellebased models, which mimic pa-
thologies in the liver in an accurate way. Specially, iPSCs differentiated into HLCs
offer a promising option for liver disease modeling in vitro allowing the study of the
mechanisms implicated and disease progression.
Many research groups have used HLCs for mimicking several liver diseases,
and in some cases, genetic editing tools have been used to correct them in order
to revert the pathology (Table 10.3). For instance, HLCs have been used to study
monogenic diseases (caused by a single mutation). It has been possible to obtain
a1-Antitrypsin (A1AT) deficiency disease cell model using HLCs from patients
andcontrols.A1ATdeficiencyisaninherited liver disease caused by a mutation
on SERPINA1, the gene that codes A1AT protein. This mutation generates a mis-
fold A1AT protein that accumulates inside the hepatocytes causing liver damage.
Rashid et al. generated HLCs from control and patients that retained the polymer
in their cytoplasm (Rashid et al., 2010). Yusa et al. corrected the mutation in
iPSCs from patients with zinc-finger nuclease approach. Corrected iPSCs
were differentiated into HLCs whose A1AT accumulation was similar to PHH
(Davidson et al., 2015;Yusa et al., 2011).
Wilson disease (WD) cell model is another example where HLCs recreate pa-
thology conditions of a human liver disease. The mutation of ATP7B gene causes
the abnormal accumulation of copper, which activates Fenton chemical reaction pro-
ducing hepatitis with progressive cirrhosis or ALF. Zhang et al. used HLCs from
WD patients and controls. HLCs from WD patients exhibited a copper-export defect,
the hallmark of the disease (Zhang et al., 2011). The treatment of the HLCs obtained
236 CHAPTER 10 Induced pluripotent stem cells in liver disease
Table 10.3 HLCs-derived iPSCs used for disease modeling.
Disease Disease hallmarks HLC model phenotype References
a1-Antitrypsin
(A1AT)
deficiency
disease (ATD)
•Mutation in SERPINA1 gene
•A1AT protein accumulation within hepatocytes
•“Proteotoxicity” in hepatocytes
•ATD-HLCs retain A1AT polymers
•Physiological phenotype rescued by gene editing
tools
Rashid et al.
(2010),Yusa
et al. (2011)
Familial
transthyretin
amyloidosis
(FTA)
•Transthyretin (TTR) gene mutation
•TTR, secreted as a tetramer from the liver, dissociates
to monomers and forms insoluble fibrils, which
accumulates in downstream target tissues
•TTR-HLCs cause a viability decrease of car-
diomyocytes and neurons
Giadone et al.
(2018)
Glycogen
storage disease
type 1a
(GSD1a)
•Glucose-6-phosphatase (G6Pase) gene mutation
•Inhibition of glucose releasing from the liver
•Glycogen and lipids metabolism impairment
•GSD1-HLCs accumulate glucose and increase
glycogen and lipids
Rashid et al.
(2010)
Alperse
Huttenlocher
Syndrome
(AHS)
•Mutations in mitochondrial DNA (mtDNA) polymerase
g(POLG)
•AHS patients are susceptible to suffer valproic acid
(VPA) hepatotoxicity
•Mutated pol ghas a reduced ability to replicate DNA
resulting in a reduced number of copies of mtDNA
•AHS-HLCs are more susceptible to
mitochondria-dependent apoptosis
•VPA treatment increases mitochondrial-
dependant apoptosis
Li et al. (2015)
Alcoholic liver
disease (ALD)
•Chronic alcohol consumption leads to ALD
•ALD progression implies steatosis to steatohepatitis,
cirrhosis, and hepatocellular carcinoma
•Lipid accumulation when HLCs are exposed to
ethanol
•In the presence of ethanol, hepatic progenitor
cells are especially sensitive to apoptosis
Tian et al. (2016)
Nonalcoholic
Fatty Liver
Disease
(NAFLD)
•NAFLD progression includes two hits: the first implies
benign lipid droplets accumulation (steatosis), whereas
the second is characterized by nonalcoholic
steatohepatitis (NASH) with inflammation and fibrosis
•Lipid overload with acute-induced ER stress
develops a steatotic phenotype in HLCs
Graffmann et al.
(2016),Parafati
et al. (2018)
Hepatitis C Viral
(HCV)
Hepatitis B Viral
(HBV)
•HCV and HBV destroy the self-regenerating ability of
the liver
•Serious associated liver diseases: cirrhosis and
hepatocellular carcinoma
•HLCs are permissive to HCV and HBV infection
•With HCV infection, HLCs express known HCV
host factors, including the liver-specific
microRNA-122 (miR-122) and entry factors
•HLCs could be infected by HBV-positive serum
Shlomai et al.
(2014),Wu et al.
(2012)
Malaria •Plasmodium spp. Sporozoite infects hepatocytes
where it develops into merozoite, which infects red
blood cells
•HLCs are more susceptible to plasmodium
infection with increasing state of cell
differentiation
Ng et al. (2015)
4. iPSC for modeling liver disease 237
from WD patients with pharmacological chaperone curcumin resulted in the resto-
ration of the location of ATP7B (Zhang et al., 2011).
Some liver metabolic disorders such as NAFLD have also been mimicked us-
ing HLCs. When HLCs are incubated with oleic acid and palmitic acid combined
with acute induced-endoplasmic reticulum stress developed a steatosic pheno-
type, characteristic of NAFLD (Parafati et al., 2018). With this model, it has
been possible to identify genes that change in NAFLD such as PLIN2, which
is upregulated, and it has been considered a suitable marker for successful induc-
tion of steatosis (Graffmann et al., 2016;Parafati et al., 2018).
Hepatotropic infectious diseases have been already studied using HLCs. For
instance, protocols for recreating hepatitis C (HCV) on HLCs have been standard-
ized and well-recapitulated. Currently, important advantages are being developed
in order to get a hepatitis B viral (HBV)-HLCs disease model. HLCs could be
infected by HBV-positive serum, but they do not have the same infection capability
as PHH (Wu et al., 2012).
AlperseHuttenlocher syndrome (AHS) is a disorder caused by mutations in
mitochondrial DNA polymerase gamma. AHS patients are susceptible to suffer val-
proic acideinduced hepatotoxicity. Li et al. (2015) recreated AHS features on HLCs
from AHS patients as the first toxicity model in a genetic disease. In AHS-HLCs,
reactive oxygen species (ROS) are increased due to transient openings of mitochon-
drial permeability transition pore (mPTP) soaring the mitochondria-dependent
apoptosis susceptibility. When AHS-HLCs are treated with valproic acid, more
mitochondrial-dependent apoptosis by mPTP opening is induced compared with
normal HLCs, relating the specific toxicity of the drug to AHS patients. Li et al.
also identified an mPTP inhibitor that counteracts the toxicity effects of valproic
acid. This model opens the possibility to use HLCs for drugs assessments in thera-
peutic research (Li et al., 2015).
5.Hepatotoxicity studies
DILI is a serious pathological condition that represents a relevant entity in med-
ical practice due to its significant morbidity and mortality. Many drugs in current
use, herbs, and dietary supplements have been associated to liver damage
(Lucena et al., 2008). DILI is becoming a major public health problem in West-
ern countries that has replaced viral hepatitis as the most frequent cause of acute
liver failure resulting in patient death or the need for liver transplantation (Lee,
2003;Ostapowicz et al., 2002;Wei et al., 2007). It is also a frequent cause of
adverse drug reactions leading to drug failure in preclinical and clinical trials
and the mean reason for withdrawal or restriction of prescription after drug
approval (Guengerich, 2011). For a few drugs (e.g., acetaminophen) hepatotox-
icity is dose-dependent and predictable for the majority of patients; however, for
most drugs, DILI episodes are due to idiosyncratic or unexpected reactions
occurring in a minority of susceptible patients at doses safe for the general
238 CHAPTER 10 Induced pluripotent stem cells in liver disease
population (Bjornsson, 2015;Gomez-Lechon et al., 2016). Clinically, DILI en-
compasses a wide spectrum of presentations of variable severity and may mimic
any form of liver disease (acute and chronic hepatitis, steatohepatitis, phospho-
lipidosis, sinusoidal obstruction, cirrhosis, etc.) (Lucena et al., 2008).
Human DILI is poorly predicted in animals, likely due to the well-known inter-
species differences in drug metabolism, pharmacokinetics, and toxicity targets
(Olson et al., 2000). Particularly, idiosyncratic hepatotoxicity is difficult to repro-
duce in animal models as many specific genetic and nongenetic patient factors
may contribute to these rare toxic reactions (Gomez-Lechon et al., 2016). Diverse
cell models extending from primary hepatocytes or hepatoma cell lines cultured
as 2D monolayers to emerging 3D culture systems or stem cellederived hepatocytes
have been proposed for hepatotoxicity studies (Gomez-Lechon et al., 2014). Despite
their potential limitations, in vitro cell-based toxicity assays provide quick and valu-
able mechanistic information on DILI.
In recent years, iPSCs have emerged as an attractive and limitless source of
cells for toxicity purposes. Transcriptomic, proteomic, and functional analysis
revealed that iPSC-derived hepatocytes exhibited many hepatic-specific charac-
teristics; however, they show a poor expression of many drug metabolizing en-
zymes and transport proteins compared to primary hepatocytes, which may
question their suitability for drug safety assessments (Baxter et al., 2015).
Despite the fact that iPSC-derived hepatocytes do not display a complete mature
hepatic phenotype, their potential to study drug-induced hepatotoxicity has been
extensively examined (Grimm et al., 2015;Lu et al., 2015;Sirenko et al., 2016;
Ware et al., 2015). Several studies evidenced that the phenotypic characteristics
of iPSC-derived HLCs resemble PHH more closely than most hepatoma cell lines
(Kang et al., 2016;Ulvestad et al., 2013). Consistent with these findings, their
response to model hepatotoxic compounds known to cause DILI through several
mechanisms of toxicity is comparable to that of PHH, showing higher correla-
tions than HepG2, the hepatoma cell line most widely used for hepatotoxicity as-
sessments (Kang et al., 2016;Lu et al., 2015;Pradip et al., 2016). Interestingly, it
was shown that gene expression profile and metabolic activities of HLCs
remained relatively stable over time in culture allowing long-term toxicity
studies (Holmgren et al., 2014;Ulvestad et al., 2013), in contrast to the sponta-
neous dedifferentiation and rapid loss of enzyme and transporter activities
observed in PHH in conventional monolayer cultures. Using a repeated-dose
approach for 14 days, drug-induced cytotoxic, phospholipidosic, and steatosic
effects were evidenced in HLCs, suggesting their utility for chronic toxicity
testing (Holmgren et al., 2014).
As indicated earlier, iPSC-derived hepatocytes show a relative low expression
of drug-metabolizing enzymes, particularly CYP enzymes. Efforts have been
made to improve maturation of iPSC-derived HLCs to obtain cells that recapit-
ulate phenotypic features of adult hepatocytes. An active area of research is
focused on the application of complex culture systems to better mimic in vivo
microenvironment of the liver. Promising results obtained using iPSC-HLCs
5. Hepatotoxicity studies 239
3D spheroids or micropatterned cocultures evidenced their potential as in vitro
platforms for high-throughput hepatotoxicity screenings (Sirenko et al., 2016;
Ware et al., 2015).
Takayama et al. (2018) reported that iPSC-HLC reproduced interindividual
differences in drug metabolism and toxicity that are due to genetic polymor-
phisms in drug-metabolizing enzymes (Takayama et al., 2018). Then, the avail-
ability of iPSC from different donors opens the possibility of generating a library
of HLCs representative of genotypic/phenotypic variability of human popula-
tion. Thus, it is possible to obtain cells reflecting a range of sensitivities to toxic
effects for more accurate and efficient preclinical safety assessments of new
drugs. This is particularly attractive for idiosyncratic DILI (iDILI), enabling
studies aimed to identify individuals or groups of patients at risk of idiosyncratic
reactions induced by a given drug as well as to dive deep into the pathophysio-
logic mechanisms involved in such rare reactions (Donato and Tolosa, 2019). By
comparison of toxicity profiles obtainedinHLCderivedfromiDILIpatientsand
safe donors, it may be possible to identify genetic polymorphisms, specific mu-
tations, or gene expression pathways potentially associated to iDILI. In this line,
an HLC-based study suggested variants of CYP1A2 as contributing factors in pa-
tient’s susceptibility to pazopanib, a tyrosine kinase inhibitor associated with
significant hepatotoxicity of unknown mechanistic basis (Choudhury et al.,
2017). These cell models provide promising platforms for interrogating
patient-specific factors that contribute to individual susceptibility.
Patient-specific iPSCs have been applied to model genetic diseases as valuable
tools to investigate disease pathology, elucidate their mechanisms, or help to develop
new therapeutic strategies. They also provide a unique opportunity to gain valuable
information on specific sensitivities to drugs associated to genetic diseases. By way
of example, Li et al. (2015) evidenced that iPSC-derived HLCs generated from
patients with AHS were more sensitive to valproic acid than cells from control
patients, in agreement with the increased hepatotoxicity of this drug reported in pa-
tients harboring this genetic disorder (see previous section). Another study showed
that iPSC-derived HLC from patients with A1AT deficiency showed increased sus-
ceptibility to known hepatotoxic compounds (acetaminophen, amiodarone, danazol,
puromycin, and aflatoxin-B) compared to HLC from a normal human cohort
(Wilson et al., 2015).
In summary, although iPSC-derived HLCs are not equivalent to primary hepato-
cytes, the hepatic phenotype that reached upon differentiation may be adequate for
hepatotoxicity studies, providing an unlimited and consistent supply of cells with
stable phenotype for high-throughput screening of huge number of compounds.
They may be obtained from multiple donors, enabling the analysis of the potential
role of genetic diversity in drug-induced hepatotoxicity and the development of
more accurate testing strategies. Finally, the generation of HLCs from patients
who have suffered a severe episode of iDILI offers the possibility of performing
retrospective personalized studies to identify patient-specific characteristics contrib-
uting to increased sensitivity to certain drugs.
240 CHAPTER 10 Induced pluripotent stem cells in liver disease
6.Conclusions
Currently, patient-derived iPSCs are a fundamental tool that allows the study of liver
disease and the mechanisms of DILI as well as being a therapeutic option alternative
to liver transplantation. Although no differentiation protocol reproduces the func-
tionality of PHH, new improvements in the protocols for obtaining HLCs derived
from iPSCs are promising, and this technology has been proved useful for creating
predictive models for rare diseases, toxicity screening, and cell therapy (Corbett and
Duncan, 2019). Advances in iPSCs technology and differentiation protocols that bet-
ter recapitulate liver development as well as scale-up procedures that permit their
mass production will allow the use of HLCs alone or in combination with other he-
patic cells for high-throughput assays for the study and treatment of multiple
diseases.
Abbreviations
A1AT a1-antitrypsin
ACLF acute-on-chronic liver failure
AHS AlperseHuttenlocher Syndrome
ALF acute liver failure
BAL bioartificial liver
CRISPR clustered regularly interspaced short palindromic repeat
CYP cytochrome P450
DILI drug-induced liver injury
ESLD end-stage liver disease
HLCs hepatocyte-like cells
HSC hepatic stellate cells
HUVECs human umbilical vein endothelial cells
iDILI idiosyncratic DILI
iPSCs induced-pluripotent stem cells
KC Kupffer cells
LSEC liver sinusoidal endothelial cells
MSCs mesenchymal stem cells
NAFLD nonalcoholic fatty liver disease
PHH primary human hepatocytes
WD Wilson disease.
Acknowledgments
This work has been supported by the Institute of Health Carlos III (Plan Estatal de IþDþi
2013e2016) and cofinanced by the European Regional Development Fund “A way to achieve
Europe” (FEDER) through grants PI16/00333, PI18/00993, and CP16/00097 and Generalitat
Valenciana (PROMETEO/2019/060). L.T. was supported by ISCIII CP16/00097. The authors
acknowledge M.A. Herrero for his support in linguistic concerns.
Abbreviations 241
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250 CHAPTER 10 Induced pluripotent stem cells in liver disease
Induced pluripotent stem
cells: potential therapeutic
application for improving
fertility in humans and
animals
11
Oscar A. Peralta
1,2
,Vı
´ctor H. Parraguez
3,4
, Cristian G. Torres
5
1
Department of Animal Production Sciences, Faculty of Veterinary and Animal Sciences, University
of Chile, Santiago, Chile;
2
Department of Biomedical Sciences and Pathobiology, Virginia-
Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, United States;
3
Department of Biological Sciences, Faculty of Veterinary and Animal Sciences, University of
Chile, Santiago, Chile;
4
Department of Animal Production, Faculty of Agrarian Sciences,
University of Chile, Santiago, Chile;
5
Department of Clinical Sciences, Faculty of Veterinary and
Animal Sciences, University of Chile, Santiago, Chile
Chapter outline
1. Biology of iPSC in mammals ...............................................................................252
2. Stem cells as candidates for in vitro germ cell derivation ....................................253
3. Potential strategies for the use of iPSC-derived germ cells in human, livestock,
and wild animal reproductive biotechnology ........................................................255
4. Paracrine control and gene signaling pathways involved in in vivo male germ
differentiation: potential approaches for in vitro derivation of germ cells from
iPSC..................................................................................................................257
5. In vitro approaches for derivation of germ cells from iPSCs .................................. 260
6. Conclusion ........................................................................................................262
Funding ..................................................................................................................262
References .............................................................................................................262
Abstract
Due to their unlimited source and high differentiation potential, induced pluripo-
tent stem cells (iPSCs) may be induced in vitro to develop into different types of
specialized cells including male gametes. In the following chapter, we describe
some of the strategies for inducing differentiation of iPSCs into germ cells, as well
as potential applications of iPSC technology for human and animal reproduction.
In this respect, we will discuss how iPSCs may be used as a potential treatment in
men, where infertility is a primary or secondary condition. We will also propose
CHAPTER
251
Recent Advances in iPSCs for Therapy, Volume 3. https://doi.org/10.1016/B978-0-12-822229-4.00009-7
Copyright ©2021 Elsevier Inc. All rights reserved.
some additional applications to animal reproduction, as an alternative method for
dissemination of elite animal genetics, production of transgenic animals, and
conservation of endangered species.
Keywords:
Bone morphogenetic protein; Cell culture; DAZL; Deleted in azoospermia-like;
FRAGILIS; Germ cell differentiation; Germ cell transplantation; Induced pluripotent stem cells;
Infertility treatment; iPSC; Retinoic acid; Spermatogenesis; STELLA; SYCP; Synaptonemal com-
plex protein; Testosterone; Transforming growth factor; VASA.
1.Biology of iPSC in mammals
Groundbreaking studies by Yamanaka and Takahashi in 2006 reported the in vitro
production of mouse induced pluripotent stem cells (iPSCs) by reprogramming of
somatic cells, using forced expression of transcription factors Oct4, Sox2, Klf4,
and c-Myc (Takahashi and Yamanaka, 2006). iPSC achieved high degree of dedif-
ferentiation and similar properties of embryonic stem cells (ESCs), including similar
morphology and in vitro capacity for differentiation into the three germ layers (ecto-
derm, mesoderm, and endoderm). Moreover, mouse iPSCs had the potential to inte-
grate in the inner cell mass and participate in the development of an embryo upon
injection into a host blastocyst. In this process, iPSCs were able to contribute to
the germline and had the potential to generate viable and fertile animals, fulfilling
criteria for ESC definition (Okita et al., 2007;Maherali et al., 2007).
Considering their biological properties, iPSCs represent an alternative source of
pluripotent cells to ESCs. In this respect, iPSCs possess the advantage of being
derived from somatic cells with an individual genetic background limiting the poten-
tial for immune rejections and serving as interesting donor candidate in autologous
transfer (Bellin et al., 2012). iPSCs also overcome ethical concerns associated to the
derivation of human ESCs from blastocysts and have the potential for a variety of
applications in patient-specific autologous cells derivation for personalized therapies
(Kimbrel and Lanza, 2015). Moreover, reprogrammed iPSCs are an interesting tool
for in vitro disease modeling and developing of screens for drug discovery and
toxicity tests.
The promise of cell therapies in medicine seems by far the most appealing appli-
cation of iPSCs. However, many obstacles will have to be overcome before iPSC-
based treatments could be introduced into the clinical practice. Despite thousands
of studies being published on cell reprogramming in human and murine species
(Reviewed by Liu et al., 2020), less than 50 studies describing cell reprogramming
are currently available in farm animal species (Reviewed by Ogorevc et al., 2016).
The majority of farm animalederived iPSCs have not been tested for pluripotency,
and the tested cells were not germline-competent and should be considered iPSC-
like cells. Moreover, current large animal iPSCs have not been able to reliably pro-
duce viable and fertile offspring, possibly due to the inability to produce stable
transgene-free iPSCs, without sustained expression of exogenous transcription fac-
tors (Du et al., 2015).
252 CHAPTER 11 Induced pluripotent stem cells
Differentiation potential of iPSC into germ cells is often mentioned as a viable
option for infertility treatments in humans; however, current protocols of differenti-
ation still do not allow full differentiation of sperm. Moreover, the reliable assess-
ment of the functional capacity of iPSC-derived germ cells is one critical issue,
especially considering that in vivo tests cannot be performed for obvious ethical
reasons. Despite more than 1500 studies being reported in germ cell differentiation
of human iPSCs (reviewed by Kurek et al., 2020), in the case of farm animal iPSCs,
the in vitro analysis of the potential germ cell derivation has not been reported.
Nevertheless, the development of this technology in humans and animals may result
in significant contributions for translational medicine, biopharming, and agriculture.
2.Stem cells as candidates for in vitro germ cell derivation
Infertility, defined as failure to conceive a clinically detectable pregnancy after
>12 months of unprotected intercourse, is a common condition reported in one of
six couples (Boivin et al., 2007). Male factor contributes to infertility in about
50% of these couples and up to 20% of the men presenting fertility difficulties
are found to be azoospermic (Boivin et al., 2007). During the last decades, several
advancements have been made in assisted reproduction technologies and now more
than 80% of couples experiencing infertility issues can conceive a child (Schlegel,
2009). However, there is still the necessity for treating infertility in couples where
assisted reproduction procedures are unsuccessful or in men where infertility is a
secondary condition after undergoing chemotherapy for cancer treatment. Risk fac-
tors for infertility in patients undergoing chemo and radiotherapy are associated to
testicular radiation, exposure to high doses of alkylating agents, and bleomycin
exposure (Wasilewski-Masker et al., 2014). These gonadotoxic effects may cause
permanent infertility by depleting germ cells or damaging the Sertoli cells, which
are then unable to support spermatogenesis (Jahnukainen et al., 2011). Additionally,
damage to Leydig cells may result in testosterone deficiency with long-term impact
(Anderson et al., 2015).
During the last few years, the in vitro derivation of germ cell lineages from stem
cells has emerged as an exciting new strategy for obtaining mature gametes (West
et al., 2013;Volarevic et al., 2014). Due to their unlimited source and high differen-
tiation potential, stem cells may be induced in vitro to develop into different types of
specialized cells including male gametes, suggesting their potential use for the treat-
ment of male infertility (Nagano, 2007). In vitro gamete derivation technology has
also expanded its potential applications to animal reproduction, as an alternative
method for dissemination of elite animal genetics, production of transgenic animals,
and conservation of endangered species.
Notwithstanding the potential impact for biomedicine, agriculture, and wild an-
imal conservation, in vitro germ cell derivation has proven technically demanding,
emphasizing the necessity for additional research. Initial experiments on germ cell
2. Stem cells as candidates for in vitro germ cell derivation 253
derivation from stem cells explored the ability of spermatogonial stem cells (SSCs)
to differentiate into male gametes in vitro and the capacity to restore male fertility
in vivo (Brinster et al., 2003;Hermann et al., 2012). SSCs reside in adult testis main-
taining spermatogenesis and continual sperm production throughout male lifespan
(Kanatsu-Shinohara et al., 2008). SSCs may be isolated from testicular parenchyma
by marker selection and cell sorting and induced to differentiate into functional
gametes by in vitro exposure to specific bioactive factors (Huleihel et al., 2007).
Furthermore, autologous and allogenic SSC transplantations into the testes of ma-
caques previously treated with chemotherapy have resulted in regeneration of sper-
matogenesis and production of functional sperms (Hermann et al., 2012). SSC
transplantation has generated great expectation on the potential use of stem cells
for donor-derived spermatogenesis in animals. However, low concentration of
SSCs in mammal testis and challenges associated with protocols for their isolation,
identification, and culturing have limited its use (McLean, 2005). Moreover, in the
case of livestock species, large amounts of SSC are required for repopulation of
recipient tubules, which has limited the usefulness of this technology (Hill and
Dobrinsky, 2006).
In order to generate an alternative to SSCs, some reports have shown that germ
cells can be derived in vitro from ESCs (Nayernia et al., 2006). ESC lines are plurip-
otent and have been derived from the inner cell mass of blastocysts from mice
(Evans and Kaufman, 1981), primates (Thomson et al., 1995), and humans (Thom-
son et al., 1998). Studies in mice first reported the in vitro differentiation of ESCs
into germ cells and functional gametes capable of giving rise to live offspring after
use of intracytoplasmatic injection (Nayernia et al., 2006). Further studies showed
the ability of human ESCs to differentiate into advanced spermatogenic stages,
including round spermatids, which are unable to fertilize the oocytes in higher-
order mammals (Easley et al., 2012). However, difficulties of germ cell derivation
from ESCs arise from other factors including ethical considerations in the ESC deri-
vation from human embryos, similarities in cell-specific marker expression between
ESC and germ cells, and potential teratogenic formation and immune rejection after
cell transplantation (Volarevic et al., 2014). In the case of animals, the unavailability
of reliable ESC lines has limited its use for germ cell production. However, the
establishment of a stable bovine ESC line has been lately reported, which may
open the opportunity for germ cell differentiation studies (Bogliotti et al., 2018).
Alternatively, the use of adult or fetal stem cells such as mesenchymal stem cells
(MSCs) for germ cell derivation has been reported (Cortez et al., 2018). MSCs
are able to differentiate until early stages of germ cell; however, the capacity to un-
dergo meiosis has not yet been demonstrated (Cortez et al., 2018;Segunda et al.,
2019).
Recently, the development of iPSCs derived from reprogrammed somatic cells
has added an additional strategy for in vitro gamete derivation. iPSCs share charac-
teristics with ESCs including similar morphology, phenotypic profile, and plasticity
(Takahashi and Yamanaka, 2006). However, iPSCs may be more suitable candidates
for in vitro gamete derivation due to several characteristics including unlimited
254 CHAPTER 11 Induced pluripotent stem cells
abundance, specific marker expression profile, and reduced risk for immune rejec-
tion after cell transplantation. Although it has been verified that mouse iPSCs can
derive in functional spermatozoa, capable of fertilizing oocytes after intracytoplas-
matic injection, and give rise to fertile offspring, iPSCs technology for that purpose
is still incipient in human and has not been reported in domestic animals.
3.Potential strategies for the use of iPSC-derived germ
cells in human, livestock, and wild animal reproductive
biotechnology
Considering the pluripotent ability, iPSCs have the potential for in vitro differenti-
ation into germ cells or in vivo induction into male gametes after transplantation
into the seminiferous tubules of recipient testis. Fibroblast-derived iPSCs with an in-
dividual genetic background may be used as donor candidates for autologous trans-
fer in patients suffering from infertility to repopulate germ cells in seminiferous
tubules and to allow production of functional sperm (Fig. 11.1). However, some
drawbacks require to be solved before transfer in patients including low cell yield
due to reduced reprogramming efficiency and potential oncogenic formation associ-
ated to oncogene use in reprogramming and presence of undifferentiated population
of cells.
Alternatively, iPSC-derived germ cells that undergo meiosis may be used in
assisted reproductive technologies for embryo production and embryo transfer.
Furthermore, germ cell transplantation has also expanded its potential applications
to animal reproduction, as an alternative method for dissemination of elite animal
genetics and production of transgenic animals (Honaramooz and Yang, 2011;Oat-
ley, 2017). Implementation of a germ cell transplantation technique would allow
the continuous supply of elite animal genetics for transplantation into seminiferous
tubules of recipient animals for donor-derived spermatogenesis, embryo production,
or sperm freezing. Despite that artificial insemination (AI) has been considered the
major driver of the massive increase in milk production in dairy cattle worldwide,
incorporation of AI technology in beef cattle has been limited due to management
of the herd under extensive range conditions. Germ cell transplantation can be
used for dissemination of elite bull genetics in extensive beef cattle grazing systems,
where animal handling is a limiting factor (Honaramooz and Yang, 2011). Since
iPSCs can self-renew, they have great potential to continuously produce germ cell
and mature gametes compared to the use of a limited source of frozen semen
from an individual donor (Honaramooz and Yang, 2011). Moreover, in vitro pro-
duced germ cells or sperm may be used for in vitro embryo production or cryopres-
ervation in a new setting for AI in livestock reproduction. In addition to applications
for genetic improvement, the transfer of transgenic donor germ cells into the recip-
ient testes may allow the production of transgenic cattle in a shorter period of time in
comparison to somatic cell nuclear transfer (SCNT) technique (Honaramooz et al.,
3. Potential strategies for the use of iPSC-derived germ cells in human 255
2003). Genetically modified cattle may have significantly economic value for the
production of biopharmaceutical proteins in milk (Keefer, 2004). As an example, re-
combinant human myelin basic protein (rhMBP) produced in the milk of transgenic
cows is currently being tested in preclinical studies as a therapeutic vaccine against
multiple sclerosis disease (Al-Ghobashy et al., 2009).
Furthermore, in vitro gamete derivation may also provide an innovative strategy
for preservation of genetic diversity in breeding programs of endangered species.
FIGURE 11.1 Potential strategies for the use of iPSC-derived germ cells in human, livestock,
and wild animal reproductive biotechnology.
(1) Fibroblast-derived iPSCs with an individual genetic background may be used as donor
candidates for autologous transfer in patients suffering from infertility to repopulate germ
cells in seminiferous tubules allowing pregnancy by intercourse. (2) Alternatively, iPSC-
derived germ cells that undergo meiosis may be used in assisted reproductive
technologies for embryo production and embryo transfer. (3) In livestock reproduction,
implementation of an iPSC-derived germ cell transplantation technique would allow the
continuous supply of elite animal genetics for transplantation into seminiferous tubules of
recipient animals for donor-derived spermatogenesis, (4) embryo production, or (5)
sperm freezing. (6) The development of germ cell from iPSC may provide an opportunity
to increase gene pools by collecting cells from wild specimens that can be turned into
sperm and used in captive breeding programs reducing the need to transfer animals
between zoos for breeding.
256 CHAPTER 11 Induced pluripotent stem cells
Several canid species are threatened by inbreeding, and artificial inbreeding tech-
niques may be one solution to exchange genetic material between wild or captive
populations. From the 36 species comprising the canid family, six are listed as
“threatened” or “endangered” by the World Conservation Union IUCN (2019).
The Darwin’s fox, Pseudalopex fulvipes, is one of the threatened canid species,
whose population located mainly in the Chiloe Island and Nahuelbuta National
Park in Chile is estimated to be less than 500 individuals (Jimenez, 2007;IUCN,
2019). In this context, AI is a valuable tool for managing this threatened wildlife
population; however, its practical application for conservation of canids is still
limited (Swanson, 2006). Semen collection in wild canids is normally performed
by electroejaculation; however, this method has been found to yield ejaculates of
small volume and contaminated with urine (Platz et al., 2001). The presence of urine
alters osmolality and pH of seminal plasma, reducing the capacity of sperm for cryo-
preservation (Platz et al., 2001). Alternatively, in vitro gamete derivation from stem
cells may be a useful strategy for conservation of endangered canid species,
providing an abundant source of germ cells that can be continually expanded, easily
frozen, and can be banked from living and deceased animals to preserve genetics.
This technology may also provide an opportunity to increase gene pools by collect-
ing cells from wild specimens that can be turned into sperm and used in captive
breeding programs reducing the need to transfer animals between zoos for breeding.
4.Paracrine control and gene signaling pathways involved
in in vivo male germ differentiation: potential
approaches for in vitro derivation of germ cells from iPSC
Gamete differentiation during embryogenesis is a well-orchestrated and highly com-
plex process that involves sequential activation of several factors associated to cell
proliferation and differentiation (Fig. 11.2). In domestic animals, male germ cells
are derived from a population of primordial germ cells (PGCs) originated in the
proximal epiblast (Lawson and Hage, 1994). PGC specification is induced by
extrinsic factors secreted by extraembryonic ectoderm, including component of
the Sma- and Mad-Related Family (SMAD) signaling pathway, bone morphogenetic
protein (BMP) 4, and transforming growth factor (TGF) b1(Surani et al., 2004).
BMP4 induces expression of the germ cellespecific marker FRAGILIS in the prox-
imal epiblast, marking the first step to the germline commitment (Saitou et al.,
2002). After gastrulation begins, a subset of presumptive germ cells positive for
FRAGILIS migrates to the extraembryonic mesoderm and starts expressing the
germ cellecompetence marker STELLA. During this process, the germline
suppresses somatic cell gene expression and promotes expression of pluripotent
genes OCT4, NANOG, and SOX2 (Hua and Sidhu, 2008). During migration through
the hindgut to the genital ridge developing into the future testis, PGCs start express-
ing VASA, a marker gene for postmigratory germ cells (Fujiwara et al., 1994).
4. Paracrine control and gene signaling pathways 257
At this time, the deleted in azoospermia-like (DAZL) protein is expressed playing
essential roles in development of PGCs and in differentiation and maturation of
germ cells (Yen, 2004). Several in vitro studies have reported that BMP4 can induce
pluripotent stem cells (Toyooka et al., 2003;Easley et al., 2012) to differentiate into
germ cells in vitro.
BMP4 activity is mediated by receptor ALK3 and transducer SMAD5, exerting
both mitogenic and differentiative effects (Pellegrini et al., 2003). Additionally, it
has been reported that TGFb1 can also induce upregulation of DAZL and formation
of spermatogonia-like morphology in ovine MSC (Ghasemzadeh-Hasankolaei et al.,
2014). Thus, supplementation of BMP4 and TGFb1 in culture media constitutes an
initial approach for in vitro derivation of male germ cells from iPSCs.
When the testis becomes morphologically distinguishable, PGCs initiate a sex-
specific development (Brennan and Capel, 2004). After migration into the devel-
oping testis, PGCs enter mitotic arrest and are reactivated after birth to initiate
FIGURE 11.2 Bioactive factors and lineage-specific genes involved in germ cell differentiation
during embryogenesis.
PGC specification, migration, and proliferation are induced by extrinsic factors secreted
by extraembryonic ectoderm, including BMP4 and TGFb1. After migration into the
developing testis, PGCs enter mitotic arrest and are reactivated after birth to initiate
spermatogenesis. The decision of meiotic entry or mitotic arrest of postmigratory PGCs is
regulated by retinoic acid (RA). Several lineage- and stage-specific genes are involved in
germ cell differentiation during embryogenesis until adulthood.
258 CHAPTER 11 Induced pluripotent stem cells
spermatogenesis. Recent studies have reported that decision of meiotic entry or
mitotic arrest of postmigratory PGCs is regulated by retinoic acid (RA) (Koubova
et al., 2006). Male PGCs do not enter meiosis because the enzyme CYP26b1
expressed in somatic cells in the male genital ridge degrades RA. Numerous studies
have shown that STRA8 is an essential factor required for germ cell entry into
meiosis (Anderson et al., 2008). RA acts inside the nucleus exerting activity during
gametogenesis through RA receptors (RAR) in Sertoli cells (RARa), round sperma-
tids (RARb), and type A spermatogonia (RARg)(Vernet et al., 2006). RA favors
spermatogonial differentiation through a direct action and an indirect effect medi-
ated by BMP4 secreted by Sertoli cells (Pellegrini et al., 2003). In addition, RA
induces meiotic initiation through controlling the RAR-dependent expression of
STRA8 in premeiotic spermatocytes (Raverdeau et al., 2012). Therefore, the effect
of RA in vivo may also be emulated in vitro to induce MSC to differentiate into late
germ cells and to undergo meiotic progression.
Nevertheless, germ cell differentiation including entry into meiosis requires
endocrine and paracrine/autocrine regulation in a specific environment, as well as
direct cell-to-cell interactions provided by the somatic cells of the testis. Testos-
terone and follicle stimulating hormone (FSH) are primary regulators of spermato-
genesis providing additive and synergistic effects for meiotic and postmeiotic
development in vivo (Haywood et al., 2003). Primary endocrine activity via FSH
and testosterone is regulated by Sertoli cells in an adluminal compartment generated
by junctional complexes. Secretion products of Sertoli cells and germ cells deter-
mine the composition of this local environment and influencing meiosis as well as
spermatocyte development (Griswold, 1995). Sertoli cells play a pivotal role by
forming niches for germ cells to reside or repopulate the seminiferous tubules
providing essential factors including BMP4 and RA for proliferation and differenti-
ation of germ cells into spermatozoa (Sofikitis et al., 1999). Use of a carefully
defined Sertoli cellegonocyte coculture system has revealed that germ cell develop-
ment likely depends on interaction with adjacent Sertoli cells (Orth et al., 2000).
Sertoli cells express receptors for androgen and are instrumental for SSC differen-
tiation, a process that critically depends on RA (Vernet et al., 2006). Combination
of testosterone and FSH in coculture of SSC with Sertoli cells induces meiosis entry
and full spermatid differentiation (Sousa et al., 2002). These findings clearly demon-
strate that environmental factors and specific interaction with Sertoli cells are natural
inducers and play a key role in germ cell differentiation.
During migration of PGCs into the undifferentiated gonads, DAZL is expressed
playing essential roles in development of PGCs and in differentiation and maturation
of germ cells (Yen, 2004). The relevance of DAZL has been demonstrated in DAZL
knockout mice, where embryos display reduced expression of germ cellespecific
genes including STELLA and VASA, and postnatal males present impairment in
progression from A to A1 spermatogonia and meiotic arrest resulting in azoospermia
and sterility (Riggiu et al., 1997;Lin and Page, 2005). DAZL is expressed
throughout most of the life of germ cells and is required for the development of
PGCs and for the differentiation and maturation of germ cells from PGCs onward
4. Paracrine control and gene signaling pathways 259
(Yen, 2004). In addition to media formulation, strategies for germ cell derivation
have included ectopic overexpression of genes involved in germ cell differentiation.
Ectopic overexpression of DAZL in goat MSC induced higher upregulation of germ
cell markers SCP3 and MVH and downregulation of NANOG mRNA, in compari-
son to RA and BMP4 treatment (Yan et al., 2015). In contrast, knockdown of DAZL
resulted in lower expression of SCP3 and MVH demonstrating that DAZL is a mas-
ter gene controlling germ cell differentiation.
5.In vitro approaches for derivation of germ cells from
iPSCs
Some of the current strategies for germ cell differentiation from iPSC include pri-
mary differentiation into PGCs followed by PGC meiosis with some variations. Veri-
fication of PGC formation has been assayed by detecting expression of markers
including c-Kit, DEAD-box helicase (DDX) 4, and SSEA1 (Mouka et al., 2016).
One crucial step in germ cell formation is confirmation of cells entering meiosis,
which is commonly evaluated by meiotic marker expression including synaptonemal
complex protein (SYCP) 3, transition protein 1 (TP1), protamine 1 (Prot 1), and
acrosin (Ishii, 2014). Moreover, a series of benchmarks for meiotic progression of
stem cells have been proposed, including synaptonemal complexes immunolabeling,
determination of markers of recombination (RAD51) and mutL homolog (MLH) 1,
visualization of DNA content by DAPI staining or a-tubulin immunolabeling of the
spindle, and finally production of viable progeny after transfer in female host (Han-
del et al., 2014).
Differentiation of human iPSCs into germ cells under in vitro conditions was first
attempted after spontaneous differentiation of embryoid bodies (EBs), using the
expression of DDX4 as the presence of germ cellelike cells (Clark et al., 2004).
This protocol allowed formation of putative germ cells expressing markers including
DAZL, developmental pluripotency-associated protein 3 (DPPA3), DDX4, and
SYCP 3. Since the EB differentiation protocols, the use of three-dimensional (3D)
culture conditions have been reported, either with a multistep approach in combina-
tion with two-dimensional (2D) culture conditions using defined matrices and feeder
cells aiming at increasing the number of differentiated cells (Fig. 11.3). The use of
gelatin (Tilgner et al., 2008) or laminin (West et al., 2008) and feeder cells has led to
the generation of germ cell-like cells expressing SSEA1, DDX4, MLH1, and
SYCP3. Thereafter, Matrigel (Panula et al., 2011;Medrano et al., 2012;Gkountela
et al., 2013;Kee et al., 2009;Julaton and Pera, 2011), as well as fetal gonadal cells
(Park et al., 2009;Gkountela et al., 2013), has been widely used in several reports.
The use of fetal gonadal cells has shown to improve the efficiency of germ cell
differentiation of iPSCs in comparison with conditioned media, adherent cultures,
and nongonadal first-trimester human stromal cells (Park et al., 2009).
Recently, iPSCs have been exposed to preinduction into pluripotent state or a
mesoderm-like state instead of jumping to direct germ cell differentiation from
260 CHAPTER 11 Induced pluripotent stem cells
primed PSCs (Irie et al., 2015;Sasaki et al., 2015). While the first study (Irie et al.,
2015) reported cultures on mouse embryonic fibroblasts (MEFs), with further prim-
ing culture on vitronectin/gelatin and final differentiation as EB, the second study
(Sasaki et al., 2015) performed their cultures on laminin 511 with priming on plasma
fibronectin and final differentiation as EBs. Furthermore, the gene expression profile
of five different human ESC lines, which were derived on human foreskin fibroblasts
(hFFs), cultured on different matrices (laminin 121 or 521 and Matrigel) was
recently compared (Albalushi et al., 2018). After nine passages on laminin 521, a
more homogeneous expression pattern of pluripotency markers POU5F1, NANOG,
SOX2, and growth differentiation factor 3 (GDF3) was observed compared to the
earlier passages (Albalushi et al., 2018). Furthermore, the feeder cells expressed
the epidermal growth factor (EGF), BMP4, and glial cellederived neurotrophic
factor (GDNF) (Yang et al., 2017;Wang et al., 2016;Li et al., 2014). iPSCs aggre-
gated with these supporting cells expressing EGF, BMP4, and GDNF display clumps
of VASA cells and cells expressing meiotic markers SYCP1, DMC1, and postmigra-
tory markers DAZL and MILI.
Although the use of coculture systems or conditioned medium may be able to
facilitate germ cell differentiation of iPSC, induction with chemically or biologically
defined factors is preferred, in order to increase safety of cells for clinical applica-
tions and to improve the reproducibility of the differentiation process (Teramura and
Frampton, 2013). Thus, supplementation of defined media with growth factors is
usually considered as an option for inducing differentiation (Teramura and Framp-
ton, 2013;Kee et al., 2006). The addition of exogenous factors to the culture media,
such as RA, BMPs, human fetal gonadal cells, testosterone, stem cell factor (SCF),
and GDNF, which can play basic roles in germ cells and gamete development
in vivo, seems to help in expanding the germ cell population and pushing them to
the meiotic process in vitro (Marques-Mari et al., 2009;Kee et al., 2006).
FIGURE 11.3 In vitro approaches for derivation of germ cells from iPSC.
Somatic cells may be reprogrammed into iPSCs by forced expression of transcription
factors Oct4, Sox2, Klf4, and c-Myc. iPSCs cultures under nonadherent conditions form
EBs and may be directed into germ-like cells using multistep approaches in combination
with two-dimensional (2D) culture conditions using feeder cells or defined matrices.
5. In vitro approaches for derivation of germ cells from iPSCs 261
Exposure of mouse iPSCs to RA or testosterone has been shown to induce dif-
ferentiation into male germ cells through EB formation and even into meiotic
progression (Li et al., 2013). These authors reported that exposure to RA and testos-
terone induced about 2%e8% of the EB cells into haploid cells, which suggests that
combination of these factors promotes differentiation efficiency of iPSCs into male
germ cells. However, RA effect in germ cell differentiation should be analyzed with
caution, since RA is also considered to be the most used morphogen to produce
neural progenitor cells (NPCs) and consequently may differentiate iPSC into nerve
cells (Sartore et al., 2011).
The use of BMP has been tested on iPSCs (IMR90) and iHUF4 cells for 7 and
14 days in the presence or absence of BMP4, BMP7, and BMP8b to evaluate its ef-
fect on germ cell differentiation (Panula et al., 2011). It was found that iPSC exposed
to BMP expressed DAZL and VASA. Surprisingly, some unexposed cells were also
positive for these markers suggesting that spontaneous differentiation may also
explain the phenotypic change of these cells. Furthermore, supplementation of
GDNF has also been evaluated in porcine iPSCs, where GDNF supplementation
induced germ cellelike cell formation and was required for SSC proliferation and
maintenance (Wang et al., 2016).
6.Conclusion
The promise of cell therapies in medicine seems by far the most appealing applica-
tion of iPSCs. However, many obstacles will have to be overcome before iPSC-based
treatments could be introduced into the clinical practice. Intense research has been
conducted during the past years in murine and human iPSCs and increasing number
of studies are conducted on iPSC-germ cell derivation. In comparison, a reduced
number of studies have been published on farm animal iPSCs, which suggests
that this potentially significant technology for animal reproduction has still a long
way for application.
Funding
Some studies presented in this chapter were funded by grants 1161251 and 1191114 from the
National Fund for Scientific and Technological Development (FONDECYT) from the Minis-
try of Education, Government of Chile.
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Induced pluripotent stem
cells in wound healing 12
Xixiang Gao
1,2
, Jolanta Gorecka
2
, Umber Cheema
3
, Yongquan Gu
1
, Yingfeng Wu
1
,
Alan Dardik
2
1
Department of Vascular Surgery, Xuanwu Hospital, Capital Medical University and Institute of
Vascular Surgery, Capital Medical University, Beijing, China;
2
Vascular Biology and Therapeutics
Program and the Department of Surgery, Yale University School of Medicine, New Haven, CT,
United States;
3
UCL Institute of Orthopaedics &Musculoskeletal Sciences, UCL Division of
Surgery &Interventional Sciences, University College London, London, United Kingdom
Chapter outline
1. Introduction ................................................................................................ .......270
2. Background .......................................................................................................271
3. Stem cell therapy in wound healing ....................................................................272
3.1 Embryonic stem cells .......................................................................... 274
3.2 Mesenchymal stem cells ...................................................................... 274
4. Induced pluripotent stem cells ............................................................................275
4.1 iPSC advantages ................................................................................. 276
4.2 iPSC-derived endothelial cells .............................................................. 276
4.3 iPSC-derived fibroblasts ....................................................................... 280
4.4 iPSC-derived mesenchymal stem cells .................................................. 281
4.5 iPSC-derived extracellular vesicles........................................................ 281
5. Challenges and solutions of iPSC in wound healing..............................................282
6. Conclusion ........................................................................................................283
7. Future directions ................................................................................................ 284
References .............................................................................................................284
Abstract
Stem cell therapy is emerging as an exciting new strategy for the treatment of
chronic wounds representing a promising approach of regenerative medicine.
Induced pluripotent stem cells (iPSC) represent an innovative, adult-derived, stem
cell source with enhanced therapeutic and translational potential. iPSC are
pluripotent stem cells derived from somatic donor cells, harvested via noninvasive
techniques from a limitless donor pool. The use of iPSC technologies allows for
generation of autologous pluripotent stem cell populations with enhanced in vivo
survival while obviating ethical issues surrounding destruction of embryos.
Because of their ability to differentiate into and repopulate all cell types found in
CHAPTER
269
Recent Advances in iPSCs for Therapy, Volume 3. https://doi.org/10.1016/B978-0-12-822229-4.00003-6
Copyright ©2021 Elsevier Inc. All rights reserved.
the skin, iPSC have the potential to enhance each phase of wound healing via
paracrine and direct cellular effects. Current methods that generate iPSC are often
inefficient, predispose cells to mutagenesis, and yield impure cellular populations.
Because undifferentiated cells carry tumorigenic potential, selection of terminally
differentiated cells and use of cell-free extracellular vesicles may improve iPSC
safety profile. While the ideal delivery platform for improving cell function and
survival is yet to be designed, advances in bioengineered materials to create next-
generation delivery scaffolds have shown promising results. Thus, despite
tremendous advantages and promise for tissue regeneration, further studies to
improve iPSC safety profile and generation methods are urgently needed prior to
widespread clinical adoption.
Keywords:
Angiogenesis; Chronic wounds; Diabetic foot ulcer; Differentiation; Extracellular
vesicles; Immune response; Induced pluripotent stem cell; Mutagenesis; Regenerative medicine;
Reprogramming; Skin regeneration; Stem cell; Stem cell therapy; Tumorigenicity; Wound healing.
1.Introduction
Wound healing is a precisely regulated physiologic response to the disruption in the
normal architecture of the skin barrier. This complex and delicate process is prone to
dysregulation secondary to local and systemic factors that can lead to the develop-
ment of chronic wounds. Important factors in wound healing include adequate nutri-
tion, smoking cessation, infection control, and management of chronic disease.
Abnormal wound healing is a significant healthcare problem, posing a large socio-
economic burden on patients, their families, and the healthcare system as a whole.
Although the psychological sequelae for patients are often understated, as they are
difficult to quantify, it has been estimated that chronic wounds affect over 6 million
Americans and cost in excess of 25 billion USD annually (Sen et al., 2009). The first
comprehensive study of Medicare spending on wound care found that wounds in the
United States alone impact nearly 15% of Medicare beneficiaries, that is, 8.2 million
patients, and a low-range estimate of their annual cost is $28 billion (Nussbaum
et al., 2018). The significant social and economic burden represented by “problem
wounds” has precipitated study of the mechanisms underlying cutaneous regenera-
tion. With the rapid growth and aging of the global population, both the number of
patients who require care and the costs of skin regeneration are increasing, adding
burden and cost to public healthcare (Fife and Carter, 2012).
Despite a multitude of available therapeutic interventions, current treatment
options for wound healing have only met moderate clinical success. Stem cell ther-
apy has emerged as an exciting wound healing therapy and has become a promising
approach in the field of regenerative medicine. Stem cells, characterized by their
multipotency and capacity for self-renewal, can mobilize and be home to injured
tissues, where they synthesize and secrete proregenerative chemokines and growth
factors. These, in turn, promote cell recruitment, angiogenesis, and extracellular
matrix (ECM) remodeling, creating a local microenvironment conducive to wound
healing. Stem cells from various sources have been used to promote wound healing
270 CHAPTER 12 Induced pluripotent stem cells in wound healing
in many preclinical and clinical trials thus far, with iPSC being the newest and most
revolutionary cell type. The following chapter reviews the process of wound healing
and the role of stem in accelerating cutaneous wound healing, with particular focus
on iPSC in animal studies. In addition, we will address the limitations that need to be
overcome to clinically translate such treatments and future directions in stem cell
therapy.
2.Background
Wound healing occurs in three overlapping yet distinct stages: inflammation, prolif-
eration, and remodeling (Gurtner et al., 2008). The first stage of wound healing
occurs immediately after tissue damage and is designed to achieve hemostasis and
initiate a cellular inflammatory response required to prevent infection and prepare
the wound bed for repair (Portou et al., 2015). Hemostasis is achieved initially by
platelet aggregation and initiation of the coagulation cascade, leading to the forma-
tion of a fibrin clot, which becomes the scaffold for infiltrating cells. Neutrophils are
then recruited to the wound in response to chemotactic signals including cytokines,
activation of complement, platelet degranulation, and products of bacterial degrada-
tion. Neutrophils remove bacteria, foreign particles, and damaged tissue via three
main pathways. They have phagocytic capacity for directly ingesting and destroying
foreign particles, they can degranulate and release various toxic substances, which
will remove bacteria and devitalized host tissue, and they can capture and kill
bacteria in the extracellular space by producing chromatin and protease traps. In
addition, oxygen-derived free radical species produced as a by-product of neutrophil
activity have bactericidal properties (Singh et al., 2017). Approximately 3 days after
injury, neutrophils decrease in number and monocytes appear in the wound and
differentiate into macrophages. Macrophages are thought to be crucial for promotion
and resolution of inflammation, host defense, removal of apoptotic cells, and support
of cell proliferation and tissue restoration (Sen et al., 2009).
The proliferative stage of wound repair predominates 2e10 days after wounding
and is characterized by cellular proliferation and influx of several different cell
types. Initially, keratinocytes migrate to the injured dermis. Granulation tissue
composed of sprouts of capillaries, fibroblasts, and macrophages replaces the fibrin
clot and forms a new matrix for keratinocyte infiltration. In the later part of this
stage, contractile cells including resident fibroblasts and bone marrowederived
myofibroblasts infiltrate the wound and initiate healing. Fibroblasts and myofibro-
blasts synthesize and deposit a collagen-rich ECM, which eventually forms the
bulk of a scar (Werner et al., 2007;Darby et al., 2014).
Remodeling is the final phase of wound healing; it begins 2e3 weeks after injury
and typically lasts for a year or more. This phase involves extension of new epithe-
lium and apoptosis of unneeded blood vessels, fibroblasts, and inflammatory cells,
resulting in maturation of scar tissue. Despite optimal healing, wounds never regain
the properties of uninjured skin (Levenson et al., 1965). Furthermore, these phases
2. Background 271
and their physiological functions occur in a regulated and precise manner, and any
discontinuities, aberrancies, or prolongations in the process can lead to failure of
healing and progression to chronicity (Guo and DiPietro, 2010).
The gold standard of wound care involves careful diagnosis of etiology, address-
ing systemic factors such as ischemia and diabetes, infection control, debridement of
nonviable tissue, selecting appropriate dressings, and reconstructive surgery using
tissue grafts and flaps (Werdin et al., 2008). Despite advances in the standard of
care, healing of chronic wounds is challenging, and treatment options are increas-
ingly limited in elderly patients, who are often less able to tolerate surgical interven-
tion (Finlayson et al., 2011). Therefore, novel and improved methods of wound
healing are urgently needed.
3.Stem cell therapy in wound healing
Stem cell therapy has become a promising new approach in the field of regenerative
medicine to improve wound healing and restore normal skin architecture (Duscher
et al., 2016). Stem cells are defined by their capacity to both self-renew and differ-
entiate into multiple cell lines, functions crucial for physiologic tissue renewal and
regeneration after injury (Behr et al., 2010;Chen et al., 2009). Their therapeutic
potential is secondary to their capability to secrete proregenerative cytokines and
growth factors. These factors promote cell recruitment, immunomodulation, ECM
remodeling and angiogenesis, which in turn allow for earlier wound closure, preven-
tion of wound contracture and scar formation, and ultimately regeneration of the
skin (Duscher et al., 2016;Cao et al., 2017).
Traditionally, stem cells have been divided into two main groups based on differ-
entiation capacity. Pluripotent stem cells (embryonic) can differentiate into every
cell of the body, whereas multipotent stem cells (adult) can differentiate into multi-
ple, but not all, cell lineages (Behr et al., 2010). Among the main sources of cells
used in wound healing are embryonic stem cells (ESC), adult stem cells, and
iPSC (Fig. 12.1). These cells have the potential to secret chemokines and growth
factors, making them an attractive option for wound healing. Although ESC have
the capacity to differentiate into all tissues of the body, they are immunogenic
and tumorigenic and rely on the destruction of embryos to produce. In contrast
with ESC, adult stem cells, especially mesenchymal stem cells (MSC), can avoid
the ethical concerns regarding fetal tissue harvest and have been widely used for
wound healing. MSC can be isolated from bone marrow, adipose tissue, and umbil-
ical cord blood. Main types of stem cells and their advantages and disadvantages are
shown in Table 12.1.
272 CHAPTER 12 Induced pluripotent stem cells in wound healing
FIGURE 12.1
Stem cell sources in wound healing therapy. Stem cells with various degrees of
differentiation capacity have been studied in the field of regenerative medicine.
Pluripotent stem cells are typically derived from the inner cell mass of the blastocyst (ESC)
or can be reprogrammed from adult somatic cells (iPSC), both of which have the capacity
to differentiate into every cell of the body. Multipotent stem cells can be isolated from
various tissues including adipose, bone marrow, and placenta and are more restricted in
their differentiated potential. ESC, embryonic stem cell; ICM, inner cell mass; iPSC,
induced pluripotent stem cell; MSC, mesenchymal stem cells.
Table 12.1 Stem cell types, advantages, and disadvantages in wound
healing.
Cell type Advantages Disadvantages
Embryonic
stem cells
Pluripotent cells capable of
differentiation into all tissues of
the body
Ethical concerns and legal
restrictions, immune rejection,
unregulated differentiation,
formation of teratomas and
teratocarcinomas
Mesenchymal
stem cells
Multilineage differentiation,
proregenerative and
immunomodulatory properties
Invasive harvesting techniques,
immunogenicity, and limited cell
survival in vivo
Induced
pluripotent
stem cells
Pluripotent as ESC without ethical
concerns, less invasive or
noninvasive harvesting, abundant
source cells, and allowing for
generation of autologous
pluripotent stem cell populations,
as well as genomic editing to
produce healthy cells from
diseased donors
Tumorigenicity, mutagenesis, and
low efficiency of reprogramming
3. Stem cell therapy in wound healing 273
3.1 Embryonic stem cells
ESC are pluripotent cells derived from the inner cell mass of the blastocyst and have
the capacity to differentiate into all tissues of the body, including cardiomyocytes,
hematopoietic progenitors, neurons, skeletal myocytes, adipocytes, osteocytes, chon-
drocytes, endothelial cells, pancreatic islet cells, and skin cells (Dor et al., 2004;
Bonner-Weir et al., 2000;Alison, 1998;Gauglitz and Jeschke, 2011;Odorico et al.,
2001). Since the successful isolation of ESCs from the inner cell mass of mouse blas-
tocysts in 1981 (Martin, 1981), their potential in cell replacement therapy and regen-
erative medicine has been widely acknowledged (Thomson et al., 1998). In culture,
ESC are able to form a multilayered epidermis coupled with an underlying dermal
compartment similar to the native skin, making them an important potential cell
source for bioengineered skin (Aberdam, 2004). To utilize the remarkable regenera-
tive potential of ESC for wound healing, Guenou et al. showed that human ESC
can be differentiated into basal keratinocytes that were subsequently used to reconsti-
tute a pluristratified epidermis (Guenou et al., 2009). These tissues were also success-
fully transplanted into immunodeficient mice to facilitate wound healing.
Despite their pluripotentiality and unlimited ability for self-renewal that make
ESC attractive for regenerative medicine, the use of ESC remains controversial, as
ethical concerns and substantial legal restrictions exist regarding the harvest of cells
from live embryos (Chen et al., 2009). Moreover, the potential for immune rejection,
unregulated differentiation, and formation of teratomas and teratocarcinomas remain
significant concerns (Wu et al., 2007a). Therefore, the clinical use of ESC-derived tis-
sue remains limited, precluding major progress in ESC-based applications.
3.2 Mesenchymal stem cells
In contrast to ESC, adult stem cells, such as MSC, avoid the ethical issues surround-
ing fetal tissue harvest. They have significant proliferative capacity, long-term self-
renewal potential, and the ability to differentiate into multiple lineages. MSC have
been isolated from various tissues including the skin, heart, liver, brain, umbilical
cord blood, adipose tissue, and bone marrow (Kern et al., 2006;Phinney, 2012).
Their therapeutic potential stems from their multilineage differentiation, large quan-
tity, ease of isolation, and the ability to migrate to injury sites (Phinney, 2012). These
cells are involved in all three phases of the wound healing process. They also
enhance wound healing by immune modulation, growth factor elaboration,
enhanced neovascularization and reepithelialization, stimulation of angiogenesis,
and acceleration of wound closure (Balaji et al., 2012).
Bone marrowederived MSC (BM-MSC) are considered the primary source of
MSC in adults and a good candidate for the treatment of different types of wounds
(Wu et al., 2007b). BM-MSC accelerate wound closure and increase reepithelializa-
tion, angiogenesis, and cellularity (Wu et al., 2007c;Assi et al., 2016). BM-MSC are
the most common stem cells currently used for the treatment of diabetic foot ulcer
(DFU) (Lopes et al., 2018). Adipose-derived MSC (ADSC) have similarly been
274 CHAPTER 12 Induced pluripotent stem cells in wound healing
shown to significantly improve wound healing and increase blood vessel formation
in many preclinical trials (Teng et al., 2014;Garg et al., 2014;Guo et al., 2018).
Together, studies have shown MSC to be promising candidates for wound healing;
however, significant challenges remain for the clinical application of MSC,
including the invasive harvesting techniques, immunogenicity, and limited cell sur-
vival in vivo (Duscher et al., 2016).
4.Induced pluripotent stem cells
A new class of stem cells that combines the advantages of MSC and ESC has recently
been described, ushering in a new era for regenerative medicine. iPSC represent a
groundbreaking innovation for adult-derived stem cells that carry enhanced therapeu-
tic and translational potential. Takahashi and Yamanaka first developed iPSC by
reprogramming adult fibroblasts into an immature, pluripotent state in 2006 (Takaha-
shi and Yamanaka, 2006). iPSC are reprogrammed into a pluripotent state in vitro by
induced expression of four transcription factors including Oct4/Sox2/c-Myc/KLF4 or
Oct4/Sox2/NANOG/LIN28 (Teng et al., 2014;Takahashi e t al. , 2 007 ;Yu et al., 2007).
Several methods currently employed for generating iPSC are shown in Ta ble 12 .2.
The use of iPSC technologies allows for the generation of autologous pluripotent
stem cell populations derived from differentiated adult tissues, thereby avoiding the
ethical issues associated with human ESC. Furthermore, autologous iPSC are nonim-
munogenic, enhancing their in vivo retention.
Table 12.2 Current methods for generating induced pluripotent stem cells.
Delivery
methods
DNA-
free Efficiency Safety
Integrative
system
Viral integrative
vectors
Retrovirus No High Low
Lentivirus No High Low
Inducible or
excisable retro
or lentivirus
No High Low
Nonviral
integrative
vectors
Plasmid/linear
DNA
No Low High
PiggyBac No High High
Sleep beauty No Low High
Nonintegrative
system
Nonintegrative
viral vectors
Adenovirus No Very low High
Sendai virus No Very high High
Nonintegrative
nonviral vectors
Minicircle No Low High
RNA Yes High High
Protein Yes Very low High
4. Induced pluripotent stem cells 275
4.1 iPSC advantages
iPSC resemble the morphology, proliferation potential, gene expression pattern, plu-
ripotency, and telomerase activity of ESC. They can differentiate into cells of all
three germ layers in vitro and in vivo (Teng et al., 2014;Garg et al., 2014). Unlike
ESC, iPSC not only eliminate ethical issues but also reduce the chances of immuno-
logical rejection in vivo (Guha et al., 2013). iPSC derived from human skin fibro-
blasts elicited a low or negligible immune response, secondary to their induction
of IL-10-secreting Treg (Lu et al., 2014). Since iPSC can be programmed in princi-
ple from any adult somatic cells, such as skin fibroblasts and keratinocytes, their
collection does not require invasive biopsies and the potential pool of source cells
is much greater than other stem cell types. Furthermore, they can be generated in
abundance from patients’ autologous somatic cells, which are nonimmunogenic,
or from a matched donor, enhancing their in vivo survival (Ojeh et al., 2015;Kirby
et al., 2015). Lastly, multiple technologies including zinc finger nucleases (ZFN),
transcription activator-like effector nucleases (TALEN), and clustered regularly
interspaced short palindromic repeats (CRISPR) technologies have been used to
successfully correct genomic mutations and produce autologous disease-free cells
(Ben Jehuda et al., 2018). Taking advantage of these characteristics, significant prog-
ress has been made in the differentiation of iPSC into skin cells including folliculo-
genic human epithelial stem cells, fibroblasts, and keratinocytes and their use in
engineered skin substitutes (Yang et al., 2014;Sugiyama-Nakagiri et al., 2016;
Hewitt et al., 2011;Bilousova et al., 2011;Itoh et al., 2011;Dash et al., 2018).
A fully functional bioengineered three-dimensional (3D) integumentary organ sys-
tem, with skin appendage organs such as hair follicles and sebaceous glands, was
developed from iPSC and did not induce tumorigenesis (Takagi et al., 2016).
Since iPSC can differentiate into cells derived from all three germ layers, they
have the potential to enhance each of the phases of wound healing via paracrine
and direct cellular effects (Singh et al., 2015). iPSC-derived cells secrete growth fac-
tors and cytokines implicated in immunoregulation, cell proliferation and migration,
neovascularization, and ECM synthesis, as well as remodeling. In addition, they
recruit macrophages, fibroblasts, and keratinocytes (Baraniak and McDevitt, 2010;
Liang et al., 2014;Clayton et al., 2018;Kim et al., 2013) and promote angiogenesis
and collagen deposition (Itoh et al., 2013;Kuzuya et al., 1995;Liu et al., 2017). The
following sections review application of iPSC-derived cells in preclinical studies of
wound healing, as well as the hurdles that need to be addressed prior to clinical
application. Major findings of studies relating to cutaneous wound healing in animal
models are outlined in Table 12.3.
4.2 iPSC-derived endothelial cells
Angiogenesis is a vital component of wound healing, since angiogenesis reestab-
lishes perfusion to injured tissues and delivers key nutrients. Unfortunately, the
nonhealing characteristic of chronic wounds is directly associated with a lack of
276 CHAPTER 12 Induced pluripotent stem cells in wound healing
Table 12.3 iPSC in wound healingdcell type, delivery method, animal model, and major findings.
Author Year Cell type Cell number Delivery method Animal model Major findings
Kim et al. 2013 hiPSC-derived
endothelial and
smooth muscle
cells
610
4
EC þ
410
4
SMC
Intradermal
injection in PBS
Full-thickness
excisional wound
Male nude mice
Nondiabetic
1. Increased
neovascularization
2. Accelerated wound healing
3. Increased in vitro VEGF,
EGF, and FGF-4
Shen et al. 2016 hiPSC-derived
endothelial
progenitors or
early vascular
cells
2.5 10
5
Topical application
Acrylated
hyaluronic acid
hydrogels
Full-thickness
excisional wound
Female nude mice
STZ-induced
diabetic
1. Accelerated wound healing
rate and reepithelialization
2. Increased vascular density
and macrophage infiltration
3. No significant difference
between healthy and type 1
diabeticdhiPSC.
Clayton
et al.
2018 hiPSC-derived
endothelial cells
510
5
Intradermal
injection
Suspended in
vehicle containing
medium and
Matrigel
Full-thickness
excisional wound
Male NOD/SCID
mice
Nondiabetic
1. Increased wound perfusion
and accelerated wound
closure
2. Increased capillary density,
collagen deposition, and
macrophage infiltration
3. Increased host angiogenic
gene expression
Nakayama
et al.
2018 iPSC-MSC of
healthy human
and RDEB
patient
Subcutaneous
1.8 10
6,
intravenous
110
6
and
310
5
Subcutaneous and
intravenous
injection
Full-thickness
excisional wound
NOD/SCID mice
Nondiabetic
1. Accelerated wound healing
and restored human type VII
collagen
Kobayashi
et al.
2018 hiPSC-derived
extracellular
vesicles
4mg Subcutaneous
injection and
topical application
Full-thickness
excisional wound
Male C57BLKS/J-
Leprdb (db/db)
mice
Genetically diabetic
1. Promote fibroblasts
proliferation and migration
in vitro
2. Promoted angiogenesis
and wound healing in vivo
Continued
4. Induced pluripotent stem cells 277
Table 12.3 iPSC in wound healingdcell type, delivery method, animal model, and major findings.dcont’d
Author Year Cell type Cell number Delivery method Animal model Major findings
Kashpur
et al.
2019 iPSC-derived
fibroblasts from
both diabetic and
nondiabetic
patients
1.6 10
4
Topical application
Self-assembled
tissues made of
polyethylene
terephthalate
membrane
Full-thickness
excisional wound
Male
immunodeficiency
mice
STZ-induced
diabetic
1. Improved migratory
properties in vitro and
increased wound closure
compared with primary DFU
fibroblasts in vivo.
2. Gene array and functional
analyses of iPSC-derived fi-
broblasts from both nondia-
betic and diabetic patients
were similar to each other
Lu et al. 2019 iPSC derived
from skin
fibroblastic cells
of rhesus
macaques, and
their exosomes
4.6 10
4
iPSC
50 mg
exosomes
Topical application Full-thickness
excisional wound
Male rhesus
macaques
Nondiabetic
1. Autologous iPSC promoted
wound healing,
reepithelialization,
angiogenesis, and collagen
deposition more effectively
compared to allogeneic
iPSC
2. Autologous iPSC exosomes
accelerated wound healing,
epithelization, and
angiogenesis more
effectively compared to
allogeneic iPSC exosomes
RDEB, recessive dystrophic epidermolysis bullosa; STZ, streptozocin.
278 CHAPTER 12 Induced pluripotent stem cells in wound healing
angiogenesis that is diminished in the hypoxic environment. Endothelial cells are
critical for vessel formation and upregulation of various angiogenic factors, and de-
livery of human-induced pluripotent stem cellederived endothelial cells (hiPSC-
EC) may be a promising approach to accelerate healing of chronic wounds
(Gallagher et al., 2007;Galkowska et al., 2006). Several mechanisms by which
hiPSC-EC improve wound healing have been identified thus far. Significantly accel-
erated wound closure with increased wound perfusion and vessel density were
observed in iPSC-EC-treated wounds in a murine model. In total, 14 days after
wounding, iPSC-EC treatment increased wound collagen deposition and
macrophage number, compared with their respective controls, suggesting sustained
macrophage activity (Takagi et al., 2016). Upregulation of key angiogenic genes
including endothelial cell adhesion molecule and vascular endothelial growth factor
(VEGF) was also observed. Cotransplantation of iPSC-derived ECs and smooth
muscle cells (SMC) promotes neovascularization and tissue repair in a murine
dermal wound model (Singh et al., 2015). Furthermore, iPSC-derived EC and
SMC enhanced in vitro tubular network formation. In vivo, coimplantation of
iPSC-derived EC and SMC led to significantly increased vascularization, acceler-
ated wound healing, and increased arteriole density in a murine full-thickness
wound model, compared to iPSC-derived EC alone.
One of the major limitations to clinical translation of stem cellebased therapies
is their reduced survival and paracrine activity in vivo. The optimal delivery plat-
form to enhance in vivo survival of iPSC has yet to be determined. Current research
focuses on using polymeric biomaterial systems that can function as a stem cell
niche and improve cell survival and secretory profile, leading to enhanced wound
healing (Shen et al., 2016;Tan et al., 2018). Several biomimetic materials have
thus far shown promise in iPSC delivery platforms. Engineered vascularized
acrylated hyaluronic-acid (AHA) hydrogel constructs containing iPSC-derived
endothelial progenitors or early vascular cells (EVC) enhanced angiogenesis and
wound closure in diabetic immunodeficient mice (Gu et al., 2015). These hydrogel
constructs accelerated recruitment of host macrophages and enhanced their integra-
tion into the wound, augmenting local angiogenic factors leading to increased angio-
genesis and accelerated wound closure. No significant difference was detected
between vascularized constructs from healthy and type-1 diabetic-derived iPSC,
suggesting that reprogrammed hiPSC might be less susceptible to diabetes-
induced impairments. However, compared to healthy controls, iPSC-EC derived
from diet-induced obese mice exhibited decreased vascular function in vitro and
reduced function and incorporation into the host vasculature in vivo (Gu et al.,
2015). Electrospun polycaprolactone (PCL)/gelatin scaffolds containing iPSC-EC
improved cellular in vivo survival, improved blood perfusion, enhanced recruitment
of macrophages, and elevated expression of proangiogenic cytokines compared to
controls (Liu et al., 2017). Lastly, in vivo survival of hiPSC-EC, as well as blood
perfusion and arteriole density, was increased after culturing cells on electrospun
PCL/gelatin scaffolds (Gallagher et al., 2007).
4. Induced pluripotent stem cells 279
Human iPSCederived EVC, including endothelial cells and pericytes, from
type-1 diabetic patients were functional both in vitro and in vivo and responded to
matrix cues to self-assemble into 3D vascular networks when encapsulated in syn-
thetic hydrogels (Chan et al., 2015). This finding confirms that diabetic patients
could receive autologous transplant, using their own cells, with equal self-renewal
capacity. Most recently, methods of differentiation of hiPSC into endothelial cells
have become faster and more efficient, paving the way for increased iPSC transla-
tional potential (Patsch et al., 2015;Prasain et al., 2014). Together, these findings
suggest that iPSC-EC are an excellent candidate for use in wound healing studies,
secondary primarily to their proangiogenic properties.
4.3 iPSC-derived fibroblasts
During wound healing, wound edge and bone marrowederived fibroblasts are stimu-
lated by macrophages to differentiate into myofibroblasts (Opalenik and Davidson,
2005). Myofibroblasts are contractile cells that, over time, bring the edges of a wound
together and play a remarkable role in the closure of the wound (Chitturi et al., 2015).
Fibroblasts and myofibroblasts interact to produce collagen, fibronectin, and proteo-
glycans that replace the initial fibrin matrix with new ECM and ultimately form the
bulk of the mature scar (Werner et al., 2007;Sugiyama-Nakagiri et al., 2016). In
response to the hypoxic wound environment, fibroblasts also release cytokines,
including fibroblast growth factor (FGF), hepatocyte growth factor (HGF), TGF-b,
epidermal growth factor (EGF), and VEGF that promote the formation of new blood
vessels, a critical component of acute wound healing (Turner and Badylak, 2015).
However, fibroblasts derived from chronic diabetic ulcers have a significantly
lower proliferation rate and an abnormal morphology in vitro, contributing to
delayed wound healing (Loots et al., 1999). Kashpur et al. found that iPSC-
derived fibroblasts from diabetic and healthy patients are more similar to each other
than the primary cells from which they are derived (Kashpur et al., 2019). Gene
ontology analysis revealed that 1304 genes involved in regulation of cell migration
and proliferation, ECM organization, response to endogenous stimuli, develop-
mental processes, and cell adhesion were differentially expressed between primary
and iPSC-derived fibroblasts. In functional assays, iPSC-derived fibroblasts showed
improved migratory properties in two-dimensional culture. In vivo, self-assembled
3D extracellular matrix tissues containing iPSC-derived fibroblasts persisted in
the wound and facilitated diabetic wound closure to a larger degree than primary
DFU fibroblasts. Similarly, in a diabetic mouse model, grafting of tissues derived
from primary, healthy foot fibroblasts significantly improved wound healing,
compared with primary fibroblasts derived from DFU.
Together, these preclinical studies confirm not only that iPSC-derived fibroblasts
accelerate wound healing, but also that fibroblasts derived from both diabetic and
healthy patients appear to be functionally equivalent. This important finding sug-
gests the potential of reconstituting a healthy fibroblast population in DFU wounds,
with autologous cells, findings of paramount importance to diabetic patients.
280 CHAPTER 12 Induced pluripotent stem cells in wound healing
4.4 iPSC-derived mesenchymal stem cells
MSC are multipotent stem cells that can differentiate into multiple mesenchymal
lineages including myogenic, chondrogenic, osteogenic, and adipogenic derivatives
(Singer and Caplan, 2011). The main mechanisms by which MSC promote wound
healing are structural repair of wounds via cellular differentiation, immunomodula-
tion, synthesis and secretion of growth factors that drive neovascularization and
reepithelialization, and mobilization of resident stem cells (Balaji et al., 2012).
MSC isolated from streptozotocin (STZ)-induced diabetic rats were inferior to
those from healthy rats in terms of proliferation, differentiation, and expression of
angiogenic factors (Kim et al., 2015). Jin et al. found that the proliferative and dif-
ferentiation properties, as well as cytokine release and antiapoptosis ability, were
significantly impaired in BM-MSC from STZ-induced diabetic rats (Jin et al.,
2010). Nakayama et al. assessed the wound healing efficacy of iPSC-derived
MSC from an epidermolysis bullosa patient and a healthy donor in a mouse model
and on microarray analysis found that the two kinds of hiPSC-derived MSC had
similar expression patterns of dermaleepidermal junction-related genes (Nakayama
et al., 2018). hiPSC-derived MSC accelerated wound healing and restored human
type VII collagen at the dermaleepidermal junction. Although it remains to be
clarified whether healthy and diabetic-derived MSC have similar wound healing
potential, iPSC-derived MSC from healthy donors have nonetheless shown prom-
ising results in wound healing studies, holding great clinical promise.
4.5 iPSC-derived extracellular vesicles
The main mechanism by which iPSC improve wound healing appears to be by syn-
thesis and secretion of growth factors and to a lesser degree direct cellular function.
Extracellular vesicles derived from iPSC contain proteins, mRNA, and miRNA that
play important roles in repairing injured tissues. The use of extracellular vesicles
eliminates the risks of teratoma formation as they do not have nuclei and thus are
independent of cell transfer, making them a new and exciting alternative among
potential wound healing therapies (Lu et al., 2019). Extracellular vesicles derived
from iPSC-derived MSC facilitated cutaneous wound healing by promoting collagen
synthesis and angiogenesis in a rat model (Zhang et al., 2015). Kobayashi et al.
found that iPSC-derived extracellular vesicles increased the migratory ability of
fibroblasts in vitro and in vivo, and administration of extracellular vesicles resulted
in a faster wound closure in a diabetic mouse model (Kobayashi et al., 2018). Lu
et al. found that autologous and allogeneic iPSC, as well as their extracellular ves-
icles, accelerated wound healing in a macaque model, as demonstrated by wound
closure, epithelial coverage, collagen deposition, and angiogenesis (Lu et al.,
2019). Autologous iPSC and their extracellular vesicles were more effective and
viable than their allogeneic counterparts. The finding that extracellular vesicles
derived from various iPSC-differentiated cells accelerate wound healing is impor-
tant, as use of cellular free vesicles could eliminate many of the potential hazards
of using pluripotent stem cells.
4. Induced pluripotent stem cells 281
5.Challenges and solutions of iPSC in wound healing
Despite iPSC holding tremendous advantages and promise for tissue regeneration,
their risk of tumorigenicity and mutagenesis must be overcome before widespread
clinical adaption (Gutierrez-Aranda et al., 2010;Zhang et al., 2011). Residual undif-
ferentiated cells have the risk of oncogenic transformation that hinders iPSC
application in clinical trials (Lee et al., 2013a). There is now strong functional
evidence indicating that pluripotent cells and their differentiated derivatives carrying
genomic abnormalities can undergo malignant transformation (Lund et al., 2012).
Interestingly, Yasuda et al. found that 10 commercially available hiPSC lines showed
remarkable variation in tumor incidence, formation latency, and volumes (Yasuda
et al., 2018).
In addition to the use of the aforementioned extracellular vesicles, multiple stra-
tegies aimed at eliminating the teratogenic potential of iPSC are currently being
investigated. Sougawa et al. showed that brentuximab vedotin, which targets
CD30, induces apoptosis in tumorigenic cells, thus increasing the safety of iPSC
therapy (Sougawa et al., 2018). Nishimori et al. suggested that morphological
analysis might be a useful method for distinguishing between undifferentiated,
tumorigenic cells and their differentiated counterparts to select for differentiated
iPSC (Nishimori et al., 2014). Pluripotent cellespecific inhibitors (PluriSIn) selec-
tively eliminate human pluripotent stem cells (hPSC) while sparing a large array of
progenitor and differentiated cells, preventing teratoma formation from undifferen-
tiated cells (Ben-David et al., 2013). Molecules such as quercetin and YM155,
agonists of antiapoptotic signals, also successfully eliminate undifferentiated cells
in a mixed population of cells in vitro (Lee et al., 2013b). Lysine-specific demethy-
lase 1 (LSD1) deregulation underlies the development of teratomas, and its inhibitor
prevented teratoma formation after hiPSC transplant into immunodeficient mice
(Osada et al., 2018).
Another potential risk of iPSC is the use of retroviral and lentiviral reprogram-
ming vectors that can integrate into the host genome, predisposing the cell to inser-
tional mutagenesis and uncontrolled modification of the genome (Pera and
Hasegawa, 2008). However, the safest reprogramming methods to generate
transgene-free iPSC are those using RNA, protein, or small particle chemical deliv-
ery; although these methods are less efficient, they have been frequently used and are
quite promising (Malik and Rao, 2013;Deng et al., 2015). However, even with
transgene-free derivation methods, iPSC safety still needs to be assessed carefully,
as the necessary reprogramming factors are oncogenes and their overexpression is
related to proliferation and development of malignant tumors (Liu, 2008). Together,
these techniques have the potential to produce iPSC in a safer and more efficient
way, thereby allowing for successful realization of the therapeutic potential of
iPSC-based therapies.
The ideal platform for cellular delivery and in vivo retention remains to be
determined (Ho et al., 2017). The use of polymer-based biomaterials for stem
cell delivery has been studied extensively over the past several years
282 CHAPTER 12 Induced pluripotent stem cells in wound healing
(Dash et al., 2018;Asti and Gioglio, 2014;Hinderer et al., 2016). These biomate-
rials can be made of natural polymers including hyaluronan, collagen, elastin,
fibrin, and silk or synthetic polymers such as poly(lactic-co-glycolic) acid, polyan-
hydrides, polyethylene glycol, etc (Sugiyama-Nakagiri et al., 2016). These poly-
meric biomaterials may act as a microenvironment to improve stem cell survival
and paracrine activity and ultimately promote wound healing (Assi et al., 2016;
Ho et al., 2017;Catanzano et al., 2015).
Regardless of the delivery method used, cellular survival in vivo following trans-
fer remains low. Increasing cell survival in vivo with hypoxic preconditioning is an
interesting possibility (Assi et al., 2016). Hypoxia, such as that occurring in the
natural stem cell niche, stimulates stem cell growth, while hypoxia-inducible factors
play a role in cellular maintenance and homing (Lekli et al., 2009). In addition,
hypoxia increases iPSC induction and differentiation efficiency and contributes to
pluripotency (Huang et al., 2018;Sugimoto et al., 2018). Preconditioning stem cells
with hypoxia prior to transfer upregulates antiapoptotic genes and increases prean-
giogenic ones, improving in vivo survival (Tang et al., 2009). Studies examining the
effect of hypoxia on iPSC activation, homing, and survival are needed to increase
iPSC regenerative potential.
6.Conclusion
Stem cellebased therapy offers a novel and powerful strategy to achieve structural
and functional skin regeneration. iPSC are a potentially abundant source of stem
cells for disease modeling, drug discovery, and regenerative medicine. They offer
a promising approach to accelerate wound healing through release of soluble growth
factors and cytokines that stimulate angiogenesis and modulate inflammation
(Gorecka et al., 2019). However, significant hurdles remain in their safety profile,
particularly in relation to tumorigenic potential, optimizing progenitor cell selection,
and efficacious mode of cell delivery. It is important to understand and improve the
reprogramming process and the differentiation potential of iPSC before their future
use in the clinical setting. Additional studies are necessary to characterize the iPSC
niche and preconditioning strategies to increase in vivo cellular survival in order to
improve iPSC wound healing potential.
While some studies show that iPSC derived from patients with chronic diseases
can function similarly to those derived from healthy donors (Opalenik and David-
son, 2005), the data remains inconclusive (Kim et al., 2015;Jin et al., 2010), sug-
gesting further studies are needed to understand the differences between iPSC
derived from diseased and healthy donors. iPSC have the potential to differentiate
into a variety of specific cell subtypes; combining iPSC with tissue engineering
approaches, one can develop more complex tissues and organs such as tissue-
engineered skin. Autologous iPSC or iPSC from matched donors may someday be
used to provide personalized therapies. Furthermore, effective delivery vehicles
are needed to protect cells within the wound environment and provide additional
6. Conclusion 283
functional enhancement. Advances in iPSC biology and biomaterials will create
exciting opportunities for wound healing.
Despite some remaining barriers to clinical implementation, iPSC have shown
great promise in preclinical models of wound healing. iPSC are pluripotent, yet
derived from adult somatic cells. They are easy to harvest, carry increased capability
to survive in vivo, and are less affected by systemic illness than their primary adult
counterparts. iPSC derivation and differentiation methods are rapidly improving in
quantity, efficiency, and purity and may eliminate the need for viral transfection.
Lastly, use of extracellular vesicles and cell selection methods has significantly
improved the safety profile of these pluripotent cells. Further translational studies
are greatly needed before clinical application in hopes of revolutionizing wound
healing and regenerative medicine in the 21st century.
7.Future directions
• Develop safer, viral-free, iPSC transfection protocols and methods for eliminating
undifferentiated cells prior to clinical use.
• Improve efficiency of iPSC differentiation protocols and improve purity of
cellular yield.
• Optimize delivery methods to promote cellular survival and differentiation.
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290 CHAPTER 12 Induced pluripotent stem cells in wound healing
Induced pluripotent stem
cells for periodontal
regeneration 13
Ryan Bloomquist
1
, Mahmood S. Mozaffari
2
1
Department of Restorative Sciences, The Dental College of Georgia, Augusta University, Augusta,
GA, United States;
2
Department of Oral Biology and Diagnostic Sciences, The Dental College of
Georgia, Augusta University, Augusta, GA, United States
Chapter outline
1. Prevalence of periodontal diseases ..................................................................... 292
2. Overview of pathogenesis of periodontitis ............................................................293
3. Current therapies for periodontitis and their limitations ........................................295
4. Development of the dental complex ..................................................................... 296
5. Stem cells and tissue regeneration ..................................................................... 298
6. iPSCs in regenerative dentistry ........................................................................... 301
7. Clinical application of iPSCs............................................................................... 305
8. Conclusions and perspective .............................................................................. 306
Acknowledgments ...................................................................................................308
References .............................................................................................................308
Abstract
Periodontal diseases are very common affecting the soft and hard tissue support of
the teeth. Periodontitis is the most severe form of periodontal diseases, which can
lead to tooth loss affecting not only esthetic and self-esteem but also nutritional
status and general heath of affected individuals. Mechanical debridement remains
the mainstay of management of periodontitis, but other therapeutic options include
local delivery of antimicrobial agents and guided tissue regeneration. Importantly,
advances in tissue regeneration technologies coupled with our increasing under-
standing of stem cell biology have raised the prospect for regeneration of the
periodontal complex. Among the variety of stem cells, induced pluripotent stem
cells are gaining increased attention given their potential application for person-
alized healthcare. Thus, we will initially review prevalence and pathogenesis of
periodontitis along with current treatment options and their limitations. Thereafter,
and in the context of development of the dental complex, we will describe the
potential of induced pluripotent stem cells, and emerging technologies, for peri-
odontal regeneration.
CHAPTER
291
Recent Advances in iPSCs for Therapy, Volume 3. https://doi.org/10.1016/B978-0-12-822229-4.00007-3
Copyright ©2021 Elsevier Inc. All rights reserved.
Keywords:
Alveolar bone; Cementum; Dental complex; Development; Induced pluripotent stem
cells; Inflammation; iPSCs; Pathogenesis; Periodontal ligament; Periodontitis; Prevalence;
Regeneration; Therapeutic options.
1.Prevalence of periodontal diseases
Periodontal diseases affect the soft and hard tissues that support and anchor the teeth
to the bone (Figs. 13.1 and 13.2). Periodontal diseases are very prevalent in both
developed and developing countries and estimated to affect about 20%e50% of
the people worldwide. In Europe, severe periodontal disease is found in 5%e20%
of middle-aged (35e44) adults and up to 40% of older individuals, 65e74 years
old (WHO). In the United States, a recent CDC report indicates that about 47%
FIGURE 13.1
The epithelium and mesenchyme of neural crest origin, through a complex process,
which is described in the text and summarized under panel A, ultimately lead to
development of the dental complex (panel B). The dental complex is composed of the
crown and the root(s) and receives nerve and vascular supplies through the apical
foramen (foramina) leading to formation of the dental pulp (panel B). Structurally, the
tooth is composed of mineralized tissues, namely enamel, dentin, and cementum. The
root(s) of the tooth is(are) anchored in its socket in the alveolar bone by the periodontal
ligament spanning the space between the root cementum and the alveolar bone (panel
B). The dental complex is the source of a variety of stem cells, several of which are
indicated on the diagram (panel B). Abbreviations: ABSCs, Alveolar Bone Stem Cells;
DPSCs, Dental Pulp Stem Cells; GDSCs, Gingival Stem Cells (GSCs); HERS, Hertwig
Epithelial Root Sheath; PDLSCs, Periodontal Ligament Stem Cells.
292 CHAPTER 13 Induced pluripotent stem cells
of adults aged 30 years or older have some form of periodontal diseases. Several fac-
tors, both modifiable and nonmodifiable, contribute to disease progression and
severity. Modifiable risk factors include inadequate oral hygiene, smoking, diabetes
mellitus, hormonal changes in female, stress, and some medications such as those
affecting salivary gland function or those promoting gingival growth. Nonmodifiable
risk factors include hereditary predilection and aging (Nazir, 2017). Indeed, peri-
odontal diseases increase with age, affecting 70% of adults 65 years or older
(CDC). Importantly, high prevalence of periodontal diseases makes it a major public
health concern because aside from adversely impacting oral health and eventually
leading to tooth loss, periodontitis is believed to increase the risk for systemic dis-
orders such as cardiovascular diseases (Bartold and Mariotti, 2017;Hirschfeld and
Kawai, 2015). Given the far-reaching consequences of periodontal diseases, the
World Health Organization recommends comprehensive and integrated public
health preventive strategies and initiatives to curtail the burden of the disease. Effec-
tive measures include meticulous oral hygiene practices, smoking cessation, dietary
consideration (e.g., vitamin sufficiency and supplementation), and topical antimi-
crobial agents (e.g., chlorhexidine mouth rinse), among others.
2.Overview of pathogenesis of periodontitis
Periodontal diseases are mainly caused by infection and inflammation of soft tissues
and the bone that surround, support, and anchor the teeth to the mandible and
maxilla; if left uninterrupted, these processes result in loss of tooth support and sta-
bility, which cause tooth mobility and eventually tooth loss (Fig. 13.2). The
FIGURE 13.2
Diagram depicts hallmark features of gingivitis (i.e., inflamed soft tissue) and periodontitis
(i.e., attachment loss) compared to the healthy dental complex. Diagnosis of periodontitis
includes measurement of pocket depth (using a periodontal probe) and radiographic
assessment of alveolar bone surrounding the tooth (red arrows).
2. Overview of pathogenesis of periodontitis 293
pathogenesis of periodontal diseases is multifaceted involving supra- and subgingi-
val microbiota, host inflammatory and immune responses as well as the influence of
environmental factors. It is now recognized that a shift from commensal to “patho-
genic” microbiota (also referred to as dysbiosis) is a component of pathogenesis of
periodontal diseases; nonetheless, bacteria are necessary but not sufficient for the ul-
timate manifestation of periodontitis (Bartold and Van Dyke, 2017;Deng et al.,
2017;Thorbert-Mros et al., 2015;Chapple et al., 2015;Kinane et al., 2017). This
has led to the increasing recognition of the pivotal roles of innate and adaptive im-
munity and inflammatory responses in the manifestation of periodontal diseases.
Accordingly, the new paradigm for pathogenesis of periodontitis proposes that
gingivitis results from a nonspecific inflammatory reaction in gingival tissues conse-
quent to supragingival accumulation of plaque. The ensuing gingival inflammation
alters the supragingival microenvironment in response to increased concentration of
proinflammatory cytokines and accumulation of bacterial and connective tissue
breakdown products (Bartold and Van Dyke, 2017;Deng et al., 2017;Kinane
et al., 2017). The prevailing conditions provide suitable environment for overgrowth
of “periodontal pathogens” within the subgingival biofilm. Should the host inflam-
matory response be sufficient, and in the presence of favorable genetic and environ-
mental influences, the lesion may be “contained” as gingivitis and not progress to
periodontitis, although it can mature into a stable inflammatory/immune lesion
(Fig. 13.2). By contrast, if the host inflammatory and immune responses do not sta-
bilize the lesion, and with coexisting genetic susceptibility and unfavorable environ-
mental factors (e.g., smoking), the condition can progress leading to the clinical
manifestation of periodontitis (Bartold and Van Dyke, 2017;Deng et al., 2017;Kin-
ane et al., 2017)(Fig. 13.2). Clearly, immune and inflammatory responses are at the
core of etiopathogenesis of periodontal diseases.
The role and contribution of innate and adaptive immunity, and their complex
interactions, to pathogenesis of periodontal diseases have been the subject of
numerous investigations (Thorbert-Mros et al., 2015;Cochran, 2008;Lazar et al.,
2016). For example, a recent pathogenic model highlights the role of T-helper
(Th)-17 cells and interleukin (IL)-23/IL-17 axis in pathogenesis of immune-
mediated inflammatory diseases such as periodontitis (Bunte and Beikler, 2019).
Accordingly, it is proposed that dendritic cells are triggered by stimuli (e.g., bacte-
ria) leading to antigen presentation and differentiation of CD4þhelper cells as well
as release of interleukin (IL)-23. In turn, IL-23 stimulates the production of proin-
flammatory cytokines from Th-17 cells (e.g., tumor necrosis factor (TNF), IL-1b,
IL-17), macrophages (e.g., IL-6, TNF, IL-1b), and dendritic cells (e.g., IL-6).
Further, interaction of IL-17 with IL-17RA/RC complex on cells that express the re-
ceptors (e.g., endothelial cells, epithelial cells, fibroblasts, osteoblasts) further in-
creases generation of inflammatory cytokines that regulate function of dendritic
cells and create a self-sustaining feedback loop via IL-23. More recently, a new class
of tissue-resident immune cells, namely the innate lymphoid cells (ILCs), have
received considerable attention as cells in the interface of innate and adaptive immu-
nity and because of their emerging and pivotal roles in several conditions including
294 CHAPTER 13 Induced pluripotent stem cells
allergy and asthma (Artis and Spits, 2015;Hazenberg, 2014;Cortez et al., 2015;
McKenzie et al., 2014;Trabanelli et al., 2015;Bostick and Zhou, 2016;Everaere
et al., 2018). ILCs are divided into three classes (i.e., ILC1seILC3s). The develop-
ment of all classes of ILCs requires cytokine signaling as well as transcription factor
Id2. Class 1 consists of natural killer cells and IFNg-producing ILCs that utilize T-
bet for lineage commitment. Class 2 consists of type 2 cytokine-producing ILCs
(e.g., IL-5, IL-13), which express the transcription factor GATA3 and exhibit IL-
33-dependent functional activity. Class 3 constitutes proinflammatory cytokines
IL-17 and/or IL-22 producing ILCs that require transcription factor RORgt for their
lineage characteristics and function (Artis and Spits, 2015;Hazenberg, 2014;Cortez
et al., 2015). Importantly, transforming growth factor-b(TGF-b) has emerged as a
molecular regulator of development of ILCs (Cortez et al., 2016). Utilizing a murine
model of ligature-induced periodontitis as well as periodontal tissues of human sub-
jects, with confirmed diagnosis of severe periodontitis, our group recently showed
the presence and upregulation of each subset of ILCs in periodontal tissues, with
the effect more marked for ILC2s, associated with upregulation of mRNA expres-
sion for relevant cytokines (Qin et al., 2017). Collectively, the results implicate a ma-
jor role for ILCs subtype in pathogenesis of periodontitis and that upregulation of
ILC2s in this condition likely suggests as a compensatory mechanism to curtail
inflammation. Importantly, being at the interface of innate and adaptive immunity
coupled with their ability to produce a variety of cytokines, ILCs are believed to
interact with other immune cells and immune responses in conditions such as infec-
tion, allergy, autoimmunity, and cancer (Ducimetie
`re et al., 2019;Flores-Borja et al.,
2016); however, their cross talk with other immune cells in the pathogenesis of peri-
odontitis remains to be established. Thus, our greater understanding of the role of
immunity in pathogenic inflammation associated with chronic periodontitis may
eventually lead to novel therapies to prevent its incidence, slow its progression,
and/or reduce its severity.
3.Current therapies for periodontitis and their limitations
The objective of periodontitis therapy is to prevent further progression of the disease
and, if possible, restore lost tissues and support the patient to maintain a healthy perio-
dontium. Thus, treatment options include techniques to change habits and behaviors via
increasing oral hygiene awareness and instructions for lifelong periodontal self-care, di-
etary recommendations, recommendation for and support of smoking cessation, me-
chanical debridement of supra- and subgingival plaque and calculus, local
pharmacotherapy as well as a variety of surgical approaches such as flap surgery as
well as tissue and bone graft, e.g., via guided-tissue regeneration approach (Graziani
et al., 2017). Nonetheless, as recently reviewed (Artzietal.,2019), shortcomings of
existing therapeutic options include: (a) while significantly reducing plaque and calcu-
lus, oral hygiene alone produces clinically insignificant improvement of periodontal
disease status; (b) chemical plaque control (triclosan or chlorhexidine-containing
3. Current therapies for periodontitis and their limitations 295
formulations) may enhance the efficacy of patient self-care but is ineffective in halting
the progression and severity of the disease; (c) professional plaque and calculus
removal, as the sole treatment modality, is not sufficient in treating periodontitis,
but it does complement patient self-care practices; (d) subgingival debridement,
with or without soft tissue flap, would not be beneficial in the absence of supragingival
plaque control by the patient; however, combination of professional subgingival instru-
mentation and supragingival plaque control by the patient provides significant benefit
as reflected in pocket depth reduction and clinical attachment gain in sites with pocket
depth of greater 4 mm; (e) while local delivery of antimicrobial agents may be useful
in some clinical situations (e.g., localized deep pocket, refractory periodontitis), sys-
temic antibiotic use must be in the context of serious considerations such as adverse
effects and contribution to antibiotic resistance given the chronic nature of periodon-
titis; (f) residual pockets associated with intrabony defects can be treated surgically
and deep intrabony defects may benefit from addition of regenerative material; how-
ever, furcation defects pose major challenge for which several therapeutic approaches
have been proposed (e.g., guided tissue regeneration and enamel matrix derivatives
(EMDs)); (g) EMDs and guided tissue regeneration result in modest, but significant,
improvement in clinical outcomes (e.g., clinical attachment gain of 1.92 and
2.27 mm, respectively) in patients with aggressive periodontitis (Artzietal.,2019).
In light of the aforementioned, coupled with high prevalence of periodontitis, it is
clear that the need to identify novel approaches to management of periodontitis remains
and that tissue regeneration relying on the use of stem cells may offer the potential to
reduce the burden of this chronic condition. However, stem cellebased periodontal
regeneration approaches can be better appreciated in the context of information
regarding development of the dental complex, which is briefly described further.
4.Development of the dental complex
The development of the dental complex is the culmination of reciprocal interactions
between epithelial and mesenchymal cells, which occur in a sequential stepwise
fashion (Balic, 2018;Li et al., 2017;Amrollahi et al., 2016;Mozaffari et al., 2019)
(Fig. 13.1). Initially, dental epithelium causes induction of tooth formation in the
mesenchyme of cranial neural crest (NC) origin (Lumsden, 1988). Accordingly, thick-
ening of dental epithelium leads to formation of dental lamina at the site of future
tooth development. Proliferation of the epithelial cells and their transition through
various shapes, ranging from bud-, cap-, to bell-like shapes, determine the three-
dimensional (3D) form and shape of the future crown. The late bell stage is associated
with cellular differentiation in preparation for the production of mineralized matrices
and subsequent development of crown of the tooth. The production of enamel and
dentin occurs at the interface between the inner dental epithelium (or inner enamel
epithelium) and dental mesenchyme (or dental papilla). Accordingly, cells of the inner
dental epithelium differentiate into ameloblasts, which produce enamel while those of
the dental papilla differentiate into odontoblasts, which produce dentin.
296 CHAPTER 13 Induced pluripotent stem cells
Completion of development of tooth crown heralds the beginning of root forma-
tion, which is achieved by joining of inner and outer enamel cells and their prolif-
eration along with their downward movement to progressively encircle dental
papilla and assuming 3D form and size of the root of the developing tooth. Similar
to crown formation, sequential and reciprocal interactions between the epithelium
and mesenchyme determine the generation of radicular dentin, cementum, and peri-
odontal tissues, involving a number of signaling pathways (e.g., Tgfb/Bmp, Wnt,
Fgf, and Shh), which coupled with multiple transcription factors ultimately mediate
tissueetissue interactions that guide root development. The epithelial cuff encircling
the dental papilla is known as the Hertwig epithelial root sheath (HERS). The inner
enamel epithelium of the root sheath ceases to differentiate into ameloblasts but con-
tinues to promote differentiation of the dental papilla cells to odontoblasts thereby
producing root dentin. HERS secrets a number of substances with important func-
tion including laminin 5, which induces the dental papilla cell attachment, growth,
migration, and differentiation while TGF-binduces differentiation of dental papilla
cells into odontoblasts and subsequent formation of root dentin. This is followed by
fragmentation of the HERS and consequent access of the ectomesenchymal cells of
the dental follicle to the root surface and their differentiation to cementoblasts,
which ultimately produce cementum (i.e., proximity of epithelial cells of HERS
and mesenchymal cells of the dental follicle leads to cementogenesis). The
cementum is composed of cellular and acellular components, which cover the apical
one-third and the rest of the root, respectively (Huang and Chai, 2012). The final
stages of root development occur during and after tooth eruption and are character-
ized by further elongation of the root and formation of periodontal tissues that pro-
vide anchorage of the tooth to the underlying bone (Fig. 13.1).
The source of periodontal tissues is the ectomesenchymal cells of the dental fol-
licle, which produce the alveolar socket and the periodontal ligament thereby con-
necting the tooth with its socket. The dental follicle gives rise to periodontal
ligaments, which become evident when interruption of HERS is detectable. Upon
interaction with HERS, profibroblasts move to the surfaces of the root and the alve-
olar bone followed by attachment of collagen fibers to the root surface and growth
into periodontal space; embedded collagen fibers in the cementum are known are
Sharpey’s fibers. During early root development, there is a disorderly arrangement
of collagen fibers; however, with progression of root development, fibroblasts, pro-
fibroblasts, and stem cells in the dental follicle are activated and the fibers become
thicker and well-organized, which bridge the root and the alveolar bone thereby sta-
bilizing the tooth in the socket, which is conducive to mastication (Huang and Chai,
2012)(Fig. 13.1).
In summary, HERS may be considered as the developmental center of root for-
mation. While HERS continuity and function promote root dentin formation, HERS
disintegration is conducive to both cementum production and periodontal ligament
formation. Importantly, similar to development of tooth crown, development of
tooth root involves reciprocal interactions of dental epithelium and the cranial NCe
derived mesenchyme, which are mediated by a series of signaling mechanisms (e.g.,
4. Development of the dental complex 297
TGF-b, bone morphogenetic protein, Wnt, Shh, FGF) and the function of homeobox
genes (e.g., Nfic, Msx1/2). It is noteworthy that remnants of HERS become a
component of the periodontal ligament (i.e., rests of Malassez), which can be the
source of some cystic jaw lesions (Li et al., 2017;Amrollahi et al., 2016;Mozaffari
et al., 2019;Huang and Chai, 2012).
5.Stem cells and tissue regeneration
Our ever-increasing understanding of developmental and stem cell biology coupled
with major advances in tissue engineering technologies has fueled much interest in
their application to repair or replace tissues that have sustained injury due to various
diseases. Given that stem cells are a critical component of tissue regeneration pro-
tocols, better understanding of stem cell biology remains a mainstay of research.
Hallmark features of stem cells include their ability for self-renewal and differenti-
ation into other cell types. Stem cells can be classified as embryonic, fetal, umbilical
cord blood, and adult stem cells (e.g., bone marrowederived hematopoietic, mesen-
chymal stem cells (MSCs), and endothelial progenitor cells as well as tooth-derived
stem cells such as those from the dental pulp and periodontal ligament, among
others). Another mode of classification is based on stem cells differentiation poten-
tial as follows: totipotency, pluripotency, multipotency, or unipotency (Mozaffari
et al., 2019;Rajabzadeh et al., 2019). A more recent introduction in the field of
stem cell research is the induced pluripotent stem cells (iPSCs).
The seminal discovery of Sir John B. Gurdon that specialization of cells can be
reversible ultimately led to identification of conditions conducive to reprogramming
of specialized adult cells to assume a stem cellelike statediPSCs (Gurdon, 1962,
2017). Initially, iPSCs were derived from virally infected mouse (embryonic or
adult) fibroblasts encoding the following transcription factors: Oct3/4, Sox2, c-
Myc, and Klf4. Mouse iPSCs display growth properties and morphology of embry-
onic stem cells (ESC) and express their marker genes; subcutaneous transplantation
of iPSCs into nude mice produces tumors containing tissues from all three germ
layers thereby establishing pluripotency of mouse iPSCs (Takahashi and Yamanaka,
2006). Subsequently, human iPSCs were introduced, which are also capable of
generating cells characteristics of all three germ layers (Yu et al., 2007). It has
been shown that only four factors (i.e., Oct4, Sox2, Nanog, and Lin28) are sufficient
to program human somatic cells (e.g., fibroblasts) to pluripotent stem cells display-
ing essential features of (human) ESCs (e.g., normal karyotypes, telomerase activity,
expression of cell surface markers and genes). While the original method for gener-
ation of iPSCs relied on the use of viral vectors (which integrate into target cell DNA
and associated increased risk of mutagenesis and tumor formation (Durcova-Hills,
2008)), more recent advances in the field of stem cell research have made it feasible
to use nonintegrating gene delivery systems such as recombinant proteins, Sendai
virus, synthetic mRNA, and episomal vectors (Wen et al., 2017;Yan et al., 2010;
Cai et al., 2013;Bang et al., 2018). Yet, another major advance relates to the use
298 CHAPTER 13 Induced pluripotent stem cells
of urine as an initial source for the ultimate objective of generation of “patient-
specific” iPSCs (Wen et al., 2017;Yan et al., 2010;Cai et al., 2013;Bang et al.,
2018;Lee et al., 2017). Thus, the advent of iPSCs has generated much interest for
a number of applications including regeneration of the dental complex or its compo-
nents (Cai et al., 2013;Lee et al., 2017).
In addition to iPSCs developed from fibroblasts or urine-derived cells, there are a
variety of multi and pluripotent cell lines that have been developed from other so-
matic cells including dental-derived iPSCs. In the stem cell literature, the argument
has been made that with multiple transformations from a somatic state, an iPSC may
have characteristics of its original features that make it more suited for one applica-
tion than another. It has been shown that iPSCs retain an “epigenetic memory” and
that specific silencing sites, histone modifications, and overall chromatin structure
must be overcome to truly induce a somatic cell into a stem cell state (Nashun
et al., 2015). For instance, it has been shown that one such methylation on histone
site H3K9 has to be removed to make the transition from a pre-iPSC state to a
true iPSC (Chen et al., 2013). Bloomquist et al. argued that in order to regenerate
dental tissues, NC-derived taste stem precursor or progenitor cells should be consid-
ered given their homology in character, their common embryonic origin, and their
likely shared evolutionary history (Bloomquist et al., 2019)(Fig. 13.3). A landmark
study published by Hiler et al. demonstrated that pluripotent stem cells generated
FIGURE 13.3
Panel shows immunofluorescent image of sagittal section of cichlid fish taste buds.
Antibodies were used to label stem cell factor Sox2 (green) protein and colabeled with
anti-BrdU (red) following a series of pulse-chase experiments to identify resident stem
cells. The double-labeled yellow cells are considered stem (arrows). For experimental
details see (Bloomquist et al., 2019, 46).
5. Stem cells and tissue regeneration 299
from rod photoreceptors (r-iPSCs) were markedly more efficient in regenerating
retina tissue than tissue derived from embryonic or fibroblast (f-iPSCs) origin (Hiler
et al., 2015). Furthermore, they showed that the end product was more complex in
tissue architecture and more closely resembled that of functioning retinal cells.
Thus, there is clear evidence that epigenetic memory plays a role in iPSCs utility
and that these considerations will play a critical part in their therapeutic applications.
With the nature of iPSCs in mind, there are a variety of craniofacial approaches
now being considered in both developing iPSCs from oral tissues and applying
iPSCs to reciprocally regenerate structures belonging to the viscerocranium,
including that of teeth and their supporting periodontium. Perhaps what makes these
tissues so interesting in the context of regeneration is their origin. As alluded to
earlier, the mesenchyme that comprises most of the viscerocranium is NC-
derived. During early development, at the specification of the neural plate, this
special subpopulation of cells arise from germ ectoderm (Meulemans and
Bronner-Fraser, 2004) and migrate to give rise to craniofacial connective, skeletal,
and muscular tissue, the peripheral nervous system, and other tissues including
placode-derived structures such as the teeth (Gans and Northcutt, 1983). Markedly,
before their migration to their destination sites, they go through a rearrangement of
their cell cytoskeleton from epithelium to mesenchyme in what is known as an
epithelial to mesenchymal transition (EMT) (Ahlstrom and Erickson, 2009). The
unique embryonic history of NC cells and their differentiation potential make
them intriguing candidates in their use in regeneration, especially in the context
of iPSCs. Notably, NC cells are prime candidates for craniofacial and nervous regen-
eration. Several different approaches have been taken to derive NC cells from human
ESCs (Menendez et al., 2013;Lee et al., 2010;Wang et al., 2011) and induced multi-
potent neural crest (iNC) has been generated from fibroblasts (Kim et al., 2014), but
the earlier arguments still hold true in regard to epigenetic memory.
A host of stem cell lines have thus been derived from dental tissues for use in
regenerative medicine and dentistry (Fig. 13.4). Specifically to the teeth, dental
pulpal stem cells (DPSCs), periodontal ligament stem cells (PDLSCs), stem cells
from human exfoliated deciduous teeth (SHED), dental follicular stem cells
(DFSCs), dental epithelial stem cells (DESCs), and stem cells from apical papilla
(SCAP) have all been isolated and have their own unique characteristics (Mozaffari
et al., 2019;Yan et al., 2010;Yildirim et al., 2016); several sources of stem cells
from the dental complex are shown in Fig. 13.1. Stem cells isolated from the shed
teeth have been previously identified as a readily available source of stem cells
that could be banked for personalized regeneration (Miura et al., 2003). Also,
DPSCs have already been used to culture the muscle, bone, and nerve (Tatullo
et al., 2015). But with the advent of iPSCs, their potentials for regenerative purposes
are increasingly being discovered. It has been demonstrated that with retroviral
transfection with as few as three stem factor genes (Oct3/4, Sox2, and Klf4),
patient-specific iPSCs could be generated with a high conversion rate (Tamaoki
et al., 2010)(Fig. 13.4). Moreover, DPSCs precursors, human immature dental
pulp stem cells (hiDPSCs)-derived iPSCs have been proven immune-privileged
300 CHAPTER 13 Induced pluripotent stem cells
and less likely to generate an immune response upon transplantation (Beltra
˜o-Braga
et al., 2011). Owing to their ability to generate all three germ lineages, these DPSCs-
derived iPSCs have been employed to produce hepatocyte-like cells for use in
clinical scenarios such as acute hepatic failure (Chiang et al., 2015) and to stimulate
cardiac tissue repair for potential application in myocardial infarction treatment
(Gandia et al., 2008). Astonishingly, SHED have also been converted into neural-
like cells in culture (Nourbakhsh et al., 2011) and DPSCs produced functionally
active neurons in avian experiments, further having been demonstrated to guide
axon growth through stromal cellederived factor-1 (CXCL12) and its receptor,
CXCR4 (Arthur et al., 2009). It has been demonstrated that multiple dental cell types
including SHED, SCAP, and DPSCs can readily be transformed to iPSCs that are
virtually indistinguishable from human embryonic stems cells and furthermore hy-
pothesized that the ectomesenchyme/NC origin of these cells promises vast potential
to regenerate many different tissue types (Yan et al., 2010)(Fig. 13.4).
6.iPSCs in regenerative dentistry
As alluded to earlier, formation of the dental complex depends on multiple cell pop-
ulations, including dental epithelium, dental mesenchyme, and NC-like cells (Cai
et al., 2013;Duan et al., 2011;Wen et al., 2012). One would expect that stem cells
from oral tissues should be better suited for regeneration of oral soft and hard tissues
(e.g., in periodontitis) given their common developmental origin, i.e., NC cells
applicability to regeneration of tissues from epithelial and mesenchymal origin.
Indeed, the applicability of oral-derived cells for tissue engineering (e.g., periodon-
tium) has been a mainstay of research and the focus of several reviews (Soda et al.,
FIGURE 13.4
Diagram shows sources of iPSCs, along with several required transcription factors for their
generation, and their utilities in tissue regeneration. Abbreviations are defined under
Fig. 13.1 legend and in the text.
6. iPSCs in regenerative dentistry 301
2019;Dave and Tomar, 2018;Ouchi and Nakagawa, 2020). For example, human
gingiva is a readily accessible and easily obtainable source of mesenchymal stem/
progenitor cells with regenerative and immunomodulatory properties (Venkatesh
et al., 2017;El-Sayed et al., 2015). Indeed, gingival-derived mesenchymal stem cells
(GMSCs), from porcine-free gingival margin, display periodontal regenerative
capacity in vivo (El-Sayed et al., 2015).Also combining GMSCs with IL-1ra-
releasing hyaluronic-acid synthetic extracellular matrix promotes periodontal regen-
eration (Fawzy El-Sayed et al., 2015). Further, transplantation of GMSCs leads to
formation of connective tissueelike structures, thus making it a preferred cell
type for extraoral cell-based clinical application and intraoral bone regenerative pro-
cedures (Venkatesh et al., 2017;El-Sayed et al., 2015).
As described earlier, the advent of iPSCs has generated a unique opportunity for
their use in tissue and organ regeneration given their ability to differentiate to cells of
three germ layers. Indeed, non-dental-derived iPSCs can lead to alveolar bone for-
mation, cementum, and periodontal ligament (PDL) regeneration in mice (Cai et al.,
2013;Duan et al., 2011). Importantly, oral tissueederived iPSCs have also been uti-
lized for regeneration of components of the periodontal complex (i.e., bone, PDL,
cementum, and vasculature); these include iPSCs derived from gingival fibroblasts,
DPSCs, apical papilla, PDL- and other dental-derived (and usually discarded) tis-
sues including third molars, oral mucosa, and exfoliated deciduous teeth (Cho
et al., 2019). For example, Yan et al. (2010) reported on production of iPSCs
from human mesenchymalelike stem/progenitor cells of dental origin. Accordingly,
four factors (i.e., Lin28/Nanog/Oct4/Sox2 or c-Myc/Klf4/Oct4/Sox2) carried by
viral vectors were used to reprogram SHED, SCAP, and DPSCs. The resulting iPSCs
exhibited higher proliferation rates than fibroblasts, a morphology indistinguishable
from human ESCs in culture and also expressed relevant markers as well as forma-
tion of embryoid bodies in vitro and teratomas in vivo containing tissues of all three
germ layers (Yan et al., 2010). Others have also utilized human immature DPSCs to
produce iPSCs, which acquired ESC-like morphology, expressed pluripotent
markers, and also possessed stable and normal karyotypes coupled with their ability
for differentiation in vitro and in vivo (Beltra
˜o-Braga et al., 2011). Further, Oda et al.
(2010) reported that MSCs derived from extracted human third molars can be
reprogrammed to iPSCs using retroviral transduction of OCT3/4, SOX2, and
KLF4 (without the use of Myc, which is considered as an oncogene); the generated
iPSCs resembled human ESCs as exemplified by the following features:
morphology, marker expression, global gene expression, epigenetic status as well
as their in vitro and in vivo differentiation to cells of the three germ layers. Indeed,
dental pulp cells have been proposed as a source for iPSCs banking given that con-
ventional three- or four-factor reprogramming effectively establishes iPSCs from
cell lines derived from human third molareobtained dental pulp stem cells (Tamaoki
et al., 2010)(Fig. 13.4).
Given the ability of iPSCs to develop into MSCs (iPSCs-MSCs), several studies
have explored the potential of this approach for periodontal tissue regeneration
(Hynes et al., 2013). Interestingly, less inflammation and bone loss were reported
302 CHAPTER 13 Induced pluripotent stem cells
for the jaws of the iPSCs-MSC-treated mouse model of Porphyromonas gingivalise
induced periodontitis than the control group (Hynes et al., 2018). Others have shown
that, in the presence of growth/differentiation factor-5 (GDF-5), iPSC-MSCs differ-
entiate into periodontal tissue-specific lineages in vitro and in vivo leading authors to
propose the potential of using recombinant GDF-5-iPSC-MSCs coupled with hydro-
gel composites as an approach in clinical settings (Yin et al., 2017). Also overexpres-
sion of TNF-alpha-stimulated gene-6 in iPSC-MSCs resulted in decreased
inflammation accompanied with reduced alveolar bone resorption in experimental
periodontitis (Yang et al., 2014). Further, injection of bone morphogenetic protein
(BMP)-6 and iPSCehydrogel complex promoted regeneration of periodontal tissue
defect in rats (Chien et al., 2018). Thus, given the unlimited differentiation potential
of iPSCs for periodontal tissue regeneration, human-derived iPSCs are promoted as
the preferred cell source because of the possibility of establishment of a cell bank
suitable for HLA match (Okita et al., 2011).
Clearly, the use of iPSCs for regeneration of the periodontium must rely on po-
tential of these cells to produce the bone, PDL, cementum, and vasculature
(Fig. 13.4). It is demonstrated that the process of differentiation of iPSCs into oste-
ocytes can be accomplished via activation of signaling pathways such as Wnt/b-cat-
enin or Notch, which serve as molecular switch for turning on/off osteogenic
differentiation; this approach has been used to induce osteogenic differentiation of
gingival fibroblastederived iPSCs (Osathanon et al., 2017). Further, a similar strat-
egy has been coupled with the use of scaffold incorporating additional conditioning
factors to direct the acquisition of certain differentiated features. Accordingly,
gingival fibroblastederived iPSCs have been incorporated into titanium discs and
conditioned with osteogenic factors, which have resulted in formation of osteogenic
cells (Choi et al., 2017); osteogenic factors that can promote differentiation of iPSCs
to osteogenic cells include bone morphogenic protein (e.g., 2 and 7), TGF-bor the
use of materials such as the inorganic phosphate polymer (Chien et al., 2018;Vo
et al., 2012;Liu et al., 2013;Hosseini et al., 2019). Further, to facilitate PDL regen-
eration, a protocol has been described to produce high number of PDL-like stem
cells from iPSCs (Hamano et al., 2018). Accordingly, iPSCs were initially induced
into NC-like cells and then captured p75 neurotrophic receptor-positive cells (iPS-
NC cells), which were subsequently cultured on extracellular matrix derived from
human PDL cells (i.e., iPS-NC-PDL cells); these cells showed reduced expression
of ESCs and NC markers compared to iPS and iPS-NC cells but exhibited increased
MSCs markers as well as multipotency and high proliferation capacity (Hamano
et al., 2018). Chien et al. (2018) utilized an animal model of maxillary molar defects
for regeneration of component of the periodontium. Accordingly, an injectable and
thermosensitive chitosan/gelatin/glycerol phosphate hydrogel was used to provide a
3D environment for transplantation of iPSCs coupled with the use of BMP-6. The
authors report that iPSCs-BMP-6-hydrogel resulted in increased mineralization
and bone volume and promoted both PDL and cementum formation in association
with reduced markers of inflammation. These observations led the authors to
conclude that reduced inflammation likely contributed to increased regeneration
6. iPSCs in regenerative dentistry 303
of the periodontium. This observation is of relevance and importance given that peri-
odontitis represents a state of dysregulated immune and inflammatory responses and
that use of iPSCs for periodontal regeneration must be in the context of due consid-
eration of these aspects. Yin et al. (2016) produced integration-free iPSCs from
human gingival fibroblasts via delivery of reprogramming factors Oct4, Sox2,
Klf4, L-myc, Lin28, and TP53 shRNA with episomal plasmid vectors; the generated
iPSCs exhibited similar morphology and proliferation characteristics as ESCs along
with expression of pluripotent markers (e.g., Oct4, Tra181, Nanog, and SSEA-4).
Further, these integration-free iPSCs maintained a normal karyotype and displayed
decreased CpG methylation ratio in the promoter regions of Oct4 and Nanog. The
acquisition of pluripotency was confirmed utilizing an in vivo teratoma formation
assay, which indicated development of tissues representative of three germ layers.
Importantly, in vitro treatment of iPSCs with EMD or growth/differentiation
factor-5 (GDF-5) increased expression of periodontal tissue markers for the bone,
periodontal ligament, and cementum (Yin et al., 2016).
Successful regeneration of periodontium must also incorporate formation of the
vasculature. Studies in the field of tissue engineering have utilized a variety of
sources for vascular forming cells including human umbilical vein endothelial
cells, human dermal microvascular endothelial cells, endothelial colony forming
cells, embryonic stem cells; however, several limitations of these cells make
them inappropriate for incorporation into engineered constructs for personalized
regeneration (Palladino et al., 2019). Thus, the advent of endothelial cells derived
from human induced pluripotent stem cells (hiPSC-ECs) has fueled interest in their
application to regenerative tissue engineering (Taur a et al. , 200 9); iPSCs-ECs
display the same plasticity compared to immature endothelial cells (Yoder,
2015). Accordingly, with respect to hiPSC-ECs, active areas of investigations
include identifying the most effective protocols to induce pluripotency, generating
cells that display endothelial phenotype, addressing safety concerns and selection
issues (e.g., to avoid teratoma formation), and exploring in vitro and in vivo pro-
tocols conducive to the formation of functional blood vessels, among others.
Importantly, exposure of iPSC-ECs to tissue-specific cues results in generation
of mature endothelial cells resembling characteristics of resident endothelial cells
(Lippmann et al., 2012;Nolan et al., 2013), an aspect of clear relevance to peri-
odontal regeneration.
Application of iPSCs to regeneration of periodontium can rely on a wealth of
knowledge emanating from research focused on mobilization of endogenous regen-
erative potentials. This research has been based on the premise that in vivo peri-
odontal regeneration could be achieved via manipulation of the cellematerial
interface, at the defect site, whereby biomaterials and biomolecules could persuade
recruitment of endogenous stem cells for repair of the defect and/or regeneration of
new tissues (Liu et al., 2019). Given varying sizes and 3D shapes of periodontal de-
fects (e.g., ranging from small intrabony defects to large horizontal bone defects),
emerging research is focused on achieving reliable and predictable regeneration us-
ing various concepts and technologies including biofabrication of scaffolds, 3D cell
304 CHAPTER 13 Induced pluripotent stem cells
technology, and 3D bioprinting coupled with the use of biomaterials, among others
(Liu et al., 2020). Aside from facilitating iPSCs transplantation, the use of biomate-
rials may minimize risks associated with residual undifferentiated iPSCs (e.g., ma-
lignant transformation) following transplantation and provide a more conducive
environment for reprogramming efficiency and delivery of other substances (e.g.,
small molecules, microRNAs, chemicals, etc.). For example, poly (N-
isopropylacrylamide)-co-poly(ethylene glycol) hydrogel has shown good efficacy
for long-term iPSCs expansion with high growth rate and fidelity of pluripotency
in a fully defined and scalable 3D culture system for expansion and differentiation
of human pluripotent stem cells (Lei and Schaffer, 2013). Further, this hydrogel sup-
ports differentiation into cells from all three germ layers (but also teratoma forma-
tion) (McKee et al., 2015). The use of 3D culture systems (as opposed to 2D
monolayers) offers several advantages including promoting cell proliferation and
function via provision of native cellecell and cellematrix interactions thereby
improving efficiency of spatiotemporal signals (Kraehenbuehl et al., 2011;Sant
et al., 2010;Han et al., 2014). Further, 3D culture systems enhance availability of
space for cell proliferation making it feasible to scale up iPSCs expansion without
triggering the formation of unfavorable clusters and yielding higher cell densities
and larger spheroids (McDevitt, 2013). On the other hand, 3D bioprinting is condu-
cive to layer-by-layer prearrangement of biomaterials, biochemicals, and cells (e.g.,
iPSCs) with accurate spatial control thereby mimicking physiological complexities
(Guillotin et al., 2010;Moon et al., 2010;Xu et al., 2013;Poldervaart et al., 2014); it
is hoped that this technology can produce 3D functional tissues and organs for clin-
ical transplantation, an aspect of particular relevance and significance for regenera-
tion of the periodontium. Indeed, with respect to periodontal tissue regeneration, a
recent study reports fabrication of a 3D complex cell sheet composed of a bonee
ligament structure by layering PDL cells and osteoblast-like cells on a
temperature-responsive culture dish followed by ectopic and orthotopic transplanta-
tion. The authors report that the complex cell sheet group exhibited regeneration of
boneeligament structure along with the functional connection of PDL-like fibers to
the tooth root and alveolar bone (Raju et al., 2020).
7.Clinical application of iPSCs
The use of stem cells for tissue repair and regeneration has been the focus of
numerous, in vitro and preclinical in vivo, studies, some of which were described
earlier. Thus, the intense research in the field of stem cells has paved the way for
exploring clinical application of stem cells for various pathologies including peri-
odontal regeneration. A recent systematic review of human controlled clinical trials
evaluated the translational potential of different cell-based strategies employed in 11
randomized controlled trials and five controlled clinical trials (Moreno Sancho et al.,
2019). The results suggest improved outcomes for alveolar ridge preservation,
lateral ridge augmentation, and periodontal regeneration. Nonetheless, best-
7. Clinical application of iPSCs 305
performing treatment modalities among the different cell-based techniques, utilized
in those studies, could not be identified. Based on clinical and histological outcomes,
extraction socket and challenging lateral and vertical bone defects (requiring bone
block grafts) were identified as strong candidates for the adjuvant MSCs therapy
(Moreno Sancho et al., 2019).
Similarly, numerous preclinical studies have explored properties and potential
application of iPSCs for tissue regeneration. Given their unique properties and po-
tentials as described earlier, the advent of iPSCs has sparked much interest for their
clinical application as exemplified by several clinical trials (Braganc¸a et al., 2019;
Haake et al., 2019). Indeed, the first clinical trial utilizing autologous iPSCs was
designed for the treatment of neovascularization accompanying age-related macular
degeneration (ARMD), a disease that can lead to loss of central vision; autologous
source for regenerative applications bypasses host immune responses. Accordingly,
patient-derived iPSCs were differentiated to generate sheets of retinal pigment
epithelial cells (RPECs) for transplantation into the eye of a patient afflicted with
ARMD; patient’s visual acuity improved without apparent untoward effects
6 months after the procedure. This promising result prompted the investigators to
similarly treat a second patient; however, recognition that two mutations exited in
the RPECs (which were not present in the original reprogrammed fibroblasts) led
to initial discontinuation of the study (but resumed several years later) (Braganc¸a
et al., 2019;Fritsche et al., 2014;Guhr et al., 2018). Importantly, in December
2019, the first National Institute of Healthesponsored clinical trial was announced,
which seeks to use patient’s own blood cells to generate iPSCs followed by program-
ming to become RPECs in preparation for transplantation to treat ARMD (DeMott,
2019). Other interventional trials include one utilizing allogeneic iPSCs-derived
MSCs for treatment of steroid-resistant acute graft versus host disease; the outcome
of phase I of this trial suggests safety of the protocol prompting phase II studies
(Haake et al., 2019;Excellent Cohort, 2018). Other clinical trials are planned to
determine the usefulness of iPSCs for the treatment of other diseases including
Parkinsonism, advanced solid tumors, and heart disease (Braganc¸a et al., 2019).
However, to our knowledge, clinical trials exploring the potential of iPSCs for regen-
eration of the human periodontium, or components thereof, have not been carried
out. Nonetheless, the ongoing clinical trials utilizing iPSCs for the treatment of other
pathologies will further lay the scientific foundation for their use to regenerate perio-
dontium thereby offering novel interventional modalities for a very prevalent human
diseasedperiodontitis.
8.Conclusions and perspective
Our knowledge of developmental biology of the periodontium coupled with our
increasing understanding of biology of iPSCs raises the prospects of achieving the
ultimate objective of regeneration of the periodontium as an interventional approach
for management of periodontitis. Thus, one can envision use of patient’s own cells
306 CHAPTER 13 Induced pluripotent stem cells
(to circumvent immunological responses) from oral environment (given their
embryological origin and suitability) to generate patient-specific iPSCs. Such cells
coupled with the use of appropriate biomaterials and emerging technologies (e.g.,
3D cell technologies and 3D bioprinting) may ultimately lead to realization of the
dream of cell-based personalized dentistry (Fig. 13.5). Nonetheless, many chal-
lenges and hurdles remain to be addressed and resolved. In general terms, these
include insufficient knowledge of in vivo functionality, scalable differentiation pro-
tocols to generate therapeutically relevant quantities of cell, reducing undifferenti-
ated iPSCs to decrease the risk of teratomas, and incorporating “suicide genes” to
improve safety, among others (Haake et al., 2019). More specifically and as applied
to regeneration of periodontium, the challenges seem arduous given the fact that
iPSCs must be programmed to generate required cell types for the generation of
bone, cementum, PDL, and vasculature in a spatiotemporal fashion in a very
confined space and for them to be functional. Aside from such consideration, the un-
derlying pathogenic mechanisms for periodontitis (i.e., dysbiosis and dysregulated
inflammatory mechanisms) must be carefully considered in any cell-based therapy,
further emphasizing the continued importance of mechanical debridement coupled
with meticulous oral hygiene to provide an oral environment more conducive to
interventional cell-based therapies. Although challenges toward clinical periodontal
regeneration seem formidable, it is noteworthy that about 15 years after the initial
report of generation of iPSCs, and founded on considerable scientific research and
discovery in the field since then, clinical trials are now underway for application
of these cells to a number of debilitating pathologies as alluded to earlier. In this
context, iPSCs-based periodontal regeneration in clinical setting, seemingly a dream
at the present time, could become a reality.
FIGURE 13.5
Conceptual framework for regeneration of periodontal structures, relying on the use of
iPSCs (e.g., oral-derived), growth factors, biomolecules, and scaffolds as well as 3D cell
culture and 3D bioprinting.
8. Conclusions and perspective 307
Acknowledgments
The authors thank Mr. Max Perim and Ms. Annie White, Department of Medical Illustration
of the Augusta University, for the artwork.
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References 313
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Current development in
iPSC-based therapy for
autoimmune diseases 14
Anil Kumar, Jugal Kishore Das, Hao-Yun Peng, Liqing Wang, Yijie Ren,
Xiaofang Xiong, Jianxun Song
Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science
Center, Bryan, TX, United States
Chapter outline
1. Introduction ................................................................................................ .......316
2. Cellular components in autoimmunity .................................................................. 318
3. Treg cells in autoimmune disease ....................................................................... 319
4. Dendritic cells (DCs) in autoimmune disease .......................................................320
5. Autoimmune disease ..........................................................................................323
6. Diabetes mellitus ...............................................................................................323
7. Rheumatoid arthritis (RA)....................................................................................325
8. Multiple sclerosis (MS) ...................................................................................... 328
9. Conclusion and future challenges .......................................................................330
Acknowledgments ...................................................................................................331
References .............................................................................................................331
Abstract
Autoimmune diseases are modern diseases characterized by destruction of own
cells by host immune cells. The initial trigger for both systemic autoimmune
disorders and organ-specific autoimmune disorders may involve the recognition of
self or foreign molecules by innate immune determinants, which in turn trigger
inflammatory responses and drive the engagement of previously undeveloped
autoreactive T cells and B cells. Dysfunctional cytokine signaling of IL-2, IL-10,
or TGF-bmay also compromise the formation or function of Treg cells leading to
severe autoimmunity. Scientific progress has led to a variety of new treatment ap-
proaches. Most of the biological approaches in clinical use rely upon the interfer-
ence with proinflammatory cytokine function and signal transduction, inhibition of
T cell activation, or depletion of B cells. These treatment regimens showed limited
efficacy with systemic side effects. Induced pluripotent stem cells (iPSCs) can be
generated from any somatic cells and can be differentiated into all cell types, which
provide potential benefit by the generation of autologous source to overcome im-
mune rejection of therapeutic cells. iPSCs have been studied more extensively in
CHAPTER
315
Recent Advances in iPSCs for Therapy, Volume 3. https://doi.org/10.1016/B978-0-12-822229-4.00001-2
Copyright ©2021 Elsevier Inc. All rights reserved.
neurodegenerative and autoimmune disorders and have shown encouraging out-
comes. iPSC technology has been utilized with potential for regeneration of specific
neural populations or for exerting an immunomodulatory effect. iPSC technology
has created an excellent clinically safe treatment option for autoimmune diseases
while also providing suitable models to study diseases and develop new therapies
in vitro. Altogether, iPSCs have revolutionized the approach of personalized
regenerative medicine and developmental biology and given an unparalleled op-
portunity in deciphering the etiology and designing a treatment regimen. Clinical
trials with iPSC-derived therapeutic cells to treat severely debilitating degenerative
diseases are underway to ignite the preliminary approach into reality for effective
treatment without compromising patient safety.
Keywords:
Autoimmune disease; Cell-based therapy; iPSC; Multiple sclerosis; Neural stem cells;
Regulatory dendritic cell (rDC); Rheumatoid arthritis (RA); Treg cells; Type 1 diabetes (T1D).
1.Introduction
The past century had witnessed a major revolution in the scientific understanding
of autoimmune diseases while also uncovering several new challenges. These chal-
lenges occur when the host antibodies or T cells attack their own cells and tissues
due to homeostatic dysregulation, self-recognition, or tolerance. Although the un-
derlying cause of autoimmune disorders is not yet fully understood, but a genetic
predisposition has been observed in many such cases. The year 1901 was arguably
the beginning of the scientific understanding of autoimmune disorders, when Paul
Ehrlich speculated that the immune cells might target host tissues (Margo and Har-
man, 2016). Since then, scientific research has identified more than 80 different
autoimmune diseases and suspects at least 40 additional diseases that are possibly
linked to autoimmunity. The incidence and prevalence of autoimmune disorders
such as Crohn’s disease, rheumatoid arthritis (RA), multiple sclerosis (MS), and
type 1 diabetes (T1D) are increasing for unknown reasons (Bach, 2002;Campbell,
2014). The National Institute of Health (NIH) estimates that 23.5 million Americans
are affected by autoimmune diseases. Indeed, autoimmune disease remains a major
burden on health systems around the world, with T1D in the United States alone
accounting annually for w$14 billion in medical costs (DiMeglio et al., 2018).
Scientific progress in the field of immunology and drug development since the
advent of understanding of autoimmune disease has led to a variety of new treatment
approaches. Most of the biological approaches in clinical use rely upon the interfer-
ence with proinflammatory cytokine function and signal transduction, inhibition of
T cell activation, or depletion of B cells. Tumor necrosis factor alpha (TNFa)is
generally considered as the master proinflammatory cytokine. In the past decades,
the introduction of anti-TNFatherapeutics, chimeric monoclonal antibodies, and
immune checkpoint blockers revolutionized the management of autoimmune
diseases such as RA, psoriatic arthritis (PsA), systemic lupus erythematosus
(SLE), ulcerative colitis (UC), and T1D. Rituximab (Rituxan) and Ofatumumab
316 CHAPTER 14 Current development in iPSC-based therapy
(Arzerra) have been used for the treatment of RA. These are chimeric monoclonal
antibodies against the CD20 molecule present on naı
¨ve and mature B cells.
Rituximab specifically depletes B cells by apoptosis, cellular cytotoxicity, and
complement activation. Certolizumab (Cimzia) and Golimumab (Simponi) are
also anti-TNFamonoclonal antibodies used in RA and PsA. However, up to 40%
of patients have no response to anti-TNF treatment and the monoclonal antibodies
directed to deplete B cells, T cells, or blocking immune checkpoint have systemic
adverse effects (Sugumar and Chari, 2009). For instance, Rituximab causes an infu-
sion reaction, characterized by fever, chills, rash, swelling (of hands, feet, and face),
bronchospasm, and hypotension. Similarly, Belimumab (binds to BLyS) treatment
causes nausea diarrhea and headaches.
Through the several decades of understanding of autoimmune disorders, it has
become apparent that the initial trigger for both systemic autoimmune disorders
and organ-specific autoimmune disorders may involve the recognition of self or
foreign molecules by innate immune determinants, which in turn trigger inflamma-
tory responses and drive the engagement of previously undeveloped autoreactive
T cells and B cells. Regulatory T (Treg) cells are conventional component of the im-
mune system and the key regulators of peripheral tolerance. Treg cells have multiple
suppressor mechanisms to mediate antiinflammatory function. Activated Treg cells
secrete antiinflammatory cytokines, such as TGF-b, IL-10, and IL-35 (Arellano
et al., 2016). These regulatory cytokines can affect multiple cell types at the site
of inflammation. Therapies for autoimmune disorders aim to inhibit the proinflam-
matory immune response by depleting specific adaptive immune cell populations or
inhibiting the activation of immune cells in target organs (Steinman et al., 2012).
Moreover, the success of adoptive T cell therapy has inspired researchers to utilize
the regulatory capabilities of Treg cells to suppress autoimmune cells in an antigen-
specific manner (von Boehmer and Daniel, 2013). In line with these observations,
adoptive cell transfer (ACT) of Treg cells has also been shown to be a promising
candidate for autoimmune diseases, such as RA, SLE, and T1D (Scalapino and
Daikh, 2009;Bluestone and Tang, 2004). Foxp3-deficient mice develop de novo
autoimmune gastritis, thyroiditis, diabetes, dermatitis, and inflammatory bowel
disease (IBD), and die around 3e4 weeks of age (Hori et al., 2003). Indeed, it
became attractive to isolate and expand Treg cells for adoptive transfer. Unfortu-
nately, in clinical setup, the source (PBMC) of Treg cells limits the scope for its
large-scale isolation and expansion. There are several other difficulties in conven-
tional Treg cellebased therapy, limiting its effective use in autoimmune diseases:
(I) availability of autologous Treg cells is limited from peripheral blood and there
are no definite surface markers for isolation of homogenous Treg cells. Additionally,
repetitive activation and expansion of Treg cells may result in loss of FoxP3, which
is the key transcription factor to maintain the stability of Treg cells, and thereby the
associated suppressive functions in autoimmunity; (II) antigen-specific T cell recep-
tor (TCR) transduction in conventional Treg cells may result in mismatched
1. Introduction 317
TCR chain pairings with endogenous TCR chains, which might cause additional
pathogenesis. Moreover, in autoimmune-prone mice or humans, contamination of
therapeutic Treg cells by effector T cells may contain pathologic autoreactive im-
mune cells. These immune cells may have the potential to aggravate autoimmune
diseases when expanded and transferred back into the donor. Therefore, ACT with
a large number of Treg cells is often not feasible owing to difficulties in generating
these cells from patients. In one study, it has been established that the transduction of
TCR and transcription factor FoxP3 and Bcl-xL in hematopoietic stem cell differen-
tiated them into stable Treg cells (Haque et al., 2010). However, the prevalent
clinical approach used in the isolation of autologous stem cells is complicated,
and therefore, a new approach to generate a large number of Treg cells has assumed
importance in immunological research.
In 2006, successful generation of pluripotent stem cells by introducing four
different genes (Oct3/4, Sox2, Klf4, c-Myc) into mouse somatic fibroblasts revolu-
tionized stem cell biology (Takahashi and Yamanaka, 2006). These genetically
reprogrammed stem cells were induced pluripotent stem cells (iPSCs). Thereafter,
the possibility of human research using pluripotent stem cells greatly expanded,
and the ethical problems associated with the use of embryonic stem cells were
largely overcome. Somatic cells can be dedifferentiated into pluripotent stem cells
with enormous capacity to expand and the ability to be reprogrammed into specific
required cellular types under appropriate condition.
2.Cellular components in autoimmunity
The loss of self-recognition and peripheral tolerance is the central dogma of autoim-
mune diseases, which results in attack on own cells and tissues by self-antibodies or
immune cells. Autoimmune disorders are categorized into two groups: (I) systemic
autoimmune disorders and (II) organ-specific autoimmune disorders. SLE is a sys-
temic autoimmune disease, which is characterized by the presence of several auto-
antibodies against DNA and other nuclear antigens. These antibodies are produced
by self-reactive B cells. Reduction of Treg cell population in this disease is also
accompanied by a reduction in the regulatory abilities of effector T cells (Teff cells)
resulting in increased inflammation, immune complex formation, and deposition in
vital organs. T1D is an organ-specific autoimmune disorder, which is characterized
by destruction of pancreatic b-cell by autoreactive T cells or autoantibody. In this
disorder, the pancreas is unable to produce adequate insulin because of
autoimmune-mediated destruction of insulin-producing bcells, which results in
altered glucose homeostasis and hyperglycemia. Similarly, RA is an inflammatory
disease of the synovial joint, which occurs due to autoreactive antibodies against
the synovial protein. The accumulation of self-reactive T cell and B cell in RA leads
to synovitis and tissue damage. Treg cells play a critical role in maintaining self-
recognition and peripheral tolerance in all these autoimmune diseases.
318 CHAPTER 14 Current development in iPSC-based therapy
3.Treg cells in autoimmune disease
Treg cells are a distinctive T cell subpopulation and the central components of the
immune system facilitating the tolerance to self-antigens. These cells express
CD4, CD25, and FoxP3 and produce antiinflammatory cytokines, such as TGF-b,
IL-10, and IL-35, which suppresses proinflammatory cells, such as TH17 cell, autor-
eactive T cells, B cells, and professional antigen-presenting cells (APCs) in order to
maintain peripheral tolerance (Theofilopoulos et al., 2017). There are two main
classes of Treg cells: naturally occurring Treg (nTreg) cells, which develop in the
thymus; and induced Treg (iTreg) cells, which are converted from Foxp3
T effec-
tors (T effs) in the periphery (Mottonen et al., 2005). T cell development in the
thymus goes through the regulated process of negative and positive selection and
thereby results in the deletion of the autoreactive T cells. However, some autoreac-
tive T cells may escape from the thymus into the peripheral immune system and their
subsequent activation causes autoimmune pathological disorders (Sakaguchi, 2005).
In the periphery, TGF-binduces the conversion of FoxP3 negative CD4 T cells to
FoxP3-positive Treg cells. Treg cells can function in the absence of TGF-bcytokine
signaling (Flores-Borja et al., 2008). Nevertheless, CTLA- 4 and TGF-bare crucial
for Treg cellular activities and likewise CTLA-4
/
or TGF-b
/
mice have a more
severe pathophysiology when compared to mice deficient in Treg (CD4
þ
CD25
þ
)
cells (Flores-Borja et al., 2008). FoxP3 is the key transcriptional regulator expressed
by the Treg cells. This transcriptional regulator is located on the X-chromosome.
Immunodysregulation, polyendocrinopathy, and enteropathy X-linked (IPEX) is X
chromosomeelinked syndrome caused by mutation in the FoxP3 gene (Wan and
Flavell, 2007). IPEX mice are deficient in Treg population and develop complex
autoimmune diseases. T cells manipulated with ectopic expression of FoxP3 acquire
Treg cell phenotype. Furthermore, destabilizing alteration in the 30UTR of the
FoxP3 mRNA leads to approximately 90% decrease in FoxP3 expression, which
results in significant reduction in Treg cellemediated suppression. It indicates
that the expression of FoxP3 protein is directly correlated with the functional activity
of Treg cells (Williams and Rudensky, 2007).
Although the precise signaling mechanisms regulating FoxP3 expression are not
fully understood, yet TGF-b, IL-2, or TCR stimulation of T cells has been shown to
increase FoxP3 expression. In many autoimmune disorders, e.g., juvenile idiopathic
arthritis, psoriatic arthritis, MS, SLE, autoimmune hepatitis, and T1D, the numbers
and suppressive activity of circulating CD4
þ
CD25
þ
Treg cells are dramatically
reduced (Derkow et al., 2007;Veldhoen et al., 2006). Treg cells are critical for main-
taining immune homeostasis, and dysregulation of Treg population or function leads
to many autoimmune disorders. The study of Treg cellular biology has therefore
gained significance for their therapeutic potential in autoimmune diseases.
Autoimmune diseases may occur due to imbalances in peripheral effector and
Treg cells arising from defects in thymic selection. Genetic defects inducing failed
Treg cell function or inadequate Treg cell activity as well as the overwhelming of
the Treg cell responses may also cause or aggravate autoimmune diseases.
3. Treg cells in autoimmune disease 319
Dysfunctional cytokine signaling of IL-2, IL-10, or TGF-bmay also compromise the
formation or function of Treg cells leading to severe autoimmunity. Similarly, APCs
play a key role in T cell activation, and consequently, defective maturation or hyper-
costimulation of these innate immune components may also lead to altered tolero-
genic autoimmunity.
The central role of Treg cells in autoimmune diseases makes it a potential target
for iPSC-mediated adoptive T cell therapy. Adoptive transfer of Treg cells to sup-
press immune response has been proven to be beneficial in SLE and other autoim-
mune disorder, such as RA and T1D (Qian et al., 2013;Haque et al., 2012,2016a).
Therefore, it has become an ideal T cell subset in the adoptive transfer therapy of
autoimmune diseases. Generation of functional iPSC-derived Treg cells is long
and complex procedure.
Although CD4
þ
CD25
þ
FoxP3
þ
Treg cells have been generated by supplementa-
tion of regulatory cytokines, such as IL-2, TGF-b, and retinoic acid (Lu et al., 2010),
retroviral transduction of FoxP3 gene followed by the stimulation of Notch ligand
DL1 accomplishes an improved induction of functional Treg cells from iPSCs
(Fig. 14.1)(Haque et al., 2016a). Importantly, in vitro stimulation of FoxP3-
transduced iPSCs with Notch ligand passes through several checkpoints of thymic
Treg cell development. During the first 2 weeks of stimulation, it becomes pre-T
cells (CD3
þ
TCRVb
þ
CD25
CD44
CD4
þ
CD8
þ
) and thereafter transitions into
CD4
þ
Treg cells (CD3
þ
TCRVb
þ
CD4
þ
CD8
CD25
þ
CD127
CTLA-4
þ
FoxP3
þ
)
(Haque et al., 2012). However, Ag-specific Treg cells express high levels of E/P-
selectin-binding ligands, multiple adhesion molecules (e.g., the integrin aEb7),
and inflammatory chemokine receptors, such as CCR4, CCR6, and CXCR3, which
stimulate their efficient migration into the inflamed tissue (Siewert et al., 2008;Van
et al., 2008).
4.Dendritic cells (DCs) in autoimmune disease
Dendritic cells (DCs) are dedicated APCs and indispensable mediators of immunity
and tolerance. These heterogeneous cell types have diverse cell-surface markers,
anatomic location, and function. DCs play prevalent role in initiating and orches-
trating the immune response by inducing naı
¨ve T cell activation and effector differ-
entiation (Martin et al., 2003, 2007). Conventional dendritic cells (cDCs) express
several Toll-like receptors (TLRs), pattern recognition receptors (PRRs) and pro-
duce proinflammatory and antiinflammatory cytokines, such TNF-a, IL-1, IL-6,
IL-12, and IL-10 upon stimulation. cDCs are highly efficient APCs and are able
to induce antigen-specific unresponsiveness in central and peripheral lymphoid or-
gans in response to specific stimuli and thereby crucial for immune tolerance
(Schmidt et al., 2012). They are characteristically able to induce not only the prolif-
eration of Treg cells, but also anergy in autoreactive effector T cells. cDCs show
different tolerogenic phenotype is response to different stimuli, which can dictate
the functional consequences of elicited Treg cells (Gordon et al., 2014). Thus, Tregs
320 CHAPTER 14 Current development in iPSC-based therapy
function can be skewed to the desired outcome by altering the tolerogenic phenotype
of applied dendritic cells for therapeutic advantage. RelB is a crucial transcription
factor that regulates the major histocompatibility complex (MHC) molecule,
CD40 expression, and antigen presentation by APCs (Li et al., 2007; O’Sullivan
et al., 2000). Deletion or iRNA-mediated silencing of RelB in dendritic cells results
in modulation of immune response and induction of tolerance by inducing Treg cells
(Andreas et al., 2019; Bracho-Sanchez et al., 2019; Li et al., 2007; Martin et al.,
2003). DCs modified for RelB and NFkB pathway have shown to suppress joint
FIGURE 14.1
Generation of autoantigen-specific Treg cells from various sources. (1) Allogeneic Treg
cells need long-term immunosuppression to evade immune rejection. (2) Autologous Treg
cells isolated from patient do not provide enough number of cells for successful adoptive
transfer and coexpress endogenous along with autoantigen-specific TCR. (3) Isolation of
stem cell from a patient is difficult. (4) Autoantigen-specific TCR-expressing Treg cells
derived from iPSCs will give plenty of monoclonal Treg cells.
4. Dendritic cells (DCs) in autoimmune disease 321
inflammation and erosion in antigen-induced arthritis in murine models (Martin
et al., 2003, 2007). RelB-deficient DCs primed with methylated bovine serum albu-
min (mBSA) induced dose-dependent tolerance and suppressed the inflammatory
response in mBSA-induced inflammatory arthritis by spontaneous accumulation
of Treg at the site of inflammation (Andreas et al., 2019; Martin et al., 2007). Pa-
tients with autoimmune disorder also have several abnormalities in dendritic cells,
such as variations in cells proportions, differences in cytokine receptor expression
particularly inhibitory receptors, and increased expression of costimulatory mole-
cules (Mackern-Oberti et al., 2014, 2015). Recent studies using regulatory DCs
have proven to reduce effector T cells in autoimmune diseases (Harry et al.,
2010;Raiotach-Regue et al., 2012).
Plasmacytoid dendritic cells (pDCs) have also been reported in the tissues of
affected organs in SLE and other autoimmune conditions. Type I interferons
secreted by pDCs activate several downstream pathways to increase maturation
and activation of dendritic cell and mediate MHC expression and antigen presenta-
tion to the lymphocytes (Wahren-Herlenius and Dorner, 2013). Type I interferonse
mediated activation of immune response upregulates inflammation and production
of interferons, which may lead to anti-self T cell activation and B cell autoantibody
production. Polymorphonuclear lymphocytes (PMNLs) of SLE patient have shown
upregulation of interferon genes, which enhanced the disease severity, and transient
depletion of pDCs ameliorated the associated pathology with decreased transcrip-
tion of interferon-induced genes in a lupus model (Rowland et al., 2014). Despite
being a promising immunomodulator, use of DCs in the treatment regimen of auto-
immune diseases is limited due to lack of adequate DCs of desired phenotypic char-
acteristics. It is imperative for isolated and expanded DCs to achieve appropriate
phenotypic profile of tolerogenic DCs. Availability of autologous DCs is limited
to peripheral blood, which curbs the scope of generation and effective use of tolero-
genic DCs in autoimmune diseases research.
Unfortunately, in clinical setup, the source (PBMC) limits the scope for its use in
ACT with a large number of DCs often not feasible owing to difficulties in gener-
ating these cells from patients. However, the prevalent clinical approach used in
the isolation of autologous stem cells is complicated, and therefore, a new approach
to obtain cells has assumed importance in immunological research. Regulatory DCs
have recently been generated from murine iPSCs with similar phenotypic function
as that of bone marrowederived DCs. The iPSC-derived regulatory DCs (iPS-
DCregs) are characterized by high CD11b/c and low CD40, CD80, CD86, and
MHC-II expressions with a high antigen uptake ability. Moreover, iPS-DCregs in-
crease the proportion of FoxP3 expressing Tregs along with its suppressive activity
on alloreactive CD4 and CD8 cells proliferation (Sachamitr et al., 2017; Zhang et al.,
2014). These cells produced IFN-gproduction in vitro and/or in vivo and inhibited
autoimmune hepatitis by regulating CD8þT cells (Joyce et al., 2018). iPSC is
encouraging source for generation of plenty of autologous iPS-DCregs for therapeu-
tic use. iPSCs can be generated from any somatic cells, which eliminate ethical com-
plications and therefore have earned importance in immunological research.
322 CHAPTER 14 Current development in iPSC-based therapy
Altogether, iPS-DCs have prospective ability to regulate the function of autoreactive
T cells and B cells and will be a potential candidate in cell-based therapy to combat
autoimmune disorders in near future.
5.Autoimmune disease
Patient-derived iPSCs could potentially be used for generating and studying several
different autoimmune disease models (Lu and Zhao, 2013). For instance, the expan-
sion of renal-specific iPSCs by using noninvasive urinary cell collection has been
previously described (Chen et al., 2013). The analysis of pathological processes
and the regeneration of tissues using iPSC-derived from both affected and unaffected
patients are possible. However, the systemic immune dysregulation in autoimmune
diseases such as SLE leads to end organ damage, and therefore, the therapeutic or
investigative targets may be better directed toward the cellular populations and an
assessment of these effects on tolerance and autoreactivity. The pathogenesis of
autoimmune diseases, such as SLE and RA, is driven by the multifactorial interac-
tion between environment and genetics, resulting in the loss of self-tolerance
(Orozco and Barton, 2010).
6.Diabetes mellitus
Diabetes mellitus is a metabolic condition in which, body’s ability to process blood
glucose is significantly impaired, resulting in microvascular and macrovascular
complications of hyper blood pressure and blood sugar. Insulin is the hormone
responsible for processing of glucose in the body. In diabetes, insulin signaling is
compromised by either lack of insulin or dysfunction or destruction of insulin-
producing bcells of pancreatic islets. There are two main subtypes of diabetes:
(1) type 1 diabetes (T1D), which arises due to lack of adequate insulin because of
autoimmune-mediated destruction of insulin-producing pancreatic bcells (Katsarou
et al., 2017); and (2) type 2 diabetes (T2D) in which receptors on cell surface do not
respond to insulin signaling. Current treatment approach for T2D is mainly based on
the reduction of insulin resistance, whereas T1D is clinically treated through regular
supplementation of exogenous insulin. Consistent and appropriate intake of insulin
is not only very difficult for individual patient management, but also has the associ-
ated risk of unstable sugar level or hypoglycemia. Engraftment of pancreatic tissue
is another way to treat T1D patients, but it requires an identical donor tissue and life-
long immunosuppression. In line with the risk factors associated with conventional
diabetes treatment regimens, the renewal of pancreatic bcells and prevention of
autoimmune destruction could be considered as an ideal strategy for the treatment
of T1D. Recent advancement in stem cell therapy has provided an elegant and novel
methodology for the replacement of pancreatic bcells. Pancreatic bcells have been
6. Diabetes mellitus 323
differentiated from embryonic stem cells with similar insulin-producing efficacy
(Schiesser and Wells, 2014;Micallef et al., 2012;Zhang et al., 2009).
However, the isolation and expansion of autologous stem cells have many ethical
and biological limitations. In the view of current scientific advancements, iPSCs
have emerged as the most relevant approach in comparison to replacement of
pancreatic bcells, in treatment of clinical autoimmune diabetes. iPSCs can be
induced from somatic cells, which differentiate into bcells. Autologous iPSC-
derived bcells overcome many ethical and immunogenicity-related limitations
and therefore pave the way for effective management of autoimmune diseases.
Moreover, iPSCs have been differentiated into mature insulin-producing cells
from patients with T1D and T2D (Teo et al., 2013;Kudva et al., 2012). These
insulin-producing cells coexpress mature bcellespecific markers, such as NKX6-
1 and PDX1, indicating a similar gene expression pattern as adult islet bcells
in vivo. Additionally, human embryonic stem cellederived pancreatic progenitor
cells have also been successfully engrafted following the study of insulin secretion
and maintenance of normoglycemia in a murine model of induced diabetes (Kroon
et al., 2008). Subsequently, iPSC-derived insulin-producing cells also have been
generated and shown to have the ability to ameliorate hyperglycemia in murine
models (Van Hoof and Liku, 2013).
It is also essential to maintain and prevent further destruction of replacing
pancreatic islet cells, after patient-specific iPSC generation and autologous transfer.
T1D is caused by autoimmune destruction of insulin-secreting bcells, raising the
possibility that iPSC-derived insulin-producing cells may lose insulin-secreting ca-
pacity over the time. Similarly, the host may lose engrafted pancreatic bcells due to
excessive inflammation or hyperactive immune cells. Although the mechanism
behind the destruction of pancreatic bcells is not known completely, autoreactive
T cells and hyperinflammatory responses may be predicted to have significant
impact on it. Pancreatic-resident Treg cells can dramatically ameliorate the delete-
rious consequences of a hyperactive immune system. iPSCs can be exploited to
obtain a renewable source of autologous Treg cells to suppress autoreactive immune
cells in T1D as they are able to produce almost all cell types, including Treg cells.
However, auto antigen-specific Treg cells differentiated from iPSCs may provide
potent and targeted immune suppression. Ag-specific Treg cells are more efficient
in suppressing local autoimmune disorders, such as RA, T1D, IBDs, and graft-
versus-host disease (GVHD) (Haque et al., 2016a,b;Song, 2016a,b;van Herwijnen
et al., 2012). Auto Ag-specific iPSC-Treg cells can be programmed to be tissue-
associated and infiltrate into local inflamed pancreatic tissue to suppress auto-
immune responses after adoptive transfer, thereby avoiding potential universal
immunosuppression from nonspecific Treg cells. The approach that the use of iPSCs
transduced with Ag-specific TCR and the transcriptional factor FoxP3, followed by
Notch signalingedriven differentiation enables iPSCs to pass hematopoietic and T
lineage differentiation checkpoints and stemming in the differentiated Ag-specific
CD4
þ
Treg cells (Haque et al., 2012,2016a;Song, 2016a). Auto Ag-specific
iPSC-Treg cells can reduce overall immunosuppression after adoptive transfer,
324 CHAPTER 14 Current development in iPSC-based therapy
which drives forward the maintenance of engrafted insulin-secreting bcells by pre-
vention of autoimmune destruction. This enhances the therapeutic potential of iPSC-
Treg cells for cell-based therapies in T1D. These evidences indicate that the optimal
treatment strategy for management of the pathophysiology of autoimmune T1D may
be achieved by the engraftment of autologous iPSC-bcells in the host, concurrently
with prevention of bcell destruction in the pancreas coupled with the suppression of
autoreactive tissue-resident immune cells (Fig. 14.2). The adoptive transfer of auto
Ag-specific iPSC-Treg cells has therefore provided clinicians with a robust tech-
nique to safely manage and treat diabetic complications.
7.Rheumatoid arthritis (RA)
RA is a chronic autoimmune disorder characterized by joint pain and inflammation
of the synovial membrane. Although the precise etiology for the development of RA
disorder is still unclear, autoreactive T cells and B cells are considered vital for the
pathogenesis of this disease. Accumulation of inflammatory cells, such as T cells, B
cells, and macrophages in the joints leads to inflamed synovial membrane and tissue
FIGURE 14.2
Schematic representation of the iPSC-based therapeutic strategy: In the early stage of T1D,
bcells can be restored by suppression of inflammation and autoreactive Tcells or B cells by
injecting autoantigen-specific Treg cells. In the late stage, iPSC-bcells can be engrafted
followed by injection of Ag-specific Treg cells to suppress preexisting inflammation and
hyperactivity of autoreactive T cells or B cells in order to restore bcell function.
7. Rheumatoid arthritis (RA) 325
destruction, especially the destruction of articular cartilage. In RA, a wide variety of
cells, including B cells, macrophages, DCs, neutrophils, fibroblasts, and granulo-
cytes, infiltrate into the normal and relatively avascular synovium. Plasma cell infil-
tration and formation of immunoglobulin aggregates have also been observed in the
joint. In 70%e80% of patients, the IgM class is the most abundant type of immune
complexes (Haque et al., 2014).
Treg cells regulate immune homeostasis and maintain peripheral tolerance
through secretion of cytokines such as TGF-b, IL-10, and IL-35 (Arellano et al.,
2016). These regulatory cytokines can affect multiple cell types at the site of inflam-
mation. Significant increase in the frequency of Treg cells (CD4
þ
CD25
þ
) had been
observed in synovial fluid from patients with RA (Mottonen et al., 2005). Despite the
presence of Treg cells in the joints of patients with RA, these cells did not retain
normal immunosuppressive activity. In RA patients, Treg cells in the synovial fluid
are exposed to a variety of inflammatory cytokines. The functional defect of Treg
cells in RA was due to higher secretion of TNF-ain inflamed synovium, which leads
to the abnormal phosphorylation and thereby reduction in the expression of FoxP3,
coupled with defective expression of CTLA-4 (Flores-Borja et al., 2008;Valencia
et al., 2006). Moreover, many therapeutic strategies used for RA are centered on
blocking the cytokines or receptors, such as TNF-a, IL-6, IL-1 receptor antagonist
and soluble CTLA-4 as summarized in Table 14.1.
The precise mechanism of action of these biological therapeutics is not
completely understood, but they have been clinically effective and safe in the treat-
ment of patients with RA. However, many patients are insensitive to anti-TNF ther-
apy and the therapeutic antibodies directed to deplete B cells, T cells, or blocking
immune checkpoint. These biological therapeutic regimens have also resulted in
systemic adverse effects. After the success of the biological therapies in RA, new
therapeutic explorations with the ultimate goal of reaching a better clinical outcome
are desirable. Adoptive transfer of engineered Treg cells decreased inflammatory
Table 14.1 Biological therapy used in rheumatoid arthritis (RA).
Commercial
name Target Function References
Adalimumab, TNF-aReduces the inflammatory effect
of TNF-a
De Stefano et al.
(2010),Mori and
Sugimoto (2012)
Tocilizumab
(mAb)
IL-6 Restores Th17/Treg ratio Navarro-Millan et al.
(2012)
Anakinra
(antagonist)
IL-1ra Restores Th17/Treg ratio Quartier et al. (2011)
Abatacept CTLA-4-
Ig
Augments the suppressive
capacity of Treg cells
Schiff (2011)
Methotrexate
(MTX)
Antifolate Suppresses purine and
pyrimidine synthesis and inhibits
DNA replication
Coury and Weinblatt
(2010)
326 CHAPTER 14 Current development in iPSC-based therapy
knee swelling and reduced Th17 cells significantly (Wright et al., 2009). Treg cells
restricted the detrimental effects of Th17 cells by reducing the accumulation at
inflamed synovium (Lohr et al., 2006). However, Treg cellebased ACT requires a
large number of cells and allogeneic Treg cells can induce immunological compli-
cations; this regimen is often not feasible owing to difficulties in isolation of these
cells from patients.
iPSCs are more desirable for adoptive cell therapy owing to their strong potential
of renewal and ability to differentiate into numbers of different cell types in the body.
Moreover, iPSCs can be easily generated from somatic cells of patients and genet-
ically manipulated to exhibit desirable characteristics, identical to those of embry-
onic stem cells. Recent studies have shown that reprogrammed functional Treg
cells derived from iPSCs can be used for adoptive immunotherapy of diabetes and
autoimmune arthritis (Haque et al., 2012,2014,2016a;Song, 2016a;Lei et al.,
2012). ACT of the iPSC-derived Treg cells has been shown to suppress the develop-
ment of arthritis in a murine model (Haque et al., 2012). Success of adoptive T cell
therapy inspired the researcher to utilize the regulatory capabilities of Treg cells to
suppress autoimmune cells in an antigen-specific manner (von Boehmer and Daniel,
2013). Antigen-specific Treg cells differentiated from iPSCs will be more effective
at targeted immunosuppression. iPSC-derived functional Treg cells are generated
using retroviral transduction of FoxP3 followed by Notch ligand DL1 and DL4
stimulation. In a recent study, a new approach has been developed to generate
antigen-specific Treg cells from iPSCs, i.e., Ag-specific iPSC-Treg cells, which
have ability to suppress autoimmunity in a murine model of RA. In this study, mu-
rine iPSCs were retrovirally transduced with a reporter construct containing genes of
MHC II (I-Ab)-restricted ovalbumin (OVA)-specific TCR and the transcriptional
factor FoxP3. Transduced iPSCs were differentiated into OVA-specific iPSC-Treg
cells with an OP9 stromal cell line expressing Notch ligands DL1, DL4, and I-A
b
in the presence of recombinant cytokines rIL-7 and rFlt3L. Adoptive transfer of
such OVA-specific iPSC-Treg cells dramatically reduced the number of Th17-
producing cells in the OVA-injected knees and reduced the inflammation, joint
destruction, cartilage proteoglycan depletion, and osteoclast activity and thereby
suppressed autoimmunity (Haque et al., 2016b;Song, 2016b). These findings
strengthen the principle of utilizing antigen-specific iPSC-Treg cells as a novel
approach for intervening autoimmune arthritis.
It is known that RA is an autoimmune disorder, but the knowledge of specific
autoantigens involved in the disease is still lacking. Autoantigen-specific iPSC-
Treg cells can be programmed to be tissue-specific and infiltrate into local inflamed
pancreatic tissue to suppress autoimmune responses after adoptive transfer, thereby
avoiding potential universal immunosuppression from nonspecific Treg cells.
Recent clinical approach targeted autoantigens that are upregulated in inflamed
synovium (Bluestone and Tang, 2004;Wieten et al., 2010). Other studies
have used heat shock protein (HSP) as autoantigen and found excellent clinical
remission in juvenile RA (Kamphuis et al., 2005). Studies using HSPs have
uncovered the selective and potent disease-suppressive activity in inflamed tissues
7. Rheumatoid arthritis (RA) 327
(van Herwijnen et al., 2012). Therefore, generation of iPSC-Treg cells specific to
HSPs will have great therapeutic potential in interventions of autoimmune arthritis.
8.Multiple sclerosis (MS)
MS is an autoimmune disorder associated with the central nervous system (CNS)
characterized by focal or multifocal inflammatory demyelination and degradation
of neural tissue, which result in neurological disability depending on the area of
the CNS involved. In MS, the immune cells damage the protective covering (myelin
sheath) of nerve fibers, which results in impairment of communication between the
brain and the rest of the body. Currently, more than 2 million people are suffering
from neurological disability MS worldwide with the number approaching
1,000,000 in the United States. MS is more prevalent in woman as compared to
the men, which account for at least 75% affected cases (Bove and Chitnis, 2014;
GBD Neurological Disorder Collaborator Group, 2017). The pathogenesis of MS
is not fully understood, and it is triggered by several factors ranging from infectious
pathogens, environmental factors, and complex interaction between genetic predis-
position resulting in immune dysregulation.
In the 1970s, it was identified that the human leukocyte antigen (HLA) class II
haplotype HLA-DRB*1501 allele is associated (Svejgaard, 2008). There are more
than 50 associated genetic loci have been identified by the Genome-wide Associa-
tion Study (GWAS), many of which encode for proinflammatory such as IL-2,IL-
7, CXCR5, IL-12A, IL-12b, and IL-12Rb1-(Hoglund and Maghazachi, 2014;
Sawcer and Hellenthal, 2011; International Multiple Sclerosis Genetics et al.,
2007). Other than genetic association of inflammatory cytokine encoding genes,
development of MS is also associated with vitamin D3 availability and Epsteine
Barr virus (EBV) infection. Increased latitude is an important geographical factor
associated with lower serum levels of vitamin D3 due to lower exposure to sun light.
Decreased serum vitamin D3 corresponds to the higher incidence and prevalence of
MS in high-latitude countries (Hoglund and Maghazachi, 2014;Simpson et al.,
2011). Recent studies have shown that vitamin D3 induced the differentiation of
T regulatory cells (Treg cells), while inhibiting Th17 cell proliferation. Moreover,
vitamin D3 promoted the secretion of the antiinflammatory cytokine, TGF-b1 and
suppressed proinflammatory cytokines such as IL-17. These observations are in
accordance with higher prevalence of MS in high-altitude countries (Prietl et al.,
2010;Zhou et al., 2017). It has also been observed that EBV seronegative individ-
uals have almost no risk of developing MS (Ascherio and Munger, 2007). This
observation leads to the hypothesis that EBV mimics myelin basic protein, which
is presented on HLA-DRB1*1501, and thereby myelin sheath was targeted by
induced inflammatory immune mediators, which provide the solid link to both
EBV infection and genetic risk factors (Hoglund and Maghazachi, 2014;Lang
et al., 2002). Vitamin D3 deficiency, Treg dysfunction, activation of inflammatory
lymphocytes, higher percentage of Th1 and Th17 cells activating CD4 T cells,
328 CHAPTER 14 Current development in iPSC-based therapy
CD8 T cells, and B cells have been predominantly reported in MS patients (Koch
et al., 2013;Viglietta et al., 2004;McFarland and Martin, 2007). Disruption of
the bloodebrain barrier has also been reported, which could be caused by inflamma-
tory cytokines and matrix metalloproteinases disrupt secreted by the activated im-
mune cells and glial cells, which further aggravate the damage to neural tissue by
initiating cascade of inflammatory events (Obermeier et al., 2013;Babbe et al.,
2000;Disanto et al., 2013;Trapp and Stys, 2009;Trapp et al., 1998).
With the limited understanding of the pathogenesis of MS, it has been ascer-
tained that damage to myelin sheath of axons and associated damages to CNS in
MS are mediated by inflammatory response initiated by proinflammatory cytokines
and activated T cells and B cells. Although remission and recovery are not fully un-
derstood but are downregulation by inflammation by suppression of myelin reactive
CD4 cells, B cells, and proinflammatory cytokines through the expansion of regula-
tory cells such as Foxp3-positive T cells and regulatory DCs (Zhou et al., 2017;
Viglietta et al., 2004;Compston and Coles, 2008). The regulatory cells that are
downregulated in autoimmune MS can suppress reactive immune cells and secretion
of inflammatory cytokines and thereby suppress the disease progression. Involve-
ment of B cells in MS immunopathogenesis is reinforced by the presence of oligo-
clonal immunoglobulins in the CSF. Moreover, depletion of B cell by monoclonal
antibodies (rituximab and more recently ocrelizumab) has shown promising efficacy
in remitting relapsing MS (RRMS) in patients with primary progressive disease
(Hoglund and Maghazachi, 2014;Michel et al., 2015).
At present, most of the biological approaches in clinical use for MS treatment
rely upon the interference with proinflammatory cytokine function and signal trans-
duction, inhibition of T cell activation, or depletion of B cells. Owing to the signif-
icant effect of MS on affected patients, it is imperative to develop better approach to
provide regenerative or immunomodulatory therapy to curb associated inflammatory
damage and regeneration of damaged tissue. Recent development in stem cell
research draws significant attention to cell-based regenerative approach to combat
complex metabolic and immune disorders. iPSCs technology provided potential
possibilities to development of regenerative therapeutic regimen to complex im-
mune disorders. iPSCs can be generated from any somatic cells and can be differen-
tiated into all cell type, which provide potential benefit by generation of autologous
source to overcome immune rejection of therapeutic cells. iPSCs have been studied
more extensively in neurodegenerative and autoimmune disorders and have shown
encouraging outcomes. IPSC technology has been utilized with potential for regen-
eration of specific neural populations or for exerting an immunomodulatory effect
(Trapp and Stys, 2009;Mattis and Svendsen, 2011). Importantly, the final common
pathway of neural injury and death is better understood in MS than for neurodegen-
erative conditions. In 2010, neural stem cells (NS cells) had been generated from
iPSCs, derived from mouse fibroblasts (NS-(f)iPS). NS-(f)iPS express neurogenic
glial marker and exhibit long-term expansion and differentiated to produce neurons,
astrocytes, and oligodendrocytes with an efficiency similar to ES-derived NS cells
(Onorati et al., 2010). iPSCs-derived NS cells (NS-(f)iPS) provide regenerative
8. Multiple sclerosis (MS) 329
autologous neural cell and glial cells to remyelinate axons after an episode of acute
demyelination, to protect axons from ongoing inflammation and eventual gliosis.
Axonal loss is responsible for the most debilitating functional deficits in the more
progressed stages of MS; this loss followed by retrograde neural degeneration and
replacement with iPSCs-derived oligodendrocyte precursor cells (OPCs) have
remyelinated the axonal damage and ameliorate the disability in experimental
autoimmune encephalomyelitis (EAE) (Filippi et al., 2003;Czepiel et al., 2011;
Tallantyre et al., 2010;Bhise and Dhib-Jalbut, 2016;Sher et al., 2008).
iPSCs-derived neural precursor cells (NPCs) have also shown immunomodula-
tory effect along with regenerative effect in EAE. Intravenous administration of
NPCs ameliorated EAE by selectively inhibition of pathogenic Th17 cell differen-
tiation. iPSC-derived NPCs produce the specific neurotrophin, leukemia inhibitory
factor (LIF), which supports the in vivo survival and differentiation of native oligo-
dendrocytes in mice with EAE (Laterza et al., 2013). LIF also inhibits the differen-
tiation of Th17 cells by antagonizing the IL-6-mediated phosphorylation of STAT3,
thus limiting CNS inflammation and hence subsequent tissue damage (Cao et al.,
2011). Neural precursor cell (NPC) therapy is considered a promising treatment mo-
dality for MS. With the electrophysiological differences, autologous iPSCs-derived
neurons overlay the way for a novel approach to the study of MS pathogenesis (Song
et al., 2012). Finally, iPSCs neural and glial cells may give unique insights to curb
the pathogenesis in neural disease regeneration of damaged tissue inflammatory dis-
eases of the CNS.
9.Conclusion and future challenges
iPSC technology has created an excellent, clinically safe treatment option for auto-
immune diseases while also providing suitable models to study diseases and develop
new therapies in vitro. Moreover, iPSCs can be generated from any somatic cells
from the patients and differentiated into any kind of the body’s cells. Autologous
iPSC-derived cells overcome many ethical and immunogenicity-related complica-
tions (Harry et al., 2010;Raiotach-Regue et al., 2012). iPSC technology has been
applied extensively in diverse disease models and organ replacement strategies.
Autoimmune diseases comprise of complex cellular and immunological events. It
requires unique immunomodulatory therapeutic strategies employing cellular com-
ponents, such as Treg cells, DCs, pancreatic bcells, and other such components that
could be engineered by iPSC technology safely. iPSC technology has enabled us to
produce, differentiate, and genetically modify large numbers of desired cells that can
be used therapeutically. Nevertheless, these novel approaches will need to have
extensive functional and safety assessments prior to their use in a clinical setting.
Although the potential benefits of autologous iPSC therapies bring forth lots of
envisions, there are challenges and limitations that need to be addressed. For
instance, the in vitro culture and expansion of iPSCs could result in the accumulation
of genetic alterations over a period. Similarly, in patient-derived autologous iPSCs
330 CHAPTER 14 Current development in iPSC-based therapy
for age-related macular degeneration, concerns regarding the genetic alterations
make it challenging to accomplish clinical-grade iPSCs (Mandai et al., 2017).
Another critical hindrance in clinical use of autologous iPSCs is the display of
immature functional characteristics. iPSC-based therapy has proven to be successful
in controlling the early-onset diseases, such as long QT syndrome and spinal
muscular atrophy (Moretti et al., 2010). Several alternate approaches are under
investigation to induce the maturation of these primitive iPSC phenotypic cells,
including treating the cells with inhibitors of protein degradation and the use of
mitochondrial stress inducers. However, these approaches have achieved only
modest maturation of iPSCs, while inducing their early aging (Studer et al., 2015).
The clinical trials show safety, feasibility, and potential therapeutic activity of
Treg cellebased therapies, but a major concern about using this approach is the
cross-reactivity of autoantigen specific Treg cells with healthy tissue (van Loenen
et al., 2010). In addition, iPSC-Treg cells usually have short-term persistence
in vivo (Kim et al., 2015). Apart from that, clinical-grade autologous iPSC produc-
tion also involves a high production cost for patient-specific iPSC-derivation by
reprogramming and differentiation into therapeutic phenotype. Successful achieve-
ment of therapeutic phenotypic cell from autologous human iPSCs is time-taking
and expensive, which limit their applicability in severe disease conditions.
Altogether, iPSCs have revolutionized the approach of personalized regenerative
medicine and developmental biology within a short time span and given an unpar-
alleled opportunity in deciphering the etiology and designing a treatment regimen
for several complex human complications, such as RA, PsA, SLE, ulcerative colitis
(UC), and T1D. Clinical trials with iPSC-derived therapeutic cells to treat severely
debilitating degenerative diseases are underway to ignite the preliminary approach
into reality for effective treatment without compromising patient safety.
Acknowledgments
This work was supported by the National Institutes of Health Grant R01AI121180,
R01CA221867, and R21AI128325 and the American Diabetes Association (1-16-IBS-281)
to J.S.
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338 CHAPTER 14 Current development in iPSC-based therapy
Index
‘Note: Page numbers followed by “f” indicate figures and “t” indicate tables.’
A
AA. See Alopecia areata (AA)
Achondroplasia, 214e215
Acrosin, 260
Acute liver failure (ALF), 226e227
Acute-on-chronic liver failure (ACLF), 226e227
AD. See Alzheimer’s disease (AD)
Adipose tissue mesenchymal stem cells
(AT-MSCs), 76e78
Adult stem cells (ASCs), 73e79
Age-related macular degeneration (ARMD), 139,
306
cellular therapies for, 159e166, 160f
diagnoses, 156
pathogenesis of, 156
retinal pigmented epithelium (RPE), 156
blooderetina barrier (BRB), 157, 158f, 159
clinical trials, 162e164
genomic instability, 164e166, 165f
induced pluripotent stem cells (iPSCs),
161e162
phototransduction, 156
AI. See Artificial insemination (AI)
Allogeneic transplantation, 183
Alopecia areata (AA), 2e4, 3f
AlperseHuttenlocher syndrome (AHS), 238
Alzheimer’s disease (AD), 51e52
dementias, 100
endogenous approach, 100
exogenous approach
embryonic stem cells (ESCs), 102
induced pluripotent stem cells (iPSCs),
102e103
mesenchymal stem cells (MSCs), 104e113,
105te108t, 116t
neuronal stem cells (NSCs), 103e104
stem cells classification, 101e102, 101f
frontotemporal dementia (FTD), 99
hippocampus, 98e99
mesenchymal stem cells (MSCs), 99
microtubule-associated protein tau (MAPT), 99
neurofibrillary tangles, 99
Amantadine, 50e51
Amyotrophic lateral sclerosis (ALS), 81e82, 104
Androgenetic alopecia, 2e4
Angiogenesis, 70e71
a1-Antitrypsin (A1AT), 236
Aquaporin (AQP), 159
Arrhythmia, 207e208
Artificial insemination (AI), 255e256
ASCs. See Adult stem cells (ASCs)
Autoimmune diseases, 316e318, 323, 330e331
cellular components, 318
dendritic cells (DCs), 320e323
diabetes mellitus, 323e325, 325f
multiple sclerosis (MS), 328e330
rheumatoid arthritis (RA), 325e328, 326t
Treg cells, 319e320, 321f
Autologous myoblasts, 207e208
Autologous transplantation, 183
B
Basal forebrain cholinergic neurons (BFCNs), 102
Basic fibroblast growth factor (bFGF), 136e137,
208e209
Bioartificial liver (BAL) systems, 233
Bioreactor system, 210e211, 210f
Blooderetina barrier (BRB), 157, 158f, 159
Bone marrow mesenchymal stem cells
(BM-MSCs), 76e78
Bone marrow stem cells (BMSCs), 78
Bone morphogenetic protein 4 (BMP4), 208e209
Brain-derived neurotrophic factor (BDNF),
51e52, 135
C
CADASIL, 84e87, 85fe88f
CD30 antigen, 213e214
Cell replacement therapy, 143
Cell therapy
clinical grade human-induced pluripotent stem
cells (hiPSCs), 30e31
dopamine (DA) cells, 29e30, 30f
efficacy tests, 27
fetal cell transplantation, 27e28
genomic stability of, 31
heart failure, 207e208
human embryonic stem cells (hESCs), 28
human-induced pluripotent stem cells (hiPSCs)
derived cell grafts, 33
liver disease, 231e233, 232t
non-induced pluripotent stem cells (iPSCs)
autologous cell sources, 29
pancreatic beta cells, 189e193
339
Cell therapy (Continued)
substantia nigra pars compacta (SNpc), 26
transplantable midbrain dopamine (mDA)
progenitors, 31e33, 32f
transplantable tissue, 27
Central nervous system (CNS), 328
Certolizumab, 316e317
Cholangiocytes, 229
Cholinesterase inhibitors, 100
Ciliary neurotrophic factor (CNTF), 135
Claudin-6, 213
Clustered regularly interspaced short palindromic
repeats (CRISPR), 134, 144e145,
276
Crohn’s disease, 316
Cryopreservation, 256e257
Current good tissue practices (cGTPs), 185
Cytokines, 207e209
Cytosine-adenine-guanine (CAG), 50
D
Dementias, 98e100
Dendritic cells (DCs), 320e323
Dental complex, 296e298
Dental pulpal stem cells (DPSCs), 300e301
Dermal papilla (DP), 4e6
Deutetrabenazine, 50e51
Diabetes mellitus, 323e325, 325f
Dickkopf-1 (Dkk-1), 136e137
3D integumentary organ system (3D-IOS), 15
Disease-in-a-dish models, 81e82
Drug-induced liver injury (DILI), 227, 238e239
Dry age-related macular degeneration (AMD), 156
Dysbiosis, 293e294
Dyskinesia, 25
E
Eectodermal precursor cells (EPCs), 10e11
Embryogenesis, 257e258
Embryoid bodies (EBs), 137, 260
Embryonic stem cells (ESCs), 252, 298e299
Alzheimer’s disease (AD), 102
Huntington’s disease (HD), 53, 55
wound healing, 274
EMIs. See Epithelial-mesenchymal interactions
(EMIs)
Enamel matrix derivatives (EMDs), 295e296
Endothelial cells (EC), 159
End-stage liver disease (ESLD), 226, 232e233
Epidermal growth factor (EGF), 260e261
Epilepsy, 104
Epithelial-mesenchymal interactions (EMIs), 4e7
F
Female pattern hair loss (FPHL), 2e4, 3f, 15e16
Fetal bovine serum (FBS), 136e137
Fetal cell transplantation, 27e28
Fibroblasts, 271
Filtering bleb, 145
Follicle stimulating hormone (FSH), 259
FOXG1, 57e58
Frontotemporal dementia (FTD), 99
G
Gamete differentiation, 257e258
Gene correction, 59
Gene signaling pathways, 257e260, 258f
Genetic diversity, 256e257
Genomic instability, 164e166, 165f
Germ cell transplantation, 255e256
Gingival-derived mesenchymal stem cells
(GMSCs), 301e302
Gingivitis, 292e293, 293f
Glaucoma, 134, 143e145
Golimumab, 316e317
GoogLeNet, 163e164
Granular osmiophilic material (GOM), 84e85
Growth and differentiation factor-11 (GDF-11),135
Growth and differentiation factor-15 (GDF-15),
135
GSX2, 57e58
H
Hair cycle, 4e6, 5f
Hair follicles (HFs)
dermal cells, trichogenic activity, 11e13, 12f
folliculogenic keratinocytes (KCs), 9e11,
10fe11f
immune responses, 16e17
induced pluripotent stem cells (iPSCs), 8
morphogenesis and morphology of, 4e6, 5f
organogenesis, 15
physiology of, 6
principles of, 8e9, 9f
regeneration, 4, 8
principles of, 6e7, 7f
sensory machinery, 2e4
structures of, 13e14, 14f
thermoregulation, 2e4
Hair loss disorders
alopecia areata (AA), 2e4, 3f
androgenetic alopecia, 2e4
cell-based therapies, 15e16
hair follicles (HFs). See Hair follicles (HFs)
therapeutic approaches, 15
340 Index
HD. See Huntington’s disease (HD)
Heart failure
cardiogenic differentiation method, 208
cytokines, 208e209
direct reprogramming, 209
small molecule-based myocardial
differentiation, 209
cell therapy, 207e208
cell transplantation therapy, 209e211, 210f
clinical applications, 213e214
drug discovery screening, 216f, 217e218, 218f
extracellular matrix (ECM), 217
matrix metalloproteinase (MMP), 217
MYBPC3 gene abnormality, 215
Myh7 gene abnormality, 215
heart transplantation, 206e207
large-scale cell culture, 209e211, 210f
left ventricular assist device (LVAD), 206e207
pathology, 206e207, 206f
proof of concept, 211e212
Heart transplantation, 206e207
Hemorrhagic stroke, 68, 70f
Hemostasis, 271
Hepatic phenotype
cocultures, 230e231
correction of, 228e229
3D cultures, 230e231
embryonic stem cells (ESCs), 228
hepatic cells, 229e230, 229f
Hepatic stellate cells (HSCs), 229
Hepatocyte-like cells (HLCs), 226e227, 227f
Hepatotoxicity, 238e240
Hertwig epithelial root sheath (HERS), 297
HFs. See Hair follicles (HFs)
Human embryonic stem cells (hESCs), 28, 135
Human leukocyte antigen (HLA), 181e182
Human umbilical vein endothelial cells
(HUVECs), 230e231
Huntingtin-associated protein (HAP), 51
Huntington’s disease (HD)
autologous transplantation, 61
embryonic stem cells (ESCs), 55
epigenetics, 51e52
HTT gene, 51, 52f
incidence of, 50
induced pluripotent stem cells (iPSCs), 55e56
transplantation, 56e59, 56f, 58t, 60t, 62f
mesenchymal stem cells (MSCs), 54
neuronal stem cells (NSCs), 54e55
protein function, 51, 52f
signs and symptoms of, 52e53, 53f
stem cell therapy for, 53e55, 54f
clinical trials, 59e61
tetrabenazine (TBZ), 50e51
Hyperglycemia, 175
Hypoglycemia, 175
Hypotension, 25
I
Idiopathic cardiomyopathy, 214
Immunogenicity, 29
Induced dopamine neurons (iDNs), 29
Induced neurons (iNs), 29
Infertility, 253
Insulin growth factor 1 (IGF-1), 136e137
Intraocular pressure (IOP), 134
In vitro differentiation process, 31e33
In vitro fertilization (IVF), 181e182
In vitro germ cell derivation, 253e255
In vivo male germ differentiation, 257e260, 258f
Ischemic cardiomyopathy, 207e208
Ischemic stroke, 73f
adult stem cells (ASCs), 73e79
angiogenesis, 70e71
embryonic stem cells (ESCs), 73
induced pluripotent stem cells (iPSCs), 73, 81,
82f, 83e84
CADASIL, 84e87, 85fe88f
drug screening, 81e82, 83f
organoids, 83
multipotent, 72
neurogenesis, 70e71
neuroprotective approaches, 69e70
neurorecovery approaches, 69e70
penumbra concept, 69e70, 72f
pluripotent, 72
risk factors, 68, 69t
therapeutic approaches, 69, 71f
totipotent, 72
types of, 68, 70f
unipotent, 73
K
Keratinocytes (KCs), 4e6, 271
folliculogenic, 9e11, 10fe11f
Kupffer cells (KCs), 229
L
Left ventricular assist device (LVAD), 206e207
Liver disease
bioartificial liver systems, 233
cell therapy, 231e233, 232t
hepatic phenotype, 227e231
hepatotoxicity, 238e240
Index 341
Liver disease (Continued)
induced pluripotent stem cells (iPSCs)
AlperseHuttenlocher syndrome (AHS), 238
animal models, 233e235, 234t
a1-antitrypsin (A1AT), 236
clinical use, 235e236
disease modeling, 236, 237t
Wilson disease (WD), 236e238
Liver sinusoidal endothelial cells (LSECs),
229e230
Liver transplantation, 233
Livestock, 255e257, 256f
M
Macrophages, 271
Magnetic resonance image (MRI)
ischemic stroke, 79, 79f
Parkinson’s disease (PD), 25
Male pattern hair loss (MPHL), 2e4, 3f, 15e16
Math5, 135
Medium spiny neurons (MSNs), 50
Mesenchymal stem cells (MSCs), 12e13,
230e231, 254
Alzheimer’s disease (AD), 105te108t, 116t
amyloid-beta peptide (Ab) deposition,
112e113
cell dosage, 119e121, 120t
homing and paracrine effects, 110
mechanisms of action, 108e110, 109f
neurodegenerative diseases, 115e117, 118t
neurogenesis, 112
neuroinflammation, 110e111
route of administration, 117e119
stem cell choice, 114e115
in vitro, 113
in vivo, 113e114
Huntington’s disease (HD), 53e54
ischemic stroke, 77f, 79fe80f
immune modulation, 75e79
neuroprotection, 75e79
neurorepair, 75e79
wound healing, 274e275
Metformin, 213
Microtubule-associated protein tau (MAPT), 99
Mild cognitive impairment (MCI), 52e53
Mitochondrial permeability transition pore
(mPTP), 238
Mouse embryonic fibroblasts (MEFs), 189,
260e261
MPHL. See Male pattern hair loss (MPHL)
Multiple sclerosis (MS), 51e52, 104, 316,
328e330
Mutant huntingtin protein (mHTT), 50
Myofibroblasts, 271
N
NAFLD. See Nonalcoholic fatty liver disease
(NAFLD)
Natural killer (NK) cells, 212
Neural progenitor cells (NPCs), 59, 74e75
Neural stem cells (NSC), 98
Neurofibrillary tangles, 99
Neurogenesis, 70e71, 112
Neuroinflammation, 110e111
Neuronal cell degeneration, 139
Neuronal stem cells (NSCs)
Alzheimer’s disease (AD), 103e104
Huntington’s disease (HD), 53e55
ischemic stroke, 74e75, 76f
Neutrophils, 271
Nitric oxide (NO), 111
3-Nitropropionic acid (3-NP), 57
Noggin, 136e137
Nonalcoholic fatty liver disease (NAFLD),
232e233
Nonparenchymal cells, 229
Normal-tension glaucoma (NTG), 134
O
Ofatumumab, 316e317
Organoids, 83
Osteodysplasia, 214e215
OTX2, 57e58
P
PACG. See Primary angle-closure glaucoma
(PACG)
Pancreatic beta cells, 189e190, 191t
current good manufacturing practice (cGMP),
190e192
tumor formation, 192e193
Paracrine control, 257e260, 258f
Parkinson’s disease (PD)
augmentative gene therapies, 25
cell therapy
clinical grade human-induced pluripotent stem
cells (hiPSCs), 30e31
dopamine (DA) cells, 29e30, 30f
efficacy tests, 27
fetal cell transplantation, 27e28
genomic stability of, 31
human embryonic stem cells (hESCs), 28
human-induced pluripotent stem cells (hiPSCs)
derived cell grafts, 33
342 Index
non-induced pluripotent stem cells (iPSCs)
autologous cell sources, 29
substantia nigra pars compacta (SNpc), 26
transplantable midbrain dopamine (mDA)
progenitors, 31e33, 32f
transplantable tissue, 27
clinical factors
cell number, 35
differentiation specificity, 34
disease expression, 34
graft survival and outgrowth, 35, 36f
implantation target site, 35
surgical technique, 35e37
variable reprogramming potential, 34
induced pluripotent stem cells (iPSCs)
optimization and standardization, 37e38
patient selection and follow-up monitoring
criteria, 38
production of, 37
regulatory and socioeconomic acceptance, 38
side effects, 25
surgical interventions, 25
PEC-01 cells, 194
Penumbra concept, 69e70, 72f
Periodontal diseases, 306e307, 307f
clinical application, 305e306
dental complex, 296e298
pathogenesis of, 293e295
prevalence of, 292e293, 292fe293f
regenerative dentistry, 301e305
stem cells, 298e301, 299f, 301f
therapies for, 295e296
tissue regeneration, 298e301, 299f, 301f
Periodontal ligament stem cells (PDLSCs),
300e301
PHHs. See Primary human hepatocytes (PHHs)
Phototransduction, 156
Pigment epithelium-derived growth factor
(PEDF), 157
Plasmacytoid dendritic cells (pDCs), 322
POAG. See Primary open-angle glaucoma
(POAG)
Polymorphonuclear lymphocytes (PMNLs),
322
Primary angle-closure glaucoma (PACG),
134
Primary human hepatocytes (PHHs), 226
Primary open-angle glaucoma (POAG), 134
Primordial germ cells (PGCs), 257e258
Prostaglandin E2 (PGE2), 111
Protamine 1 (Prot 1), 260
Psoriatic arthritis (PsA), 316e317
R
Reactive oxygen species (ROS), 238
Recombinant human myelin basic protein
(rhMBP), 255e256
Recombinant tissue plasminogen activator (rtPA),
69
Reperfusion therapy, 69
Retinal cell fate specification
regulatory mechanisms, 134e135
retinal organoid differentiation, 137e139, 138f
stem cell, 136e137, 136f
Retinal cell transplantation, 139
induced pluripotent stem cells (iPSCs), 143
primary retinal ganglion cell transplantation,
139e140
retinal organoid transplantation, 140e143, 142f
stem cell-derived retinal ganglion cell
transplantation, 140, 141f
Retinal ganglion cells (RGCs), 134, 143e144
Retinal pigmented epithelium (RPE), 156, 306
blooderetina barrier (BRB), 157, 158f, 159
clinical trials, 162e164
genomic instability, 164e166, 165f
induced pluripotent stem cells (iPSCs), 161e162
phototransduction, 156
Retinal progenitor cells (RPCs), 135
Retinal translocation, 160
Retinitis pigmentosa (RP), 139, 157
Retinoic acid (RA), 9e10, 137
Rheumatoid arthritis (RA), 316, 325e328, 326t
Riluzole, 50e51
Rituximab, 316e317
RNA-sequencing techniques, 138e139
RPE. See Retinal pigmented epithelium (RPE)
S
Sertoli cells, 259
Single nucleotide polymorphisms (SNPs),
182e183
Somatic cell nuclear transfer (SCNT), 255e256
Sox4, 135
Sox11, 135
Spermatogenesis, 259
Spermatogonial stem cells (SSCs)
testicular parenchyma, 253e254
transplantation, 253e254
Stargardt’s disease, 157
Stem cell factor (SCF), 140e141
Stem cell therapy
Alzheimer’s disease (AD). See Alzheimer’s
disease (AD)
Huntington’s disease (HD), 53e55, 54f
Index 343
Stem cell therapy (Continued)
periodontal diseases, 298e301, 299f, 301f
in vitro germ cell derivation, 253e255
Stroke. See Ischemic stroke
Subgranular zone (SGZ), 98
Substantia nigra pars compacta (SNpc), 26
Subventricular zone (SVZ), 98
Survivin, 213
Synaptonemal complex protein 3 (SYCP 3), 260
Systemic lupus erythematosus (SLE), 316e317
T
Tau protein, 99
T cellemediated immunity, 111
T1D. See Type 1 diabetes (T1D)
Testicular parenchyma, 253e254
Testosterone, 259
Tetrabenazine (TBZ), 50e51
Thermoregulation, 2e4
Thoracotomy, 207e208
Trabeculectomy, 145
Traditional machine learning (TML), 163e164
Transcription activator-like effector nucleases
(TALEN), 276
Transepithelial electrical resistance (TEER), 164
Transition protein 1 (TP1), 260
Treg cells, 319e320, 321f
Trichogenic dermal cells, 11e13, 12f
Tumor necrosis factor alpha (TNFa), 316e317
Type 1 diabetes (T1D), 316
complications of, 174
human induced pluripotent stem cells (hiPSCs),
183e184
culture and expansion, 189
reprogramming, 187e189
human pluripotent stem cells (hPSCs)
allogeneic vs. autologous transplantation, 183
cell therapy clinical trials, 176, 177te180t
clinical use, 185e186, 185fe186f
genotype of donors, 182e183
human embryonic stem cells (hESCs),
181e182
human induced pluripotent stem cells
(hiPSCs), 181e182
pancreatic beta cells, 189e193, 191t
PEC-01 cells, 194
reprogramming, 187e189
Semma Therapeutics, 196
somatic cell isolation, 186e187
tissue acquisition, 186e187
VC-01 clinical trial, 194e195
VC-02 clinical trials, 195e196
hyperglycemia, 175
hypoglycemia, 175
total insulin replacement, 175
U
Ulcerative colitis (UC), 316e317
Umbilical cord mesenchymal stem cells
(UC-MSCs), 76e78
Unified Huntington Disease Rating Scale
(UHDRS), 52e53
V
Valproic acid, 238
Vascular endothelial cells (VECs), 85e87
Vascular endothelial growth factor (VEGF), 156,
279
Vascular smooth muscle cells (VSMCs), 85e87
W
Wet age-related macular degeneration (AMD),
156
Wild animal reproductive biotechnology,
255e257, 256f
Wilson disease (WD), 236e238
Wound healing, 270e271
induced pluripotent stem cells (iPSCs), 282e283
advantages of, 276
endothelial cells, 276e280
extracellular vesicles, 281
fibroblasts, 280
mesenchymal stem cells (MSCs), 281
inflammation, 271
proliferation, 271
remodeling, 271
stem cell therapy, 272
embryonic stem cells (ESCs), 274
mesenchymal stem cells (MSCs), 274e275
X
X-ray diffraction analysis, 211
Y
Yamanaka factors, 8, 188
Z
Zinc finger nucleases (ZFN), 276
344 Index
