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Explore new dimensions in
3D cell modeling
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All trademarks are t he proper ty of Thermo Fish er Scienti fi c and its subsidia ries unless otherwi se specifi ed. COL 23163 1218
Find out more about culturing and analyzing organoids
and spheroids at thermofi sher.com/organoid
As we look to the future of innovation, creating more physiologically relevant models
through the study of organoids and spheroids is becoming increasingly important.
These three-dimensional (3D) models not only mimic cell functions, they also allow for
more robust testing of compounds and a more representative analysis.
That’s why we’ve compiled a collection of tested products, protocols, and seminal
publications to help you accelerate the development of more realistic cellular models.
3D Cell Culture and Analysis
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1
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Welcome
Dear Colleagues,
On behalf of the International Society for Stem Cell Research (ISSCR), we warmly welcome you to beautiful
Amsterdam for the International Symposia, “Stem Cells & Organoids in Development & Disease.” A leader
in life science and health technologies, Amsterdam was recently ranked as one of the top academic and
biomedical communities in Europe. Amsterdam’s culture of education and innovation makes it the perfect
setting for this timely and important meeting.
Research using organoids is rapidly advancing the eld of stem cell science. Recent work has demonstrated
that stem cells retain self-organizing properties in culture, recapitulating in vivo processes and forming organ-
like tissues. This remarkable ability of stem cells and organ progenitors to form organoids has the potential
to revolutionize regenerative medicine. Organoids can help illuminate organ development, degeneration, and
cancer progression, and patient-derived stem cells can be used to model specic disease variances and to
test drug responses. Additionally, this technology could potentially be used in the future for cell replacement
therapy, which would bypass the issue of immune rejection.
We have created a diverse, engaging program that will explore this cutting-edge technology that is pushing
the boundaries of stem cell science. During this symposium you will hear from scientists using organoids to
model a wide variety of systems, to uncover novel therapeutics, and to repair organs in vivo. The symposium
will be bookended by a forum on the policy and ethics of this regenerative medicine and a panel discussion
by leaders of the eld on the challenges, limitations, and promise of organoids. In addition, the latest tools
and technologies will be on display by innovative companies at the Exhibit Hall, a vibrant hub of meeting
activities.
This International Symposium presents many opportunities to learn about new areas of research and new
approaches to stem cell science, expand your network of colleagues and friends, and build new collabora-
tions that can help your research progress. We hope this meeting will inspire you with new ideas and foster a
broader appreciation of the eld and its exciting potential. Thank you for your continued support of the ISSCR,
and your important work that is increasing our understanding of development and disease and advancing the
eld of regenerative medicine.
Sincerely,
The Amsterdam Organizing Committee
Paola Arlotta, Harvard University, USA
Eduard Batlle, Institute for Research in Biomedicine, Spain
Nissim Benvenisty, The Hebrew University of Jerusalem, Israel
Meritxell Huch, University of Cambridge, UK
2
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Mission Statement
The mission of the International Society for Stem Cell
Research (ISSCR) is to promote excellence in stem cell
science and applications to human health.
History & Philosophy
With about 4,100 members from over 60 countries, the
ISSCR is the preeminent transnational, cross-disciplinary
science-based organization dedicated to stem cell
research.
Formed in 2002, the Society promotes global collabora-
tion among talented and committed stem cell scientists
and physicians, and plays a catalyzing role in the develop-
ment of effective new medical treatments.
The Society brings together investigators who are en-
gaged in both fundamental and applied research. Their
investigations include the use of pluripotent stem cells
and stem cells within adult organs and tissues to create
applications in specic therapeutic settings.
The ISSCR represents academia and industry on a broad
range of issues that affect the well-being of patients
and their families, and strives to educate the public and
government regulators on the basic principles of stem
cell science and the realistic potential for new medical
treatments and cures.
Theleadership of the ISSCRis acutely aware of the
responsibility the Society bears to promote the highest
scientific and ethical standards, and is dedicated
to integrity in the rigor and quality of the research
community’s scientific work, the public policy stands it
takes on stem cell related issues, and the organization’s
relations with its key constituents and the public. Only
such an abiding commitment to integrity can ensure
that as the ISSCR grows, it will continue to serve a fair
and trusted advocate by both its internal and external
stakeholders.
Contact Us
The International Society for Stem Cell Resarch
5215 Old Orchard Road, Suite 270
Skokie, Illinois 60077, USA
+1-224-592-5700
www.isscr.org
www.acloserlookatstemcells.org
www.facebook.com/isscr
www.twitter.com/isscr
ABOUT THE ISSCR
PLATINUM SPONSOR GOLD SPONSOR
Meeting Sponors
3
AMSTERDAM NETHERLANDS
Table of Contents
GENERAL INFORMATION ................................4-5
Venue Information .......................................... 4
Registration Information ..................................... 4
Internet Access ............................................. 4
Special Events .............................................. 4
Exhibits Information ......................................... 5
Poster Set-Up and Take-Down ................................ 5
Recording Policy ............................................ 5
ISSCR Policies .............................................. 5
BOARD OF DIRECTORS,
ORGANIZERS, AND REVIEWERS .......................6-7
PROGRAM SCHEDULE ................................ 8-14
Wednesday, 20 February ...................................8-10
Thursday, 21 February ....................................11-12
Friday, 22 February .......................................13-14
SPONSORS AND EXHIBITORS .....................17-19
SPEAKER ABSTRACTS ..............................20-30
Wednesday, 20 February ..................................20-23
Thursday, 21 February ....................................23-28
Friday, 22 February .......................................28-30
POSTER ABSTRACTS .............................31-106
AUTHOR INDEX ....................................107-108
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
General Information
4
VENUE
Stem Cells & Organoids in Development & Disease, an
ISSCR 2019 International Symposium, takes place at the
K.I.T. Royal Tropical Institute, Mauritskade 64, 1092 AD
Amsterdam, Netherlands.
Program sessions will take place in the Queen Máxima Hall
(Auditorium) of the K.I.T. Royal Tropical Institiute.
Registration and coat check can be found by the entrance
of the K.I.T. Royal Tropical Institute. Refreshment breaks,
lunches, poster sessions, reception, and the Exhibit Hall
are located in the foyer spaces of the venue.
REGISTRATION AND
BADGE PICKUP
Pick up your attendee name badge at the ISSCR Registration
Desk found by the entrance of the K.I.T. Royal Tropical
Institute.
Name badges are required for admission to all sessions,
poster presentations, and social events. Since the meeting
badge serves as proof of participation, all attendees,
speakers, and exhibitors are required to wear their badges
at all times. Access to events may be refused if the meeting
badge is not displayed.
Registration Desk and Badge Pickup Hours
Wednesday, 20 February 11:30 – 17:00
Thursday, 21 February 08:30 – 17:00
Friday, 22 February 08:30 – 16:00
INTERNET ACCESS
Complimentary access to the internet is available within the
K.I.T. Royal Tropical Institute during the International
Symposium.
Network name: Conference & Events
Password: welcometokit
As a courtesy to speakers, please be sure to silence any
mobile phones and devices and refrain from using the
internet during sessions. Please note that the bandwidth
of this connection might be limiting.
SPECIAL EVENTS
Poster Presentations
Poster presenters will be at their posters for discussion at
these times:
Odd numbered posters presented Thursday, 21 February
from 13:00 to 14:00
Even numbered posters presented Friday, 22 February from
13:00 to 14:00
Refreshment Breaks
Refreshment breaks will be available in the Marble Hall
(Auditorium Foyer) of the venue. Complimentary coffee
and tea will be served during the following days and times
(times are subject to change):
Wednesday, 20 February 15:30 – 16:00
Thursday, 21 February 08:30 – 09:00
10:30 – 11:00
15:30 – 16:00
Friday, 22 February 08:30 – 09:00
10:30 – 11:00
Lunch Hours
Thursday, 21 February 12:00 – 14:00
Friday, 22 February 12:30 – 14:00
Welcome Reception
Sponsored by Thermo Fisher Scientic
The Welcome Reception will take place at
Pompstation, Zeeburgerdijk 52, 1094 AE,
Amsterdam, Netherlands.
Wednesday, 20 February 17:30 – 20:00
Networking Reception
A networking reception will take place at the K.I.T. Royal
Tropical Institute, in the Marble Hall (Auditorium Foyer).
Thursday, 21 February 17:00 – 18:30
AMSTERDAM NETHERLANDS
5
General Information
EXHIBITS INFORMATION
The Exhibition Hall features leading suppliers and vendors.
Please support the Exhibitors who help make this Sympo-
sium possible.
Exhibit Hours
Wednesday, 20 February 11:30 – 17:00
Thursday, 21 February 08:30 – 18:30
Friday, 22 February 08:30 - 14:00
POSTER SET-UP
AND TAKE-DOWN
Poster presenters are responsible for displaying their poster
at the appropriate times and removing them at the end of
their presentation hour.
Odd numbered posters: displayed Wednesday, 20 February
before 16:00 until Thursday, 21 February at 14:00
Even numbered posters: displayed Thursday, 21 February
after 15:30 until Friday, 22 February at 14:00
Posters not removed by February 22 at 14:00 will be
discarded by the organizer.
RECORDING POLICY
Still photography, video, and/or audio taping of the sessions,
presentations, and posters at the International Symposium
is strictly prohibited. Communicating or disseminating
results or discussion presented at ISSCR events is STRICTLY
PROHIBITED until the start of each individual presentation.
Thank you for your cooperation.
ISSCR CODE OF CONDUCT
The ISSCR is committed to providing a safe and productive
meeting environment that fosters open dialogue and
discussion and the exchange of scientic ideas, while
promoting respect and equal treatment for all participants,
free of harassment and discrimination. All participants are
expected to treat others with respect and consideration,
follow venue rules, and alert staff or security of any danger-
ous situations or anyone in distress. Attendees are expected
to uphold standards of scientic integrity and professional
ethics. These policies apply to all attendees, speakers,
exhibitors, staff, contractors, volunteers, and guests at the
meeting and related events.
ISSCR prohibits any form of harassment, sexual or other-
wise. Incidents should immediately be reported to security
and ISSCR meetings staff at isscr@isscr.org.
6
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Board of Directors, Organizers,
and Reviewers
OFFICERS
President
Douglas A. Melton
Harvard University, USA
Past President
Hans C. Clevers
Hubrecht Institute, Netherlands
President Elect
Deepak Srivastava
Gladstone Institutes, USA
Clerk
Amander T. Clark
University of California,
Los Angeles, USA
Vice President
Christine L. Mummery
Leiden University Medical
Center, Netherlands
Treasurer
Arnold R. Kriegstein
University of California,
San Francisco, USA
DIRECTORS
Roger A. Barker
University of Cambridge, UK
Marianne E. Bronner
California Institute of Technology,
USA
Fiona Doetsch
Biozentrum at the University of
Basel, Switzerland
Valentina Greco
Yale Stem Cell Center,
Yale Medical School, USA
Konrad Hochedlinger
Massachusetts General Hospital
and Harvard University, USA
Jane Lebkowski
Patch Technologies, USA
Ruth Lehmann
NYU School of Medicine, USA
Urban Lendahl
Karolinska Institute, Sweden
Haifan Lin
Yale University School of Medicine,
USA
Melissa H. Little
Murdoch Children’s Research
Institute and University of
Melbourne, Australia
Charles E. Murry
Institute for Stem Cell and Regenerative
Medicine, University of Washington,
USA
Martin F. Pera
The Jackson Laboratory, USA
Hans R. Schöler
Max Planck Institute for Molecular
Biomedicine, Germany
Takanori Takebe
Cincinnati Children’s Hospital Medical
Center, USA/Tokyo Medical and
Dental University and Yokohama City
University, Japan
Joanna Wysocka
Stanford University, USA
Ex Ofcio Members
Sally Temple
Neural Stem Cell Institute, USA
Leonard I. Zon
Boston Children’s Hospital, USA
BOARD OF DIRECTORS
7
AMSTERDAM NETHERLANDS
Paola Arlotta
Harvard University, USA
Eduard Batlle
Institute for Research
in Biomedicine, Spain
Ori Bar-Nur
ETH Zurich, Switzerland
Chris Barry
International Society for Stem Cell
Research, USA
Sina Bartfeld
University of Wurzburg, Germany
Barak Blum
University of Wisconsin, USA
Sylvia Boj
Hubrecht Organoid Technology,
Utrecht, Netherlands
Henner Farin
University of Frankfurt, Germany
Anne Grapin-Botton
DanStem, University of Copenhagen,
Denmark and Max Planck Institute
of Molecular Cell Biology and
Genetics, Germany
Board of Directors, Organizers,
and Reviewers
ORGANIZING COMMITTEE
Nissim Benvenisty
The Hebrew University of Jerusalem, Israel
Meritxell Huch
University of Cambridge, UK
ABSTRACT REVIEWERS
Marisa Karow
LMU Munich, Germany
Bon-Kyoung Koo
IMBA Institute of Molecular
Biotechnology, Austria
Calvin Kuo
Stanford University, USA
Melissa Little
Murdoch Children’s Research Institute,
Melbourne, Australia
Simona Lodato
Humanitas University, Italy
Matthias Lutolf
EPFL, Switzerland
Tom Nowakowski
University of California, San Francisco,
USA
Julie Perlin
International Society for Stem Cell
Research, USA
Alexander Pollen
University of California, San Francisco,
USA
Giorgia Quadrato
University of Southern California, USA
Nadav Sharon
Harvard University, USA
Mark Skylar-Scott
Harvard University, USA
Elly Tanaka
Institute of Molecular Pathology, Austria
Achia Urbach
Bar-Ilan University, Israel
James Wells
Cincinnati Children’s Hospital Medical
Center, USA
8
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Wednesday, 20 February
08:20 – 11:30 PRE-SYMPOSIUM FORUM
Ethical and policy challenges of organoid research and regenerative
medicine applications
08:20 – 08:35 WELCOME AND OVERVIEW
Melissa Little, Murdoch Children’s Research Institute, Australia
08:35 – 09:05 KEYNOTE ADDRESS
Wolfgang Burtscher, European Commission, Belgium
09:05 – 09:20 Roger Barker, University of Cambridge, UK
09:20 – 09:35 Ana Soa Carvalho, Institute of Bioethics, Catholic University
of Portugal, Portugal
09:35 – 09:50 Arnold Kriegstein, University of California San Francisco, USA
09:50 – 10:15 REFRESHMENT BREAK
10:15 – 10:30 Jeremy Sugarman, Johns Hopkins University, USA
10:30 – 10:45 Annelien Bredenoord, University Medical Center Utrecht, Netherlands
10:45 – 10:50 Daniel Klimmeck, EMBO Journal, Germany
10:50 – 11:30 PANEL DISCUSSION
10:50 – 11:30 Moderator: Hans Clevers, Hubrecht Institute, Netherlands
Co-organized with EMBO
9
AMSTERDAM NETHERLANDS
Wednesday, 20 February
13:00 – 14:00 OPENING KEYNOTE SESSION
13:00 – 13:15 WELCOME REMARKS
Hans Clevers, Hubrecht Institute, Netherlands
Nancy Witty, ISSCR CEO
Meritxell Huch, University of Cambridge, UK
13:15 – 14:00 KEYNOTE ADDRESS
Hans Clevers, Hubrecht Institute, Netherlands
STEM CELL-BASED ORGANOIDS AS AVATARS IN HUMAN DISEASE
14:00 – 15:30 RECAPITULATING DEVELOPMENTAL PROCESSES I
Chair: Nissim Benvenisty, The Hebrew University of Jerusalem, Israel
14:00 – 14:30 Anne Grapin-Botton, DanStem, University of Copenhagen, Denmark
and Max Planck Institute of Molecular Cell Biology and Genetics, Germany
PANCREAS ORGANOIDS TO DECONSTRUCT DEVELOPMENTAL
MECHANISMS
14:30 – 14:45 Aparna Bhaduri, University of California, San Francisco, USA
EVALUATING THE FIDELITY OF HUMAN CEREBRAL ORGANOIDS TO
DEVELOPING HUMAN CORTEX
14:45 – 15:00 Nicole Repina, University of California, Berkeley, USA
OPTOGENETIC CONTROL OF CANONICAL WNT SIGNALING,
DIFFERENTIATION, AND EARLY EMBRYOGENESIS IN HUMAN
PLURIPOTENT STEM CELLS
15:00 – 15:30 James Wells, Cincinnati Children’s Hospital, USA
PLURIPOTENT STEM CELL-DERIVED GASTROINTESTINAL ORGANOIDS
AS NEW MODELS TO STUDY HUMAN DEVELOPMENT AND DISEASE
15:30 – 16:00 REFRESHMENT BREAK
10
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
16:00 – 17:00 RECAPITULATING DEVELOPMENTAL
PROCESSES II
Chair: Helen Blau, Stanford University, USA
16:00 – 16:15 Nicole Prior, University of Cambridge, UK
LGR5+ STEM/PROGENITOR CELLS RESIDE AT THE APEX OF AN EMBRYONIC
HEPATOBLAST POOL
16:15 – 16:30 Killian Hurley, Royal College of Surgeons in Ireland, Ireland
SINGLE-CELL TRANSCRIPTOMIC PROFILING OF TYPE 2 ALVEOLAR
EPITHELIAL CELL DIFFERENTIATION FROM HUMAN INDUCED PLURIPOTENT
STEM CELLS: AN EMERGING TOOL TO MODEL INTERSTITIAL LUNG DISEASES
16:30 – 17:00 Paola Arlotta, Harvard University, USA
UNDERSTANDING BRAIN DEVELOPMENT AND DISEASE: FROM THE EMBRYO
TO BRAIN ORGANOIDS
17:30 – 20:00 WELCOME RECEPTION
Sponsored by Thermo Fisher Scientic
Location: Pompstation, Zeeburgerdijk 52, 1094 AE Amsterdam
Wednesday, 20 February
11
AMSTERDAM NETHERLANDS
Thursday, 21 February
09:00 – 10:30 ORGANOIDS AND TISSUE REGENERATION I
Chair: James Wells, Cincinnati Children’s Hospital, USA
09:00 – 09:30 Meritxell Huch, University of Cambridge, UK
ADULT LIVER CHOLANGIOCYTES, ORGANOID CULTURES, AND TISSUE
REGENERATION
09:30 – 09:45 Karl Koehler, Indiana University School of Medicine, USA
GENERATION OF HAIR-BEARING SKIN ORGANOIDS FROM HUMAN
PLURIPOTENT STEM CELLS
09:45 – 10:00 Marije Koning, Leiden University Medical Centre, Netherlands
EFFICIENT VASCULARIZATION AND MATURATION OF HUMAN IPSC-
DERIVED KIDNEY ORGANOIDS UPON TRANSPLANTATION IN THE
COELOMIC CAVITY OF CHICKEN EMBRYOS
10:00 – 10:30 Botond Roska, Institute of Molecular and Clinical Ophthalmology, Switzerland
TITLE TBA
10:30 – 11:00 REFRESHMENT BREAK
11:00 – 12:00 ORGANOIDS AND TISSUE REGENERATION II
Chair: Calvin Kuo, Stanford University, USA
11:00 – 11:15 Marta Roccio, University of Bern, Switzerland
IN VITRO EXPANSION OF EPCAM+/CD271+ HUMAN FETAL COCHLEAR
DUCT CELLS AND DIFFERENTIATION INTO FUNCTIONAL HAIR CELLS IN
3D ORGANOIDS
11:15 – 11:30 Weng Chuan Peng, Stanford University, USA
TISSUE REGENERATION SIGNALS FOR THE IN VITRO CULTURE OF MOUSE
PRIMARY HEPATOCYTES
11:30 – 11:45 Todd McDevitt, Gladstone Institutes, USA
MEDULLARY HINDBRAIN ORGANOID DEVELOPMENT FROM HUMAN
PLURIPOTENT STEM CELLS
11:45 – 12:00 Andras Nagy, Lunenfeld-Tanenbaum Research Institute - Sinai Health System, Canada
TREATING DISEASE WITH ORGANOIDS REQUIRES SOLUTIONS FOR TWO
MAJOR HURDLES
12:00 – 14:00 LUNCH AND POSTER VIEWING
12
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Thursday, 21 February
12:45 – 13:45 MEET THE EDITORS OF STEM CELL REPORTS
Location: Meet at the front of the auditorium.
14:00 – 15:30 MODELING DISEASE I
Chair: Eduard Batlle, Institute for Research in Biomedicine, Spain
14:00 – 14:30 Melissa Little, Murdoch Children’s Research Institute, Australia
ADVANCING THE APPLICATIONS OF KIDNEY ORGANOIDS FOR DISEASE
MODELLING
14:30 – 14:45 Oded Kopper, The Hebrew University of Jerusalem, Israel
HUMAN DERIVED ORGANOID PLATFORM FOR OVARIAN CANCER CAPTURE
INTRA- AND INTER PATIENT HETEROGENEITY
14:45 – 15:00 Jerome Mertens, The Salk Institute for Biological Studies, USA
AGE-EQUIVALENT AND ADULT-LIKE INDUCED NEURONS REVEAL
DE-DIFFERENTIATED NEURONAL STATE IN ALZHEIMER’S DISEASE
15:00 – 15:30 Toshiro Sato, Keio University, Japan
MODELING OF GASTROINTESTINAL DISEASE USING ORGANOIDS
15:30 – 16:00 REFRESHMENT BREAK
16:00 – 17:00 MODELING DISEASE II
Chair: Paola Arlotta, Harvard University, USA
16:00 – 16:15 Hugo Snippert, University Medical Center Utrecht, Netherlands
SINGLE-CELL DRUG RESPONSE IN PATIENT-DERIVED TUMOR ORGANOIDS
16:15 – 16:30 Alexander Rialdi, Icahn School of Medicine at Mount Sinai, USA
A PHARMACOGENOMIC APPROACH USING PRECISION MODELS OF HCC
TO IDENTIFY NOVEL THERAPEUTICS
16:30 – 17:00 Arnold Kriegstein, University of California, San Francisco, USA
CEREBRAL ORGANOIDS AS MODELS OF HUMAN-SPECIFIC BRAIN
DEVELOPMENT, EVOLUTION, AND DISEASE
17:00 – 18:30 NETWORKING RECEPTION
Location: KIT Royal Tropical Institute, Marble Hall (Auditorium Foyer)
13
AMSTERDAM NETHERLANDS
Friday, 22 February
09:00 -10:30 MODELING CELL INTERACTIONS
Chair: Meritxell Huch, University of Cambridge, UK
09:00 – 09:30 Shahin Rai, Weill Cornell Medical, USA
VASCULARIZATION OF TISSUE-SPECIFIC NORMAL AND MALIGNANT
ORGANOIDS WITH ADAPTABLE ENDOTHELIAL CELLS
09:30 – 09:45 Elisa Giacomelli, Leiden University Medical Center, Netherlands
HUMAN IPSC-DERIVED CARDIAC FIBROBLASTS ENHANCE STRUCTURAL
AND FUNCTIONAL CARDIOMYOCYTE MATURATION IN 3D MICROTISSUES
09:45 – 10:00 Arti Ahluwalia, University of Pisa, Italy
ALLOMETRIC SCALING OF ORGANOIDS
10:00 – 10:30 Calvin Kuo, Stanford University, USA
MODELING THE TUMOR IMMUNE MICROENVIRONMENT IN ORGANOIDS
10:30 – 11:00 REFRESHMENT BREAK
11:00 -12:30 TISSUE ENGINEERING
Chair: Shahin Rai, Weill Cornell Medical, USA
11:00 – 11:30 Matthias Lutolf, Ecole Polytechnique Federale de Lausanne, Switzerland
ENGINEERING ORGANOID DEVELOPMENT
11:30 – 11:45 Shukry Habib, King’s College London, UK
ENGINEERING A HUMAN OSTEOGENIC TISSUE MODEL
11:45 – 12:00 James Hudson, QIMR Berghofer Medical Research Institute, Australia
HIGH-CONTENT SCREENING IN HUMAN CARDIAC ORGANOIDS IDENTIFIES
KEY PROLIFERATIVE PATHWAYS WITHOUT FUNCTIONAL SIDE-EFFECTS
12:00 – 12:30 Helen Blau, Stanford University, USA
REJUVENATING STEM CELL FUNCTION TO INCREASE MUSCLE STRENGTH
14
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDSAMSTERDAM NETHERLANDS
12:30 – 14:00 LUNCH AND POSTER VIEWING
14:00 – 15:00 PANEL DISCUSSION: The Challenges, Limitations,
and Promise of Organoids
Moderator: Hans Clevers, Hubrecht Institute, Netherlands
Panelists: Arnold Kriegstein, University of California, San Francisco, USA
Anne Grapin-Botton, DanStem, University of Copenhagen, Denmark
and Max Planck Institute of Molecular Cell Biology and Genetics,
Germany
Botond Roska, Institute of Molecular and Clinical Ophthalmology,
Switzerland
Melissa Little, Murdoch Children’s Research Institute, Australia
15:00 – 16:00 CLOSING KEYNOTE SESSION
15:00 – 15:45 Juergen Knoblich, IMBA-Institute of Molecular Biotechnology, Austria
CEREBRAL ORGANOIDS: MODELLING HUMAN BRAIN DEVELOPMENT AND
TUMORIGENESIS IN STEM CELL DERIVED 3D CULTURE
15:45 – 16:00 Eduard Battle, Institute for Research in Biomedicine, Spain
CLOSING REMARKS
Friday, 22 February
AMSTERDAM NETHERLANDS
The goal of this Cell Symposium is to bring scientists studying organoids
and organ engineering together with bioengineers to discuss the exciting
opportunities and challenges for engineering complexity in higher-order
organ-like systems and to foster collaborations and synergize efforts toward
generating cellular platforms that can address a myriad of unmet needs.
Speakers
Hans Clevers, The Netherlands
Fred Gage, USA
Penney Gilbert, Canada
Tracy Grikscheit, USA
Juan Carlos Izpisua Belmonte, USA
Rudolf Jaenisch, USA
Jürgen Knoblich, Austria
Madeline Lancaster, UK
Wendell Lim, USA
Melissa Little, Australia
Matthias Lutolf, Switzerland
Guo-Li Ming, USA
Hiromitsu Nakauchi, USA
Laura Niklason, USA
Milica Radisic, Canada
Jeremy Rich, USA
Toshiro Sato, Japan
Barbara Treutlein, Germany
Gordana Vunjak-Novakovic, USA
cell.com/symposia
cell-symposia.com/organoids-2019
Engineering Organoids
and Organs
August 25–27, 2019 — Paradise Point, San Diego, USA
Organizers
Hans Clevers, Hubrecht Institute for Developmental Biology and Stem Cell
Research, The Netherlands
Juan Carlos Izpisua Belmonte, Salk Institute, USA
Anh Nguyen, Scientific Editor, Cell Stem Cell
Sri Devi Narasimhan, Senior Scientific Editor, Cell
May 3,
2019
Abstract
submission
deadline:
June 14,
2019
Early
registration
deadline:
17
Sponsor and Exhibitor Directory
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
PLATINUM SPONSOR
Thermo Fisher Scientic
5791 Van Allen Way
Carlsbad, CA 92008
+1- 800-955-6288
www.thermosher.com/organoid
As the world leader in serving science, Thermo Fisher Scien-
tic supplies trusted and innovative solutions for the rapidly
growing application of 3D cell biology. Through our Gibco,
Invitrogen, and Thermo Scientic brands, researchers can
access a broad range of products, services, and educational
resources to support the reliable growth and analysis of 3D
models including organoids and spheroids.
GOLD SPONSOR
StemCell Technologies
1618 Station St
Vancouver, BC
V6A1B6 Canada
+1-604-675-7575
www.stemcell.com
At STEMCELL, science is our foundation. Driven by our mis-
sion to advance research globally, we offer over 2,500 tools
and services supporting discoveries in stem cell research,
regenerative medicine, immunotherapy and disease research.
By providing access to innovative techniques like organoid
culture and gene editing, we’re helping scientists accelerate
the pace of discovery. Inspired by knowledge, innovation and
quality, we are Scientists Helping Scientists.
EXHIBITORS
3Brain
Einsiedlerstrasse 30
Wadenswil, Switzerland 8820
+39 333 4224119
www.3brain.com
3Brain is the world’s rst company to design and produce
high-resolution microelectrode arrays (HD-MEAs). Featuring
thousands of microelectrodes, these HD-MEA platforms
offer unparalleled precision and outperform current market
leaders. The Swiss company’s latest technology features 3D
microelectrodes which streamline the collection of high-den-
sity electrophysiological data and boost research in brain
organoids, slices and tissues, as well as drug discovery.
Axion BioSystems
1819 Peachtree Road NE
Atlanta, GA 30309
+1-404-477-2557
www.axionbio.com
Developing advanced electrically active cells is challenging.
Enter Maestro Pro and Edge, the world’s most advanced
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Bio-Techne
19 Barton Lane
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BioLamina
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Sundbyberg, Sweden 17266
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www.biolamina.com
BioLamina offers a wide variety of chemically dened and
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18
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Sponsor and Exhibitor Directory
BrainXell
455 Science Dr., Suite 210
Madison, WI 53711
+1 608-438-8501
www.brainxell.com
BrainXell provides a range of high-purity, iPSC-derived human
neurons for research and development with a focus on drug
discovery. From motor neurons to cortical sub-types, we
can provide specic models for both disease or normal drug
screening. We are dedicated to delivering the highest quality
products from both our off-the-shelf neurons as well as any
custom service projects.
ChemoMetec
Gydevang43
Allerod, Denmark 3450
www.chemometec.ocm
ChemoMetec is a Danish founded company, which spe-
cializes in the development, manufacturing and sales of
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Corning Life Sciences
Fogostraat 12
Amsterdam, Netherlands 1060 - LT
+31 20 659 6051
www.corning.com/lifesciences
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GE HEALTHCARE
De Random 8
Eindhoven, Netherlands 5612AF
https://gelifesciences.com/cellanalysis
GE Healthcare Life Sciences is at the forefront of new tech-
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MaxWell Biosystems
Mattenstrasse 26
Basel, Switzerland 4058
+41 61 551 1070
www.mxwbio.com
MaxWell Biosystems provides advanced high-resolution elec-
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MIMETAS
JH Oortweg 19
2333 CH, Leiden
The Netherlands
+31 (0) 858883161
www.mimetas.com
MIMETAS develops better predictive human tissue and
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19
AMSTERDAM NETHERLANDS
Sponsor and Exhibitor Directory
NIPPON Genetics EUROPE & Ajinomoto
Binsfelder StraBe 77
Dueren NRW, Germany 52351
+49 2421 55 496 11
www.nippongenetics.eu
NIPPON Genetics is a Japanese Life-Tech company, which
focuses on the development of cutting-edge products. NIP-
PON Genetics EUROPE is now cooperating with Ajinomoto
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the Center for iPS Cell Research and Application at Kyoto Uni-
versity in 2014 and has been providing it to related facilities
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regenerative medicine.
PeproTech
29 Margravine Road
London, United Kingdom W6 8LL
+44(0)20 7610 3062
www.peprotech.com
PeproTech creates the building blocks of your life science
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vance scientic discovery and human health. Since 1988,
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Philips Molecular Pathway Dx
High Tech Campus 11
Eindhoven, Netherlands 5656 AE
oncosignal@philips.com
www.philips.com/oncosignal
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32 Tonlegee Drive, Raheny
Dublin, Ireland D05Y1K1
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20
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Speaker Abstracts
WEDNESDAY, 20 FEBRUARY 2019
OPENING KEYNOTE
STEM CELL-BASED ORGANOIDS AS AVATARS
IN HUMAN DISEASE
Clevers, Hans C.
Hubrecht Institute, Utrecht, Netherlands
Stem cells are the foundation of all mammalian life. Stem
cells build and maintain our bodies throughout life. Two
types of stem cells are discerned. 1) Embryonic stem cells
(ES cells) are briey present in the early human or mouse
embryo, a few days after fertilization. These ES cells can be
grown indenitely in the lab and have the potential to build
each and every tissue in our body. Because of this
‘pluripotency’, ES cells hold great promise for therapeutic
application in the eld of regenerative medicine. However,
derivation of ES cells leads to the destruction of the (mouse/
human) embryo. This has caused intense debates from
ethical, religious and logistical perspectives. A recent
development circumvents the destruction of embryos. It is
now possible to take skin cells (or other cells) from adults
and convert these in the lab into cells with ES properties, so
called iPS cells. Many of the hurdles that ES cell technology
have faced, do not exist for iPS cells. 2) Adult stem cells.
Every organ in our body is believed to harbor its own
dedicated stem cells. These adult stem cells replace tissue
that is lost due to wear and tear, trauma and disease. Adult
stem cells are highly specialized and can only produce the
tissue in which they reside; they are ‘multipotent’. Examples
are bone marrow stem cells that make all blood cells, skin
stem cells and gut stem cells. Even the brain is now known
to harbor its specialized stem cells. The adult stem cells
allow us to live 80-90 years, but this comes at a cost: they
are the cells that most easily transform into cancer cells. We
have identied a gene (lgr5) that marks a series of known
and novel adult stem cells, in organs such as the gut, the
liver, the lung and the pancreas. We have learned to grow
these stem cells in a dish into mini-versions of the human
organs from which they derive. This so called organoid
technology opens a range of avenues for the study of
development, physiology and disease, and for personalized
medicine. In the long run, cultured mini-organs may replace
transplant organs from donors and hold promise in gene
therapy.
Keywords: Adult/tissue stem cell; Lgr5; Organoid
RECAPITULATING
DEVELOPMENTAL PROCESSES I
PANCREAS ORGANOIDS TO DECONSTRUCT
DEVELOPMENTAL MECHANISMS
Grapin-Botton, Anne1,2
1DanStem, University of Copenhagen, Denmark, 2Max Planck
Institute of Molecular Cell Biology and Genetics, Dresden,
Germany
To understand pancreas development, as a complement
to in vivo investigations, we designed simplied in vitro
systems that can be monitored and manipulated better than
the whole embryo. We established 3D culture conditions
that enable the efcient expansion, differentiation and
morphogenesis of dissociated mouse embryonic pancreatic
progenitors. We will discuss how we used these systems to
understand the mechanisms of differentiation of acinar and
endocrine cells and the formation of the ductal network that
transports digestive enzymes. Focusing on the initial condi-
tions leading to these organoids, we observed that the
organoids formed if enough cells were clustered and
identied a cooperative community effect. Assembling
dened numbers of Notch active and inactive cells shows
that their interaction is needed to initiate organoid formation
and fuel growth. We will also discuss how the culture medium
can be used to control the differentiation trajectory of cells
towards acinar or endocrine cells and the associated morpho-
genetic changes. Developing the model to study human
development and model disease, we will present recent data
showing the robust expansion, differentiation and morpho-
genesis of human pancreatic organoids derived from embry-
onic stem cells and their similarity to human fetal pancreas.
Keywords: pancreas; fetus; morphogenesis
EVALUATING THE FIDELITY OF HUMAN CEREBRAL
ORGANOIDS TO DEVELOPING HUMAN CORTEX
Bhaduri, Aparna1, Andrews, Madeline G.1, Shin, David2,
Allen, Denise2, Nowakowski, Tomasz2
1Regeneration Medicine, University of California,
San Francisco, CA USA, 2Anatomy, University of California,
San Francisco, CA, USA
Cerebral organoids are an exciting in vitro model that enable
otherwise inaccessible long term vital culture and experimen-
tal manipulation of human brain development. To evaluate the
delity of cerebral organoids to primary developing human
cortex, we performed single-cell RNA sequencing of 200,000
cortical organoid cells across developmental stages generat-
ed with a range of protocols, from undirected signaling to
strongly directed forebrain protocols. Using this dataset, as
well as published sequencing datasets from cortical organ-
oids, we compared cell type identity and molecular signatures
of the organoid model to primary developing human cortex
single-cell RNA sequencing data obtained in our laboratory as
part of the NIH BRAIN Initiative. Molecular trajectories indicate
that while cerebral organoids effectively recapitulate neuronal
differentiation programs that characterize the transition from
radial glia progenitor cells to neuronal identity, the precise
21
AMSTERDAM NETHERLANDS
Speaker Abstracts
specication of radial glia and neuronal subtypes observed in
normal human development is obscured in the organoid
model. Comparisons of molecular maturation states between
organoids and primary samples indicate that the cortical
organoid models mature substantially faster than primary
developing cortical counterparts, and cortical organoid
progenitor cells exist in a much smaller physical space
compared to the span in primary tissue between the ventricular
zone and cortical plate. Interestingly, area-specic neuronal
signatures are a hallmark of primary human newborn neurons,
and in some cases the organoid newborn neurons recapitulate
these identities, but in most cases, they express no previously
characterized area-specic neuronal transcriptomes. Across all
organoid datasets explored, we nd a signicantly higher
expression of markers of glycolysis and endoplasmic reticulum
stress in cortical organoids compared to primary developing
cortex that we subsequently validate with immunohistochem-
istry. Together, these ndings highlight that although there are
important benets to in vitro cerebral organoid models, the
differences between organoids and primary tissue should be
accounted for when designing and interpreting experiments.
Funding Source
NIH awards F32NS103266 to A.B. and U01MH114825 and
R35NS097305 and California Institute of Regeneration
Medicine GC1R-06673-A to A.R.K.
Keywords: Cortical organoids; Single-cell RNA sequencing;
Developmental trajectories
OPTOGENETIC CONTROL OF CANONICAL WNT
SIGNALING, DIFFERENTIATION, AND EARLY
EMBRYOGENESIS IN HUMAN PLURIPOTENT
STEM CELLS
Repina, Nicole A.1, Bao, Xiaoping2, Schaffer, David1
1Bioengineering, University of California, Berkeley, CA, USA,
2Chemical Engineering, University of California,
Berkeley, CA, USA
Embryonic development is governed by dynamic, spatially and
temporally varying signals that guide stem cell differentiation,
migration, and tissue morphogenesis to ultimately create the
adult organism. However, traditional genetic approaches and
current in vitro methods do not allow for precise, dynamic,
spatiotemporal control of a signaling pathway and are thus
insufcient to readily study how the dynamics of a signal
impacts cell and tissue development. Fortunately, unlike
soluble proteins, small molecule agonists, or protein-coated
beads, optogenetics offers the opportunity to control the
location, timing, and intensity of a signal. We present an
optogenetic system, named optoWnt, to control the Wnt/B-cat-
enin signaling pathway in human embryonic stem cells
(hESCs) and mimic signaling patterns of early embryonic
development. Using newly designed illumination devices, we
activate the canonical Wnt pathway in hESC lines engineered
with a CRISPR knock-in of a Wnt co-receptor fusion with A.
thaliana photoreceptor Cryptochrome 2. Wnt pathway activa-
tion is induced in a light dose-dependent manner, has a high
dynamic range (~1000-fold induction), and displays no
detectable dark-state activity or phototoxicity effects. Optoge-
netic stimulation induces hESC differentiation into mesendo-
derm, as indicated by fate marker expression such as
Brachyury, gain of epithelial-to-mesenchymal transition
markers, and loss of pluripotency. To mimic the spatial
signaling gradients present in the early mammalian embryo, we
use patterning of illumination to spatially localize mesendo-
derm differentiation and nd that differentiating cells adopt a
migratory phenotype, migrating beyond the regions of illumina-
tion, and assemble into dened structures. Furthermore, mixed
co-cultures of wild-type and optoWnt hESCs self-segregate
upon illumination, creating sharp boundaries between the two
cell populations in a process accompanied by changes in cell
adhesion protein expression. Thus, our optogenetic platform
and illumination device are a robust system for activating Wnt
signaling in hESCs and can be used for studying how spatial
and temporal signaling patterns affect gastrulation, primitive
streak formation, and tissue self-assembly in early human
development.
Keywords: Optogenetics; Gastrulation; Wnt signaling
PLURIPOTENT STEM CELL-DERIVED GASTROIN-
TESTINAL ORGANOIDS AS NEW MODELS TO
STUDY HUMAN DEVELOPMENT AND DISEASE
Wells, James M.
Cincinnati Children’s Hospital, Cincinnati, OH, USA
Successful efforts to direct the differentiation of human
embryonic and induced pluripotent stem cells (PSCs) into
specic organ cell types in vitro have largely been guided
by studies of embryonic organ development. We have used
principles of organogenesis to generate complex, three-
dimensional human gastrointestinal organ tissues from PSCs
in vitro. We have done this by focusing on the signaling
pathways that drive anterior-posterior and dorsal-ventral
patterning of the developing endoderm. We can now generate
organoids representing all of the organs of the gastrointestinal
tract including esophagus, gastric fundus, gastric antrum,
small intestine and colon. GI organoids contain complex
epithelial structures and diverse cell types that are unique to
their representative organ; esophageal organoids develop a
stratied squamous epithelium, gastric organoids have a
glandular epithelium that secrete digestive enzymes, hor-
mones, and acid, and intestinal organoids additionally absorb
nutrients. While the rst generation of GI organoids had
epithelium and mesenchyme, they were lacking important cell
types and functions. We have now engineered additional
cellular complexity into organoids, such as small intestinal
organoids with a functional enteric nervous and colonic
organoids with functional immune cells capable of triggering
an inammatory cascade in response to pathogenic bacteria.
Ongoing studies include PSC-derived organoids to identify the
underlying mechanisms behind birth defects including
Hirschsprung’s disease and esophageal atresia, to identify
new pathologies in patients with complex GI diseases. Lastly
we are using human organoids to investigate the how the GI
endocrine system modulates a broad array of metabolic
functions including nutrient sensing and absorption.
Funding Source
P01HD093363, R01DK098350, R01DK092456,
U01DK103117, U19 AI116491.
Keywords: Human Organoids, pluripotent stem cells,
stomach, esophagus, pancreas, intestine, colon, diabetes,
digestive disease, embryonic development
22
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Speaker Abstracts
RECAPITULATING
DEVELOPMENTAL PROCESSES II
LGR5+ STEM/PROGENITOR CELLS RESIDE AT THE
APEX OF AN EMBRYONIC HEPATOBLAST POOL
Prior, Nicole1, Hindley, Christopher1, Rost, Fabian2,
Meléndez, Elena1, Lau, Winnie 3, Göttgens, Bertie3,
Rulands, Steffen2, Simons, Benjamin1, Huch, Meritxell1
1Gurdon Institute, University of Cambridge, UK, 2Max Planck
Institute for the Physics of Complex Systems Dresden,
Germany, 3CIMR, University of Cambridge, UK
During mouse embryogenesis, progenitors within the liver
bud known as hepatoblasts give rise to the adult liver
epithelial hepatocyte and cholangiocyte cells. Contrary to
previous reports, we show that the hepatoblast pool is
functionally heterogeneous and that a sub-population of
E9.5 hepatoblasts exhibit a previously unidentied early
commitment to cholangiocyte fate. Importantly, we also
identify a sub-population of hepatoblasts which at E9.5
express the adult stem cell marker LGR5 and contribute to
liver development by generating both hepatocyte and
cholangiocyte progeny that persist for the life-span of the
mouse. Using a combination of lineage tracing and single
cell RNA sequencing, we show that LGR5 marks bi-potent
liver progenitors residing at the apex of a hierarchy of the
hepatoblast population. Notably, isolated LGR5+ hepato-
blasts can be clonally expanded in vitro into embryonic liver
organoids, which can commit to hepatocyte or cholangio-
cyte fates dependent upon the culture conditions. Our study
represents the rst functional demonstration of heterogene-
ity within E9.5 liver progenitors and identies LGR5 as a
marker for a sub-population of truly bi-potent hepatoblasts
which contribute to the postnatal liver.
Keywords: Liver stem/progenitor cells; LGR5; Hepatoblast
SINGLE-CELL TRANSCRIPTOMIC PROFILING OF
TYPE 2 ALVEOLAR EPITHELIAL CELL DIFFERENTI-
ATION FROM HUMAN INDUCED PLURIPOTENT
STEM CELLS: AN EMERGING TOOL TO MODEL
INTERSTITIAL LUNG DISEASES
Hurley, Killian J.1, Jacob, Anjali2, Ding, Jun3, Herriges,
Michael J.2, Lin, Chieh3, Hawkins, Finn2, Vedaie, Marally2,
Villacorta-Martin, Carlos2, Bar-Joseph, Ziv3, Kotton, Darrell2
1Department of Medicine, Royal College of Surgeons in
Ireland, Dublin, Ireland, 2Center of Regenerative Medicine,
Boston University and Boston Medical Center, Boston, MA,
USA, 3Computational Biology Department, School of Comput-
er Science, Carnegie Mellon University, Pittsburgh, PA, USA
Type II alveolar epithelial cells (AEC2) play key roles in
the response of lung epithelial cells to injury and the
pathogenesis of interstitial lung diseases (ILD). Mutations
affecting genes highly expressed in AEC2s, such as
surfactant protein (SFTP) C, SFTPB, and ABCA3, result in
neonatal respiratory distress or early-onset ILD. Access to
human tissue to study AEC2s is difcult, therefore,
generating a robust in vitro model of AEC2s from an
alternative inexhaustible source could provide insights into
disease pathogenesis. We have previously published a
directed differentiation protocol to generate putative
AEC2 from induced pluripotent stem cell (iPSC), however,
we have further shown that the resulting cells are
transcriptomically heterogenous. We therefore sought to
identify at the single-cell level the developmental pathways
involved in the generation of AEC2s. We targeted uorescent
reporters into the endogenous NKX2-1 and SFTPC loci of a
human iPSC line. Using a directed differentiation approach,
we generated lung progenitors followed by putative AEC2s in
3D ‘alveolospheres’. Characterization of the development of
putative AEC2 was carried out over a time course by
single-cell RNA sequencing (scRNA-seq) and conrmed by
bulk qPCR and ow cytometry. Machine learning analysis of
the transcriptional prole was carried out using
forced-directed layouts of k-nearest-neighbor visualization
and continuous lineage trajectory analysis of transcription
factors (TF)s. k-nearest neighbor analysis of the time series
scRNA-seq reveals early transcriptional heterogeneity with
WNT low dependent pathways leading to lung (NKX2-1 high)
and WNT high leading to non-lung (NKX2-1 low) cell lineag-
es. Putative AEC2 are found exclusively in the WNT low
pathways and were enriched for surfactant processing
genes, SFTPB, LPCAT1 and CLDN18. Reconstructing the
regulatory networks of TFs reveals that early withdrawal of
WNT agonist during differentiation improves the yield of
SFTPC expressing cells and suggests important TFs in the
differentiation of progenitors to AEC2. Our results provide
key transcriptional proles of engineered AEC2 at single-cell
resolution, revealing fundamental mechanisms underpinning
alveolar development and provide a guide for improving
directed differentiation to AEC2.
Keywords: Induced Pluripotent Stem Cells; Single-cell RNA
sequencing; Interstitial lung disease
UNDERSTANDING BRAIN DEVELOPMENT
AND DISEASE: FROM THE EMBRYO TO BRAIN
ORGANOIDS
Arlotta, Paola1,2, Velasco, Silvia1,2, Kedaigle, Amanda J.1,2,3,
Simmons, Sean K.2,3, Nash, Allison1,2, Rocha, Marina 1,2,
Quadrato, Giorgia1,4, Nguyen, Lan3, Adiconis, Xian2,3, Regev,
Aviv3,5, Levin, Joshua Z.2,3
1Department of Stem Cell and Regenerative Biology, Harvard
University, Cambridge, MA, USA, 2Stanley Center for
Psychiatric Research, Broad Institute of MIT and Harvard,
Cambridge, MA, USA, 3Klarman Cell Observatory, The Broad
Institute of MIT and Harvard, Cambridge, MA, USA, 4Eli and
Edythe Broad CIRM Center for Regenerative Medicine and
Stem Cell Research , University of Southern California, Los
Angeles, CA, USA, 5Howard Hughes Medical Institute, Koch
Institute of Integrative Cancer Research, Department of
Biology, MIT, Cambridge, MA, USA
Much remains to be understood regarding the cellular and
molecular principles that govern the development of the
mammalian brain, and how these events are affected in
23
AMSTERDAM NETHERLANDS
Speaker Abstracts
neurodevelopmental disease. I will present some of our
work on the generation and long-term development of
human brain organoids; their developmental trajectory,
cellular diversity and neuronal network features. I will then
discuss our most recent progress in developing human brain
organoids that are capable of generating the large diversity
of cell types found in the cerebral cortex while achieving
unprecedented levels of organoid-to-organoid reproducibility
in cellular composition and their extended developmental
trajectories. Through this work, I will discuss the challenges
and opportunities of modeling human brain development
within 3D human brain organoids, and the promise that
organoids offer to understand complex neurodevelopmental
disease.
Keywords: Brain organoids; Neurodevelopment; Cerebral
cortex
THURSDAY, 21 FEBRUARY 2019
ORGANOIDS AND
TISSUE REGENERATION I
ADULT LIVER CHOLANGIOCYTES, ORGANOID
CULTURES AND TISSUE REGENERATION
Huch, Meritxell
The Wellcome/CRUK Gurdon Institute, University of
Cambridge, UK
The adult liver exhibits low physiological turnover,
however excels for its extensive proliferative capacity and
regeneration ability after damage. In conditions where
hepatocyte proliferation is impaired, resident ductal cells
de-differentiate and acquire a proliferative and
progenitor-like state that allows the tissue to regenerate
both hepatocytes and ductal cells. In vivo, after tissue
damage, proliferating ductal cells increase the expression
levels of the stem-cell markers such as Lgr5, FoxL1 and
Trop2. Similarly, in vitro, we will show that mouse and human
ductal cells recapitulate this process when grown in
3D as liver organoid cultures. Thus, differentiated ductal
cells cultured as liver Organoids acquire a bi-potential
progenitor state that enables them to self-renew and
differentiate into hepatocyte-like cells in vitro and in vivo,
upon transplantation. However, the molecular mechanism
behind the de-differentiation and acquisition of a
proliferative state is largely unknown. Here I will discuss our
recent ndings on how the regulation of the epigenome is
critical for organoid formation and tissue regeneration.
We anticipate that epigenetic regulation to be a general
mechanism by which adult cells exit dormancy as
response to injury/regeneration.
Keywords: liver organoids ; epigenetic regulation; regenera-
tion
GENERATION OF HAIR-BEARING SKIN ORGAN-
OIDS FROM HUMAN PLURIPOTENT STEM CELLS
Koehler, Karl R.
Otolaryngology - Head and Neck Surgery, Indiana University
School of Medicine, Indianapolis, IN, USA
Over one hundred million people worldwide suffer from
injury or loss of skin due to burns, diseases, or genetic
defects. Skin is essential for protecting body by regulating
uid retention and temperature, guarding against external
stresses, and mediating touch and pain sensation.
Human skin develops from coordinated interactions
between multiple cell lineages and is vulnerable and
difcult to be reconstructed once damaged. Despite
repeated attempts for decades, however, a method of
reproducing the full cellular diversity of skin in tissue
cultures or in bioengineered skin equivalents has been
elusive. Here we report a skin organoid culture system that
generates complex skin from human pluripotent stem cells
and recapitulate key features of skin development. We found
that skin organoids are composed of stratied epidermis,
fat-rich dermis, dermal condensate, pigmented hair follicles
equipped with sebaceous glands and bulge stem cells, and
sensory neuronal cells forming synapses with Merkel cells in
organoid hair follicles, mimicking human touch circuitry.
Furthermore, the skin organoids are comparable to human
fetal facial skin and capable of reconstituting hairy skin in a
xenograft mouse model. Together, our results demonstrate
that the skin organoids produced in our culture system are
the most functional and fully equipped human skin tissue
that can be generated in vitro, to date. We anticipate our
study provides a foundation for using skin organoids in
studying skin development, modeling skin disease
processes, or supplying cell source for skin regeneration
and transplantation.
Funding Source
Ralph W. and Grace M. Showalter Trust, the Indiana CTSI
(core pilot grant UL1 TR001108), and the Indiana Center for
Biomedical Innovation (technology enhancement grant).
Keywords: Skin Organoid; Human pluripotent stem cells;
Hair follicle
EFFICIENT VASCULARIZATION AND MATURATION
OF HUMAN IPSC-DERIVED KIDNEY ORGANOIDS
UPON TRANSPLANTATION IN THE COELOMIC
CAVITY OF CHICKEN EMBRYOS
Koning, Marije1, Avramut, Cristina2, Lievers, Ellen1, Wiersma,
Loes E.1, Jaffredo, Thierry3, van den Berg, Bernard M.1,
Howden, Sara E.4, Little, Melissa H.4, van den Berg,
Cathelijne W.1, Rabelink, Ton J.1
1Internal Medicine - Nephrology, Leiden University Medical
Centre, Leiden, Netherlands, 2Department of Cell and Chemical
Biology, Leiden University Medical Centre, Leiden, Netherlands,
3Developmental Biology Laboratory, Sorbonne Université,
Paris, France, 4Paediatrics, University of Melbourne, Australia
Human induced pluripotent stem cell (hiPSC) derived kidney
organoids closely resemble in vivo kidney tissue, offering
unprecedented possibilities for modeling disease and
24
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Speaker Abstracts
development. Despite recent advances in kidney organoid
generation, currently available protocols still yield organoids
that lack a functional vasculature. As kidneys are highly
vascularized organs that depend on the presence of
vascular endothelial cells and blood ow for their correct
development and functionality, it is not surprising that these
organoids remain immature. Recently, it was shown that
transplantation of hiPSC-derived kidney organoids under the
renal capsule of mice leads to functional vascularization
and maturation of the organoids. However, this model is
labor-intensive and time consuming, requiring 28 days for
optimal maturation. Here, we describe a new model where
we transplanted kidney organoids inside the coelomic cavity
of early chicken embryos. We observed rapid vascularization
by host vasculature as well as reorganization of organoid-
derived human endothelial cells. Through intravascular
injection of uorescently-labeled lectins, we show function-
ality of and anastomosis between the chicken- and human-
derived blood vessels. Upon 8 days of transplantation we did
not observe misdirected differentiation to stromal tissue.
Furthermore, unbiased wide eld transmission electron
microscopy analysis of these organoids demonstrated
glomerular maturation with the formation of podocyte foot
processes, slit diaphragms and a glomerular basement
membrane. Tubular structures also showed signs of
maturation, with the appearance of cell junctions and
microvilli. These data demonstrate the suitability and accessi-
bility of this model for studying vascularization and show
that intracoelomic transplantation inside chicken embryos is
an efcient method to rapidly vascularize and mature kidney
organoids.
Keywords: Kidney organoid; Vascularization; Chicken
embryo
Roska, Botond
Institute of Molecular and Clinical Ophthalmology, Switzerland
Abstract not available at time of printing.
ORGANOIDS AND TISSUE
REGENERATION II
IN VITRO EXPANSION OF EPCAM+/CD271+
HUMAN FETAL COCHLEAR DUCT CELLS AND
DIFFERENTIATION INTO FUNCTIONAL HAIR CELLS
IN 3D ORGANOIDS
Roccio, Marta1, Perny, Michael1,2, Ealy, Megan3,4, Widmer,
Hans Ruedi5, Heller, Stefan3, Senn, Pascal6
1Department of Biomedical Research, University of Bern,
Switzerland, 2Institute of Infectious Diseases, University of
Bern, Switzerland, 3Department of Otolaryngology, Head &
Neck Surgery, Institute for Stem Cell Biology and Regenerative
Medicine, Stanford University, Stanford, CA, USA, 4Department
of Biology, Drury University, MO, USA 5Experimental Neurosur-
gery, University of Bern and University Hospital Insel, Bern,
Switzerland, 6Department of Otorhinolaryngology, Head &
Neck Surgery, Hôpitaux Universitaires de Genève, University
Hospital Geneva,
Switzerland
Sensory hair cells located in the organ of Corti are essential
for cochlear mechanosensation. Their loss is irreversible in
humans resulting in permanent hearing loss. The
development of therapeutic interventions for hearing loss
requires fundamental knowledge about similarities and
potential differences between animal models and human
inner development as well as the establishment of human
cell based-assays. We have carried out a systematic
analysis of the fetal human inner ear in a temporal window
spanning from week 8 to week 12 post conception, when
cochlear hair cells become specied. We analyzed gene and
protein expression of the developing cochlear duct, the spiral
ganglion and vestibular tissue. We have identied surface
markers for the cochlear prosensory domain, namely
EPCAM and CD271, that allow to purify postmitotic hair cell
progenitors. When placed in culture in three-dimensional
organoids these regained proliferative potential and at the
same time maintained marker expression and features of
the native tissue. We could further differentiate these tissue
resident progenitors into hair cell-like cells in vitro, displaying
expression of hair cell markers such as BRN3C and MYO7A,
F-Actin and Espin positive hair bundles and showing active
MET-channel transduction. The competence of the
generated hair cells to uptake aminoglycosides antibiotics
such as gentamycin provides now a platform to test ototox-
icity and regeneration. While the expansion and differentia-
tion yields are still not suitable for drug screening purposes,
we have started to analyze approaches to optimize both
based on “chemical reprogramming” of cochlear progeni-
tors. These results provide a foundation for comparative
studies with otic cells generated from human pluripotent
stem cells and for establishing novel cell based platforms
for drug validation.
Funding Source
EU-FP7 Health, grant number 603029.
Keywords: Inner Ear Organoids; Human Fetal Cochlea
Development; Differentiation fetal hair cell progenitors
TISSUE REGENERATION SIGNALS FOR THE
IN VITRO CULTURE OF MOUSE PRIMARY
HEPATOCYTES
Peng, Weng Chuan1, Grompe, Markus3, Li, Bin3, Nusse, Roel2
1HHMI, Institute for Stem Cell Biology and Regenerative
Medicine, Stanford University, Mountain View, CA, USA, 2
Developmental Biology, Stanford University, Stanford, CA,
USA, 3Oregon Stem Cell Center, Oregon Health & Science
University, Portland, OR, USA
In the healthy adult liver, most hepatocytes proliferate
minimally. However, upon physical or chemical injury to the
liver, hepatocytes proliferate extensively in vivo under the
direction of multiple extracellular cues, including Wnt and
pro-inammatory signals. Currently, liver organoids can be
generated readily in vitro from bile-duct epithelial cells, but
not hepatocytes. Here we show that TNFa, an injury-induced
25
AMSTERDAM NETHERLANDS
Speaker Abstracts
inammatory cytokine, promotes the expansion of hepato-
cytes in 3D culture and enables serial passaging and
long-term culture for almost a year, at the time of writing.
Single-cell RNA sequencing reveals broad expression of
hepatocyte markers. Upon the withdrawal of expansion
signal Wnt and inammatory signal TNFa, hepatocytes
upregulate the expression of genes related to liver function.
Strikingly, in vitro-expanded hepatocytes engrafted, and
signicantly repopulated, the injured livers of Fah-/- mice.
We anticipate tissue repair signals can be harnessed to
promote the expansion of otherwise hard-to-culture cell-
types, with broad implications.
Funding Source
Howard Hughes Medical Institute (HHMI), California
Institute for Regenerative Medicine (CIRM).
Keywords: Liver hepatocyte; Inammatory Cytokine;
Tissue repair/regeneration signals
MEDULLARY HINDBRAIN ORGANOID DEVELOPMENT
FROM HUMAN PLURIPOTENT STEM CELLS
McDevitt, Todd1, Butts, Jessica1, Mihaly, Eszter1, Joy, David1,
Lai, Michael1, Yackle, Kevin2
1GICD, Gladstone Institutes, San Francisco, CA, USA,
2Physiology, University of California, San Francisco, CA, USA
The hindbrain is the most conserved region of the CNS and
controls critical autonomic functions, such as respiration.
Specically, excitatory interneurons (INs) of the pre-Bötzing-
er complex, a cluster of several thousand neurons in the
ventral medulla, generate the pace of breathing. Organoids
created from human pluripotent stem cells (PSCs) have
been used to mimic various regions of the developing brain,
but hindbrain development has lagged signicantly behind
fore- and midbrain models. We previously generated
excitatory caudal INs from PSCs using a combination of
neuroinductive morphogenic signals. Subsequent analysis
of HOX gene expression along with single cell RNA-Seq
revealed that the neuronal population consisted predomi-
nantly of 3 neurons normally found in the hindbrain:
PHOX2B/PHOX2A+ (chemosensitive), LHX5/PAX2+ (V0 IN),
and CHX10/SOX14+ (V2a IN). Based on these ndings, 3D
aggregates of iPSCs (WTC line; 104 cells/aggregate) were
differentiated using a similar protocol in suspension culture
for 1 year. Multiple neuroepithelial cystic structures formed
within individual aggregates and doubled in size during the
rst 2 weeks of differentiation. By day 17, coincident
emergence of V0, V2a and chemosensing neurons was
observed in spatially distinct patterns. The relative propor-
tion of V0:V2a INs was controlled by varying the concentra-
tion of purmorphamine (pur), a Shh agonist, analogous to
the ventral-to-dorsal SHH gradient in the developing neural
tube. Organoids cultured for 100 days exhibited increased
neuronal maturation based on NeuN, Tau, VGlut2, and
synaptophysin expression. Although glial cells (GFAP+) were
not readily detected until day 50, they increased in number
thereafter. Starting at ~40 days, organoids from GCaMP6-ex-
pressing iPSCs displayed rhythmic Ca++ transients that
rapidly dissipated within 2 minutes in normoxic conditions,
potentially suggestive of C02 sensing. The patterns of Ca++
waves continued but became less periodic over time (after
day 60) as the cellular composition and structure of the
organoids changed. These results provide the rst description
of a human iPSC-derived hindbrain organoid that replicates
elements of normal neuronal circuitry and physiologic
function, and therefore could be used to model hindbrain
development ex vivo.
Keywords: hindbrain; pre-Botzinger; interneurons
TREATING DISEASE WITH ORGANOIDS REQUIRES
SOLUTIONS FOR TWO MAJOR HURDLES
Nagy, Andras1,2, Liang, Qin3, Nagy, Kristina1, Monetti, Claudio1,
Yang, Huijuan1, Harding, Jeffrey1
1Lunenfeld-Tanenbaum Research Instittute, Sinai Health
System, Toronto, ON, Canada, 2Australian Regenerative
Medicine Institute, Monash University, Melbourne, Australia,
3Harvard Stem Cell Institute, Harvard University, Cambridge,
MA, USA
Organoids have proven to be powerful tools for studying
organ development and disease processes. Organoids are
also instrumental in evaluating disease treatments and they
hold promise for the treatment of illness by transplantation
into patients. The most obvious examples of such a promise
are the pancreatic islet organoids faithfully responding to
blood glucose levels with insulin secretion and the liver
organoids that rescue liver failure. Here, we report solutions
for the two major hurdles faced by transplant recipients,
which are the graft safety and the long-term-tolerated alloge-
neic organoid products. We provide a denition and quantita-
tion of safety by calculating the odds of accidentally generat-
ing potentially dangerous therapeutic cells and aim to
ameliorate these odds through gene-editing. We introduced
an inducible “kill-switch” into the genome thus making the
unwanted proliferating cells sensitive to ablation while leaving
quiescent cells resistant. The kill-switch was placed into the
3’UTR of a cell division-essential gene in a homozygous
manner. This precise genome editing created a bicistronic
gene encoding for two proteins; the division essential factor
(i.e. Cdk1) and a negatively selectable marker (i.e. HSV-TK)
and achieved a high level of protection of the kill-switch
(Fail-SafeTM cell system). To obtain allogeneic tolerance, we
identied eight immune modulatory cell surface or local
acting genes. Transgenic over-expression of this set of genes
was sufcient to achieve long-term induced allogeneic cell
tolerance (iACT) without the need for systemic immune
suppression. By combining the Fail-SafeTM and the iACT
systems, we are in the position to provide a single pluripotent
cell line to serve as a source for therapeutic cells and organ-
oids for all humankind. *All authors contributed equally.
Funding Source
This work was funded by Canadian Institutes for Health
Research, Medicine by Design, and The Foundation Fighting
Blindness, Canada.
Keywords: Safe Cells; Allogeneic Cell Tolerance; Cell and
Organoid Therapy
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Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Speaker Abstracts
MODELING DISEASE I
ADVANCING THE APPLICATIONS OF KIDNEY
ORGANOIDS FOR DISEASE MODELLING
Little, Melissa H.1, Hale, Lorn1,2, Forbes, Thomas1, Howden,
Sara1, Er, Pei1, Kynan, Lawlor1, Khan, Shahnaz1, Ghobrial,
Irene1, Combes, Alexander1, Phipson, Belinda1, Zappia, Luke2,
Oshlack, Alicia2
1Murdoch Children’s Research Institute, Victoria, Australia,
2Bioinformatics, Murdoch Children’s Research Institute,
Melbourne, Australia
The development of protocols for the differentiation of
human pluripotent cells to complex multicellular organoids
provides novel opportunities for stem cell medicine. We have
developed a protocol for the generation of multicellular
human kidney organoids from human pluripotent stem cells.
The application of kidney organoids for disease modelling
will require robustness, transferability between line, develop-
mental accuracy, appropriate controls and an appropriate
and tractable readout. Single cell RNAseq has facilitated the
direct comparison between human fetal kidney and kidney
organoids, revealing strong conservation of cell type.
Comprehensive evaluation of transcriptional variation
between differentiation experiments has identied technical
variation as the major source of differences between organ-
oids. CRISPR-Cas9 gene editing was then used to generate
isogenic patient lines representative of heritable monogenic
kidney disease. Using these lines to create kidney organoids,
we are able to replicate tubular and glomerular disease
phenotypes and demonstrate causative mutations. Together,
this is moving us closer to the application of
patient-derived kidney organoids for disease modelling.
Funding Source
National Institute of Health USA, National Health and
Medical Research Council
Keywords: kidney organoid; disease modelling; pluripotent
stem cell
HUMAN DERIVED ORGANOID PLATFORM FOR
OVARIAN CANCER CAPTURE INTRA- AND INTER
PATIENT HETEROGENEITY
Kopper, Oded1, De Witte, Chris J.2, Lõhmussaar, Kadi3,
Espejo Valle-Inclan, Jose2, Hami, Nizar2, Kester, Lennart3,
Balgobind, Anjali3, Korving, Jeroen3, Proost, Natalie4,
Begthel, Harry3, van Wijk, Lise M.5, Van De Ven, Marieke4,
Vrieling, Harry5, Vreeswijk, Maaike P.G5, Jonges, Trudy6,
Zweemer, Ronald P.7, Snippert, Hugo8, Kloosterman, Wigard P.2
1The Azrieli Center for Stem Cells & Genetic Research, The
Hebrew University of Jerusalem, Israel, 2Center for Molecular
Medicine, University Medical Center Utrecht, Netherlands,
3Hubrecht Institute, Utrecht, Netherlands, 4Preclinical
Intervention Unit of the Mouse Clinic for Cancer and Ageing,
Netherlands Cancer Institute, Amsterdam, Netherlands,
5Department of Human Genetics, Leiden University Medical
Centre, Leiden, Netherlands, 6Department of Pathology,
University Medical Center Utrecht, Netherlands, 7Department of
Gynaecological Oncology, University Medical Center Utrecht,
Netherlands, 8Molecular Cancer Research, University Medical
Center Utrecht, Netherlands
Ovarian cancer (OC) is a heterogeneous disease usually
diagnosed at a late stage. Experimental in vitro models that
faithfully capture the hallmarks and tumor heterogeneity of
OC are limited and hard to establish. We present a novel
protocol that enables efcient derivation and long-term
expansion of OC organoids. Utilizing this protocol, we have
established 56 organoid lines from 32 patients, representing
the spectrum of ovarian neoplasms, including non-malignant
borderline tumors, as well as mucinous, clear-cell,
endometrioid, low- and high-grade serous carcinomas.
OC organoids recapitulate histological and genomic
features of the pertinent lesion from which they were derived,
illustrating intra- and inter-patient heterogeneity, and can be
genetically modied. We show that OC organoids can be
used for drug screening assays and capture different tumor
subtype responses to the gold standard platinum-based
chemotherapy, including acquisition of chemoresistance in
recurrent disease. Finally, OC organoids can be xenografted,
recapitulating in vitro drug sensitivity. Taken together, this
demonstrates their potential application for research and
personalized medicine.
Keywords: Ovarian cancer; Organoids; Disease modeling
AGE-EQUIVALENT AND ADULT-LIKE INDUCED
NEURONS REVEAL DE-DIFFERENTIATED NEURO-
NAL STATE IN ALZHEIMER’S DISEASE
Mertens, Jerome1, Herdy, Joseph1, Böhnke, Lena2,
Traxler, Larissa2, Schäfer, Simon1, Galasko, Douglas3
1Laboratory of Genetics, The Salk Institute for Biological
Studies, La Jolla, CA, USA,
2
Department of Genomics, Stem
Cell Biology and Regenerative Medicine, University of
Innsbruck, Austria,
3
ADRC, University of California,
San Diego, CA USA
Sporadic forms of Alzheimer’s Disease (AD) exclusively
affect people at old age and represent the overwhelming
majority of all AD cases, as genetically dened familial
cases are the rare exception. Still, most research on AD has
been performed on genetic causes and their directly related
pathways, also because we were in lack of models that can
reect complex human genetics, physiology, and age in an
appropriate human neuronal context. While patient-specic
iPSC-based models represent an attractive solution, iPSC
reprogramming results in cellular rejuvenation and thus
yields phenotypically young neurons. By contrast, direct
conversion of old patient broblasts into induced neurons
(iNs) preserves endogenous signatures of aging. To control
for the involvement of aging in human neuronal models for
AD, we took advantage of combining both technologies and
generated age-equivalent broblast-derived iNs, as well as
rejuvenated iPSC-derived neurons from a large cohort of AD
patients and controls. In addition to their rejuvenated state,
27
AMSTERDAM NETHERLANDS
Speaker Abstracts
we found that iPSC neurons transcriptionally resemble
prenatal developmental stages, while iNs reect adult-like
neuronal stages and show little correlation with the prenatal
brain. Thus not surprisingly, only age-equivalent adult-like
iNs, but not rejuvenated prenatal-like iPSC neurons, revealed
a strong AD patient-specic transcriptome signature,
which shows high concordance with previous human
p
ost-mortem AD studies, and highlights functional gene
categories known to be involved in neurodegeneration. Based
on AD patient-specic transcriptional, functional, and
epigenetic changes, we found that AD iNs display a more
de-differentiated neuronal state than control iNs, which might
underlie many of the here and previously observed changes
in AD. These data show that iNs represent a unique tool for
studying age-related neurodegeneration, and support a view
where a partially de-differentiated state of old cells might
permit the loss of the specialized neuronal tness in AD.
Funding Source
National Institute on Aging R01-AG056306-01 and
K99-AG056679-01;H2020-MSCA-IF-2017 797205; Paul G.
Allen Family Foundation; Shiley-Marcos Alzheimer’s Disease
Research Center at the University of California San Diego.
Keywords: Induced neurons (iNs) ; Alzheimer’s Disease;
Aging
MODELING OF GASTROINTESTINAL DISEASE
USING ORGANOIDS
Sato, Toshiro
Department of Organoid Medicine, Keio University, Tokyo, Japan
The biological understanding of gastrointestinal cancer
requires faithful disease modeling that recapitulates the
disease heterogeneity and pathobiological traits of original
cancers. We optimized stem cell niche factor medium for
gastrointestinal tumor organoids and established over 100
patient-derived organoid lines from various tissue origins
and histological subtypes including previously uncultured rare
tumors. Tumor organoids reproduced the histopathological
grade and differentiation capacity of parental tumors in vitro
and upon xenografting. Integrated molecular and biological
analyses of tumor organoids revealed variable degree of
niche independency along with accumulating genetic
mutations. In general, cancer organoids acquire niche
independent growth capacity through corresponding signal
mutations, however, we noted that some cancers do not
follow this rule. For instance, pancreas cancers often gained
Wnt-niche independency through epigenetic reprogramming
such that they no longer required Wnt signal activation for
their long-term self-renewal. In this session, we would like to
show our recent progress of disease modeling using
patient-derived cancer organoids and share our biological
understanding how cancers acquire their niche independency.
Keywords: Intestinal stem cells; Colorectal cancer; Gastric
cancer
MODELING DISEASE II
SINGLE-CELL DRUG RESPONSE IN PATIENT-
DERIVED TUMOR ORGANOIDS
Snippert, Hugo, Ponsioen, Bas, Post, Jasmin, Buissant des
Amorie, Julian
Center for Molecular Medicine, University Medical Center
Utrecht, Netherlands
Patient-derived tumor organoids recapitulate the
histopathological features of native tumors, including patient
specic responses towards therapies. Moreover, organoids
develop cellular heterogeneities in respect to cell fate and
signaling activities, providing a platform to study single
cell drug responses towards targeted inhibitors in the
context of population dynamics and therapy resistance. To
understand functional heterogeneities of RAS signaling
activity in colorectal cancers (CRC), we established
quantitative single-cell activity measurements of the
downstream effector ERK in patient-derived CRC organoids.
Using improved sensors and calibration strategies, we can
scale recorded proles of all individual cells within an CRC
organoid to the full physiological ERK activity range. We use
patient-derived CRC organoids of variable genetic back-
grounds to monitor heterogeneity between ERK activity
proles. Moreover, we CRISPR-engineered isogenic tumor
organoids where we introduced common mutations of the
RAS pathway to study mutation specic signaling
characteristics. Intriguingly, we reveal markedly different
proles and drug-responses among individual cells within
clonal organoids. Of note, we identify ERK re-activation,
a signaling rewiring phenomenon that is generally hold
responsible for therapy resistance, to occur only in a subset
of tumor cells. Together, single-cell measurements of
real-time RAS signaling activity in CRC organoids shows
great potential to improve our understanding of cellular drug
responses and the nature of resistant cells that give rise to
cancer recurrence.
Funding Source
KWF; SU2C; ERC starting grant; HFSP young investigator
grant.
Keywords: Organoids; Colorectal cancer; Real-time imaging
A PHARMACOGENOMIC APPROACH USING
PRECISION MODELS OF HCC TO IDENTIFY NOVEL
THERAPEUTICS
Rialdi, Alexander, Scopton, Alex, Rosemann, Felix, Duffy,
Mary, Molina-Sanchez, Pedro, Lujambio, Amaia, Dar, Arvin,
Guccione, Ernesto
Oncological Sciences, Icahn School of Medicine at Mount
Sinai, New York, NY, USA
Hepatocellular carcinoma (HCC) presents unique challenges
for the identication of drug targets and therapeutics. The
most prevalent mutations, amplications, or transcriptional
de-regulations are within tumor suppressors or yet-to-be
drugged oncogenes. Only small number of kinase inhibitors
(KIs) are approved for advanced HCC, which only extend life
28
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Speaker Abstracts
by ~3 months and cause a variety of side effects. Most
importantly, there have been no attempts to stratify HCC
patients for personalized medicine approaches. The
aforementioned data highlight the urgent need for: (I) the
identication of novel clinical compounds, and (II) the
understanding of genetic and epigenetic drivers that impact
drug efcacy on tumor cells. Herein, we describe a
multidimensional approach to target validation and drug
discovery that focuses on kinase networks within the
context of sophisticated HCC murine models and tumor
organoids. We have used chemical biology to generate
libraries of KIs, which are built off known HCC-approved
drugs thereby retaining drug-like properties and allowing us
to optimize activity in multiple genetic systems. To do so,
we have adopted a precision system to quickly induce
autochthonous and mosaic liver tumors that are driven by
genetic alterations observed in the human population.
Tumor organoids are derived from each model to be used
for drug screening and for the transcriptomic and epigenomic
understanding of drug response mechanisms. This
pharmacogenomic approach has been scaled to ve
common genetic alterations in HCC: CTNNB1 activation,
TP53 loss, TERT amplication, PTEN loss, and KMT2C loss.
Our data suggests that individual driver alterations establish
unique chromatin landscapes, which in turn, drive
transcriptomic modulation of targetable kinomes. Our KI
library screening approach has revealed a number of
chemicals that are active across all models, but more
importantly, chemicals that act in a genotype-specic
manner. To the best of our knowledge, we are the rst to
demonstrate a scalable organoid platform for chemical
genetic screening of HCC therapeutics. We believe that
informed modications to KIs will lead to stratied and
more effective therapeutics for HCC.
Keywords: Hepatocellular carcinoma; Pharmacogenomics;
Drug discovery
CEREBRAL ORGANOIDS AS MODELS OF HUMAN-
SPECIFIC BRAIN DEVELOPMENT, EVOLUTION, AND
DISEASE
Kriegstein, Arnold R.1, Pollen, Alex2, Andrews, Madeline2,
Thomas, Nowakowski3
1Eli and Edythe Broad Center of Regeneration Medicine and
Stem Cell Research, University of California San Francisco, CA,
USA, 2Neurology, University of California San Francisco, CA,
USA, 3Anatomy, University of California San Francisco, CA, USA
The human cerebral cortex has the largest number of
neurons among living mammals. The features of cortical
development that underlie the evolutionary expansion of the
human cortex as well as susceptibility to disease are largely
unexplored. We have identied outer radial glia (oRG) as a
neural stem cell population that appear to generate a
signicant number of human cortical neurons, and have
sequenced mRNA from single human progenitor cells and
young neurons for unbiased classication of cell identity and
for detection of activated signaling pathways. oRG cells are
enriched in genes related to extracellular matrix production,
epithelial-mesenchymal transition, and stem cell maintenance,
suggesting mechanisms by which human oRG cells actively
maintain their neural stem cell niche. Additionally, our genomic
data has informed a novel model of primate corticogenesis,
suggested a relationship between oRG cells and brain tumors
and provided insights into the specic cell types affected by
genetic forms of lissencephaly. Our census of cell types and
lineages along with their molecular and physiological proper-
ties also supports comparisons of the same cell subtypes
generated from pluripotent stem cells in in vitro models of
development. To identify human-specic features of cortical
development and disease, we leveraged recent innovations
that permit generating pluripotent stem cell-derived cerebral
organoids from human and non-human primates. We system-
atically evaluated the delity of organoid models to primary
human and macaque cortex, nding organoid models preserve
gene regulatory networks related to cell types and develop-
mental processes but exhibit increased metabolic stress. Our
molecular insights also show that human oRG cells are
dependent upon mTOR signaling, and are thus likely to be
targeted by disease causing mutations of this signaling
pathway, as for example, in autism.
Funding Source
Supported by grants from the NINDS, the NIMH, and the
California Institute of Regenerative Medicine.
Keywords: organoid; cortex; mTOR
FRIDAY, 22 FEBRUARY 2019
MODELING CELL INTERACTIONS
VASCULARIZATION OF TISSUE-SPECIFIC NORMAL
AND MALIGNANT ORGANOIDS WITH ADAPTABLE
ENDOTHELIAL CELLS
Rai, Shahin and Palikuqi, Brisa
Division of Regenerative Medicine, Ansary Stem Cell Institute,
Weill Cornell Medicine, New York, NY.
Normal organoids and tumoroids are ideal biological replica of
the organ-specic tissues. Nonetheless, most of the organoid
cultures are devoid of interactive niches, which has diminished
the physiological and translational relevance of this technology.
Tissue-specic vascular endothelial cells (VECs) represent
essential niche cells, that by supplying key paracrine angiocrine
signals regulate the metabolic, morphogenic and patterning
during organ regeneration and tumorigenesis. However, current
sources of adult VECs fail to arborize, adapt and interact with
the various organoids in three-dimensional (3D) co-cultures.
Here, we show that transient expression of the embryonic-re-
stricted ETS variant 2-transcription factor (ETV2) along with
dened extracellular matrix, “Reset” adult human mature
endothelial cells into adaptable VECs (R-VECs) capable of
remodeling into long-lasting compliant tissue-specic vessels.
In dened Laminin, Entactin, CollagenIV (L.E.C) extracellular
matrix, without the constraints of articial scaffolds or enforced
perfusion, R-VECs acquire the molecular and cellular attributes
29
AMSTERDAM NETHERLANDS
Speaker Abstracts
of interactive and malleable VECs. Upon seeding into dened
L.E.C matrix, R-VECs undergo harmonized tubulogenesis
and self-assemble into lumenized vascular network. In 3D
co-cultures, R-VECs avidly arborize and co-opt tissue-specic
organoids and tumoroids supporting in vivo and in vitro
organogenesis and tumorigenesis. R-VECs serve as an
adaptive vascular niche cells that by deploying key angiocrine
factors accelerate intestinal, islet, and glomerular epithelial
organoid expansion and patterning. By contrast, R-VECs
maladapt to various tumor organoids forming abnormal
vessels supporting aberrant tumor cell growth and invasive
behaviour. Deciphering the cross-talk among R-VECs and
co-opted organoid cells could facilitate identication of
factors that determine tissue/tumor-specic EC heterogene-
ity, enable physiological chemical screens and set the stage
for functional organ regeneration and tumor targeting.
Keywords: Adaptable endothelium, Organoid vascularization,
Tumuroid vascularization
HUMAN IPSC-DERIVED CARDIAC FIBROBLASTS
ENHANCE STRUCTURAL AND FUNCTIONAL
CARDIOMYOCYTE MATURATION IN 3D MICRO-
TISSUES
Giacomelli, Elisa1, Meraviglia, Viviana1, Campostrini, Giulia1,
Garcia, Ana Krotenberg1, Slieker, Roderick2, van Helden,
Ruben WJ1, Jost, Carolina2, Mulder, Aat2, Mei, Hailiang3,
Koster, Abraham AJ2, Tertoolen, Leon GJ1, Orlova, Valeria1,
Bellin, Milena1
1Anatomy and Embryology, Leiden University Medical Centre,
Leiden, Netherlands, 2Cell and Chemical Biology, Leiden
University Medical Centre, Leiden, Netherlands, 3Sequencing
analysis support core, Leiden University Medical Centre,
Leiden, Netherlands
Human induced pluripotent stem cell-derived cardiomyo-
cytes (hiPSC-CMs) in vitro are structurally and functionally
immature, unless incorporated into engineered tissues or
forced to undergo cyclic contraction, thus limiting their use
in many potential applications. Previously, we described
that scaffold-free, miniaturized three-dimensional (3D)
cardiac microtissues (MT-CMECs), containing hiPSC-CMs
and hiPSC-derived cardiac endothelial cells, exhibited
some features of maturation. Here, we increased microtis-
sue complexity through inclusion of cardiac broblasts
derived from hiPSC-epicardium (MT-CMECFs) and we
showed an enhancement of structural, electrical, mechani-
cal and metabolic maturation in hiPSC-CMs compared to
MT-CMECs. MT-CMECFs were larger and showed more
homogeneous endothelial cell distribution compared to
MT-CMECs. Direct coupling occurred between hiPSC-CMs
and cardiac broblasts and intercellular paracrine signals
appeared to contribute to hiPSC-CM maturation. hiP-
SC-CMs in MT-CMECFs showed enhanced sarcomere
alignment, T-tubule formation, electrophysiology, contractil-
ity, and canonical responses to pharmacological regulators
of inotropy compared to MT-CMEC. Primary adult cardiac-
but not dermal broblasts could replace hiPSC-cardiac
broblasts. Our results indicate that cardiac broblasts
play crucial rol
es in enhancing CM maturation. Using just 5000
cells per microtissue we were thus able to induce structural,
electrical, mechanical and metabolic maturation of hiPSC-CMs. In
conclusion, this system represents a scalable and affordable
platform to study cardiovascular development, disease and
cardiotoxicity in vitro.
Funding Source
European Research Council; European Community’s Seventh
Framework Programme; European Union’s Horizon 2020 re-
search and innovation Programme; Netherlands
Organ-on-Chip Initiative (NWO Gravitation project).
Keywords: Three-dimensional culture model; Cardiac microtis-
sue; Cell-cell interactions
ALLOMETRIC SCALING OF ORGANOIDS
Ahluwalia, Arti, Magliaro, Chiara
Centro di Ricerca E.Piaggio, University of Pisa, Italy
Despite their remarkable resemblance to downscaled
mammalian organs, organoids are often reported to possess
non-viable cores due to oxygen diffusion limits. To assess their
potential as physiologically relevant in vitro models, we
determined a working window in which quarter power allome-
tric scaling as well as a minimum threshold oxygen concentra-
tion is maintained during organoid growth. Oxygen consump-
tion and diffusion in organoids at different stages of growth
were computed using three-dimensional nite element models.
The results show that it is impossible to establish mature
organoids free of necrotic cores using current protocols.
However, micro-uidic oxygen delivery methods can be
designed to ensure that oxygen levels are above a minimum
viable threshold throughout the constructs and that they follow
physiological metabolic scaling laws. The results provide new
insights into the signicance of
allometric scaling in systems
not equipped with a resource- supplying network. They may also
be used to guide the
exp
erimental set up and design of more predictive and physio-
logically-relevant in vitro models, providing an
effective alternative to animals in research.
Keywords: Computational modelling; Allometric scaling;
Oxygen
MODELING THE TUMOR IMMUNE MICROENVIRONMENT
IN ORGANOIDS
Kuo, Calvin, MD, PhD
Department of Medicine, Stanford University School of Medicine,
Stanford, CA
The tumor microenvironment contains diverse populations
including broblasts, vasculature and immune cells, all of
which critically regulate tumor progression. A major historical
impediment to basic and translational cancer studies has been
the lack of in vitro methods that allow tumor epithelium to be
robustly cultured together with stromal components. This talk
will describe advances in 3D tumor organoid culture that allow
tumor cells to be grown in a manner that preserves their native
interaction with diverse tumor-inltrating immune cells and
AMSTERDAM NETHERLANDS
30
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Speaker Abstracts
recapitulates immune checkpoint inhibition. Such organoid
modeling of the tumor immune microenvironment within
human clinical cancer biopsies will facilitate basic and
translational immunooncology studies, allow a more holistic
in vitro modeling of cancer biology and potentially facilitate
determination of personalized treatment responses.
Keywords: Organoids, Immunotherapy, checkpoint inhibition,
PD-1
TISSUE ENGINEERING
ENGINEERING ORGANOID DEVELOPMENT
Lutolf, Matthias P.
Ecole Polytechnique Federale de Lausanne, Switzerland
Organoids form through poorly understood morphogenetic
processes in which initially homogeneous ensembles of stem
cells spontaneously self-organize in suspension or within
permissive three-dimensional extracellular matrices. Yet, the
absence of virtually any predened patterning inuences such as
morphogen gradients or mechanical cues results in an extensive
heterogeneity. Moreover, the current mismatch in shape, size and
lifespan between native organs and their in vitro counterparts
hinders their even wider applicability. In this talk I will discuss
some of our ongoing efforts in developing programmable
organoids that are assembled by guiding cell-intrinsic self-pat-
terning through tissue engineering.
Keywords: Organoid; Self-organization; Tissue engineering
ENGINEERING A HUMAN OSTEOGENIC TISSUE
MODEL
Habib, Shukry J.
Centre for Stem Cells and Regenerative Medicine, King’s
College, London, UK
Tissue engineering focuses on the development of tissue and
organ substitutes by generating a controlled 3D biological and
biophysical environment. Stem cell-based therapies that
integrate tissue-engineering technologies and biomaterials
science are
a fundamental pillar of regenerative medicine. Wnt signalling
regulates, with high spatial control, the function of skeletal stem
cells (SSCs) and is essential for bone remodelling. We have
recently pioneered a novel engineered system that recapitulates
a human bone niche. Our system consists of an immobilised
Wnt-platform that maintains primary human SSC population
and directs the production of multilayers of increasingly
differentiated cells with an osteogenic phenotype in an organ-
ised fashion within one week. We have characterised the
mechanism of the formation of the Wnt induced osteogenic
tissue model (WIOTM). Our ndings indicate that the immobil-
ised Wnt polarises the Wnt/beta-catenin machinery, controls
the spindle orientation and induces asymmetric cell division of
the SSCs in 3D. This implicates the role of spatially conned
Wnt signals in the maintenance of stem cells and directed cell
differentiation. Therefore, spatial presentation of Wnt signals
to cells in a 3D context can be used for tissue engineering
purposes. Our ultimate goal is to mimic basic cellular, signal-
ling, and mechanical elements of the bone environment by
generating a controlled microsystem of stem cells and a
directed differentiation into oestrogenic cells in 3D culture.
Funding Source
Sir Henry Dale Fellowship and the UK Regenerative Medicine
Platform (Medical Research council).
Keywords: Bone regeneration and tissue engineering;
Asymmetric stem cell division, Wnt-bandage; Biomaterials
HIGH-CONTENT SCREENING IN HUMAN CARDIAC
ORGANOIDS IDENTIFIES KEY PROLIFERATIVE
PATHWAYS WITHOUT FUNCTIONAL SIDE-EFFECTS
Hudson, James
Cell and Molecular Biology, QIMR Berghofer Medical
Research Institute, Brisbane, Australia
Human pluripotent stem cell-derived cardiomyocytes are
emerging as a powerful platform for cardiovascular drug
discovery and toxicology. However, standard 2D cultures
are typically immature, which limits their capacity to predict
human biology and disease mechanisms. To address this
problem, we have recently developed a high-throughput
bioengineered human cardiac organoid (hCO) platform,
which provides functional contractile tissue with biological
properties similar to native heart tissue including mature,
cell cycle-arrested cardiomyocytes. Here, we take advantage
of the screening capabilities of our mature hCO system to
perform functional screening of 105 small molecules with
pro-regenerative potential. Our ndings reveal a surprising
discordance between the number of pro-proliferative
compounds identied in our mature hCO system compared
with traditional 2D assays. In addition, functional analyses
uncovered detrimental effects of many hit compounds on
cardiac contractility and rhythm. By eliminating compounds
that had detrimental effects on cardiac function, we identied
two small molecules that were capable of inducing cardio-
myocyte proliferation without any detrimental impacts on
function. High-throughput proteomics on single cardiac
organoids revealed the underlying mechanism driving
proliferation, which involved synergistic activation of the
mevalonate pathway and a cell cycle network. In vivo valida-
tion studies conrmed that the mevalonate pathway was shut
down during postnatal
heart maturation in mice and statin-mediated inhibition of the
pathway inhibited proliferation and heart growth during the
neonatal window. This study highlights the utility of human
cardiac organoids for pro-regenerative drug development
including identication of underlying biological mechanisms
a
nd minimization of adverse side-effects.
Funding Source
National Health and Medical Research Council and The
National Heart Foundation of Australia
Keywords: Cardiac Tissue Engineering; Regeneration; Drug
Screening
31
AMSTERDAM NETHERLANDS
Poster Abstracts
AMSTERDAM NETHERLANDS
REJUVENATING STEM CELL FUNCTION TO
INCREASE MUSCLE STRENGTH
Blau, Helen
Microbiology and Immunology Department, Stanford
University, Stanford, CA, USA
Regenerative medicine holds great promise for local
enhancement of skeletal muscle repair to treat muscular
dystrophies and aging-associated muscle wasting. Muscle
stem cells (MuSCs) are a potent population that resides
within muscle tissues, poised to repair muscle damage
throughout life. However, the therapeutic utility of MuSCs is
currently limited by their rarity and their inefcient survival,
self-renewal, and differentiation after injection into muscle
tissue. We have devised bioengineering strategies and
discovered novel molecular regulators to surmount these
hurdles. By dening the myogenic stem cell progression by
single cell mass cytometry (CyTOF), we can target metabolic
functions that dictate cell fate transitions. By fabricating
biomimetic hydrogels with differing elasticity matching
muscle tissue,
we can overcome the loss of stem cells on traditional plastic
cultureware. Fibrosis, which causes dysfunction and
ultimate failure of numerous tissues with aging, is character-
ized by increased tissue stiffness. We are developing a
dynamic material platform to enable mechanistic studies of
cellular dysfunction as brosis progresses in real time. Cell
autonomous defects in MuSC function accompany aging. By
targeting these molecular pathways, we can rejuvenate stem
cell function. As an alternative to cell therapy, we are seeking
to stimulate the function of endogenous quiescent satellite
stem cells within muscle tissues. Through an in silico
screen, we identied a potent regulator that robustly aug-
ments stem cell function and may serve as a novel therapeu-
tic agent to induce muscle regeneration and counter
debilitating muscle wasting in the elderly.
Keywords: rejuvenation ; muscle stem cell; hydrogel micro-
environment
CLOSING KEYNOTE
CEREBRAL ORGANOIDS: MODELLING HUMAN
BRAIN DEVELOPMENT AND TUMORIGENESIS IN
STEM CELL DERIVED 3D CULTURE
Knoblich, Juergen A.
IMBA-Institute of Molecular Biotechnology, Vienna, Austria
The human brain is unique in size and complexity, but
also the source of some of the most devastating human
diseases. While many of these disorders have been
successfully studied in model organisms, recent experi-
ments have emphasized unique features that cannot easily
be modeled in animals. We have therefore developed a 3D
organoid culture system derived from human pluripotent
stem cells that recapitulates many aspects of human brain
development. These cerebral organoids are capable of
generating several brain regions including a well-organized
cerebral cortex. Furthermore, human cerebral organoids
display stem cell properties and progenitor zone organiza-
tion that show characteristics specic to humans. We have
used patient specic iPS cells to model microcephaly, a
human neurodevelopmental disorder that has been difcult
to recapitulate in mice. This approach reveals premature
neuronal differentiation with loss of the microcephaly
protein CDK5RAP2, a defect that could explain the disease
phenotype. More recently, we have been able to generate
organoid based models for human brain cancer and demon-
strated their feasibility for drug testing. Our data demon-
strate an in vitro approach that recapitulates development of
even this most complex organ, which can be used to gain
insights into disease mechanisms.
Keywords: Organoids; tumorigenesis; stem cells
32
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Poster Abstracts
001
GENERATION AND CHARACTERIZATION OF
TASMANIAN DEVIL (SARCOPHILUS HARRISSI)
INDUCED PLURIPOTENT STEM CELL-DERIVED
MESENCHYMAL STEM CELLS
Weeratunga, Muhandiram P.1, Shahsavari, Arash 1,
Fennis, Evelien2, Ovchinnikov, Dmitry3, Whitworth, Deanne1
1School of Veterinary Sciences, The University of Queensland,
Brisbane, Australia, 2Faculty of Veterinary Medicine, Utrecht
University, Utrecht, Netherlands, 3Australian Institute for
Bioengineering and Nanotechnology, The University of
Queensland, Brisbane, Australia
Marsupials have long attracted scientic interest because
of their unique biological features and their position in
mammalian evolution. Mesenchymal stem cells (MSCs)
have attracted considerable research interest in translational
medicine due to their immunomodulatory, anti-inammatory,
regenerative, migratory and homing properties. MSCs have
been harvested from various tissues in numerous eutherian
species; however, there are no descriptions of MSCs derived
from a marsupial. In this study, we have generated Tasmanian
devil (Sarcophilus harrisii) MSCs from devil induced
pluripotent stem cells (iPSCs), thus providing an unlimited
source of devil MSCs circumventing the need to harvest
tissues from live animals. Devil iPSCs were differentiated
into MSCs (iMSCs) through both embryoid body formation
assays (EB-iMSCs) and via inhibition of the transforming
growth factor beta (TGF-β)/activin signalling pathway
(SB-iMSCs). Both EB-iMSCs and SB-iMSCs are highly
proliferative, express the specic MSC surface markers
CD73, CD90 and CD105, in addition to the pluripotency
factors OCT4, SOX2 and NANOG. Moreover, devil iMSCs
readily differentiate along the adipogenic, osteogenic and
chondrogenic pathways in vitro, conrming their trilineage
differentiation potential. Importantly, while in vitro teratoma
assays conrmed the pluripotency of devil iPSCs, iMSCs
only form derivatives of the mesodermal germ layer.
Therefore, devil iMSCs will be an indispensable tool for
further studies on marsupial biology but, perhaps most
signicant, is their potential in the development of an
effective MSCs based treatment strategy against Devil
Facial Tumour Disease (DFTD) which is threatening
Tasmanian devils with extinction.
Funding Source
This study was funded by a Morris Animal Foundation (USA)
grant offered to Deanne J. Whitworth (Grant Number:
D14ZO-080) and an Australian Government Research
Training Program Scholarship offered to Prasanna Weera-
tunga.
Keywords: Mesenchymal stem cells; marsupial;
Devil Facial Tumour Disease
003
INVESTIGATING THE HUMAN TEASHIRT GENE
TSHZ3 IN PANCREATIC AND ENDOCRINE LINEAGE
COMMITMENT USING GENOME MODIFIED HUMAN
EMBRYONIC STEM CELLS (HESC)
Eng, Shermaine, Dunn, Norris Ray, Tsuneyoshi, Norihiro
Endodermal Development and Differentiation, A*STAR
Institute of Medical Biology, Singapore
PDX1 (Pancreatic and Duodenal Homeobox 1) encodes an
evolutionarily-conserved homeobox transcription factor
expressed during the earliest stages of human pancreatic
development. PDX1 transcripts label early pancreatic
progenitor (ePP) cells that are multipotent, giving rise to all
components of the adult organ. Strikingly, loss of PDX1
results in complete pancreatic agenesis in man and mice, a
drastic phenotype that emphasizes PDX1’s critical role in
coordinating the morphogenesis of this indispensable organ.
In the Dunn laboratory, a human embryonic stem cell (hESC)
differentiation protocol was developed that tightly adheres to
developmental logic and yields abundant PDX1+ ePP cells
after roughly 17 days of in vitro culture. These ePP cells
display a molecular signature that signicantly overlaps with
the developing pancreatic primordium in vivo. PDX1 knock-
out hESC lines were also generated using TALEN gene
editing technology, and it was found that PDX1 null cells
expectedly fail to activate the pancreatic transcriptional
program and divert to alternate fates in vitro. This nding
mirrors the human congenital disorder of pancreatic agene-
sis that results from the loss of the PDX1 gene. Unpublished
microarray studies comparing wild-type and PDX1 knock-out
hESC lines that are available in the laboratory reveal a list of
differentially expressed genes, which are hypothesized to
play key roles in orchestrating pancreatic development
downstream of PDX1. Corresponding microarray studies of
PDX1 null mutant hESC with PDX1 Chromatin Immunoprecip-
itation-Sequencing data revealed a novel list of candidate
PDX1 transcriptional targets including TSHZ3, the human
homolog of the Drosophila homeotic gene teashirt, which is
involved in gut and limb development. The gene’s functions
are analyzed in detail for its effect on pancreatic differentia-
tion through loss-of-function cell lines generated using
CRISPR/Cas9 genome-engineering. Functionalizing the
candidate gene will greatly expand our knowledge of human
pancreatic development and impact our understanding of
pancreatic homeostasis and disease. Our results provide
insights into the importance of TSHZ3 in endocrine commit-
ment and pancreatic differentiation, and interestingly, the
gene’s inuence on neural development.
Keywords: Pancreatic differentiation; TEASHIRT; PDX1
ODD NUMBERED POSTERS PRESENTED THURSDAY, 21 FEBRUARY FROM 13:00 TO 14:00.
EVEN NUMBERED POSTERS PRESENTED FRIDAY, 22 FEBRUARY FROM 13:00 TO 14:00.
ABSTRACTS ARE INCLUDED ON PAGES 31–106.
TO SEARCH FOR AUTHORS ALPHABETICALLY, PLEASE REFER TO THE AUTHOR INDEX AT THE END OF
THIS PROGRAM BOOK.
33
AMSTERDAM NETHERLANDS
Poster Abstracts
004
BART-SEQ: COST-EFFECTIVE MASSIVELY
PARALLEL TARGETED SEQUENCING FOR SINGLE
CELL TRANSCRIPTOMICS
Uzbas, Fatma1, Opperer, Florian1, Shaposhnikov, Dmitry1,
Angerer, Philipp2, Sass, Steffen2,
Sonmezer, Can3,
Theis, Fabian J.4, Muller, Nikola2, Drukker, Micha1
1Institute of Stem Cell Research, Helmholtz Center Munich,
Germany, 2Institute of Computational Biology, Helmholtz Center
Munich, Germany, 3Genome Biology Unit, European Molecular
Biology Laboratory, Heidelberg, Germany, 4Department of
Mathematics, Technical University of Munich, Germany
The introduction of techniques for single cell transcriptomics
by next-generation sequencing (NGS) has remarkably
accelerated our understanding of stem cell regulation.
Majority of these, however, suffer from shallow coverage
because they are global (unbiased), thus require numerous
reads to cover the gene repertoire. Targeted approaches can
remove this bottleneck by lowering the number of analyzed
genes (thus, increase the coverage), but current methods are
not compatible with gene expression measurements of single
cells. We developed the rst highly sensitive and quantitative
barcoding technique for enriching selected transcripts from
thousands of samples/single cells cost-effectively, using
common laboratory equipment and reagents, and multiplex-
ing them for conventional NGS. The novel workow, named
Barcode Assembly foR Targeted Sequencing (BART-Seq), is
based on a simple method for synthesizing differentially
barcoded forward and reverse primers, which are assembled
as matrices to generate sample-specic combinatorial
amplicon labels. Moreover, we developed tools for designing
the primers and barcodes, and decoding the barcoded NGS
reads. We demonstrated the technique by analyzing the
expression of selected pluripotency markers in human
embryonic stem cells (hESCs) grown on different media
(mTeSR, E8, and bFGF), which might account for the varia-
tions in multi-lineage differentiation capacities of the cells
maintained with them. Furthermore, we have observed
distinguishable subpopulations emerging from hESCs upon
72 hours of Wnt/b-catenin pathway activation using CHIR,
ectopic expression of constitutively active b-catenin, or
recombinant Wnt3a protein. Taken together, BART-Seq will
complement low-sensitivity global single cell transcriptomics
approaches for discovering the mechanisms of stem cell
regulation, by cost-effective analysis of thousands of single
cells across the dynamic range of gene expression.
Keywords: Targeted single cell RNA sequencing; Human
pluripotent/embryoic stem cells; Barcoding
005
HDSPC-DERIVED CONDITIONED MEDIUM PLAYS A
PROTECTIVE ROLE AGAINST UVA IRRADIATION IN
NORMAL HUMAN EPIDERMAL KERATINOCYTES
Shim, Joong Hyun
Faculty of Cosmetics and Beauty Biotechonology, Semyung
University, Jecheon, Korea
This study was performed to dene the epidermal
moisturizing effects of human dermal stem/progenitor
cell-derived conditioned medium (hDSPC-CM) on normal
human epidermal keratinocytes (NHEKs). To investigate
epidermal moisturizing effect of hDSPC-CM on NHEKs, I
measured superoxide scavenging activity, cell viability assay,
gene expressions, and hyaluronic acid (HA)-ELISA assay. In
this study, I could elucidate the effects of hDSPC-CM on
AQP3, HAS2, KRT1, and KRT10 mRNA expressions and
hyaluronic acid production. Quantitative Real-time RT-PCR
presented that hDSPC-CM increased gene expression levels
of HAS2, AQP3, KRT1, and KRT10 and HA-ELISA also
revealed that hDSPC-CM increased HA production in NHEKs.
I identied the epidermal moisturizing effects of hDSPC-CM,
and this outpot showed that the hDSPC-CM can be a potent
moisturizing cosmetic ingredient for epdiermal moisturizing.
Based on this study, I anticipated further studies about the
mechanisms of hDSPC-CM on epidermal keratinocytes to
develop not only cosmetics but for healthcare medicines.
Funding Source
This work was supported by the National Research
Foundation of Korea (NRF) grant funded by the Korea
government (MSIP; Ministry of Science, ICT & Future
Planning) (No. 2017R1C1B5076217).
Keywords: hDSPC-CM, Moisturizing; HAS2; AQP3
006
OVOL1 INFLUENCES THE DETERMINATION
AND EXPANSION OF IPSC REPROGRAMMING
INTERMEDIATES
Kagawa, Harunobu1, Shimamoto, Ren2, Kim, Shin-Il1,
Oceguera-Yanez, Fabian1, Yamamoto, Takuya1,
Schroeder, Timm 2, Woltjen, Knut1
1Department of Life Science Frontiers, Center for iPS Cell
Research and Application (CiRA), Kyoto University, Kyoto,
Japan, 2Department of Biosystems Science and Engineering,
ETH Zurich, Basel, Switzerland
During somatic cell reprogramming to induced pluripotent
stem cells (iPSCs), broblasts undergo dynamic cellular and
molecular changes. These changes include a mesenchy-
mal-to-epithelial transition (MET) and gradual gain of gene
expression regulating pluripotency; processes which are
highly inuenced by Yamanaka factor (Oct3/4, Sox2, Klf4,
and c-Myc) stoichiometry. For example, in early reprogram-
ming, high Klf4 levels are correlated with the induction of
MET genes which sustain expression in iPSCs, as well as
MET genes with transient expression, whose functions in
reprogramming remain undened. Here, we identied the
cell surface protein TROP2 as a marker for cells with
transient MET gene expression in the high-Klf4 condition. We
observed the emergence of cells expressing the pluripotency
marker SSEA-1+ mainly from within the TROP2+ fraction.
Upon isolation by cell sorting, double-positive cells which
subsequently lost TROP2 displayed a high reprogramming
potential. Using TROP2 as a marker in CRISPR/Cas9-mediat-
ed candidate screening of MET genes, we identied the
transcription factor Ovol1 as a potential regulator of an
alternative epithelial cell fate characterized by the expres-
sion of non-iPSC MET genes and low cell proliferation. Our
study sheds light on how reprogramming factor stoichiome-
try alters the spectrum of intermediate cell fates, ultimately
inuencing reprogramming outcomes.
Keywords: Mouse somatic cell reprogramming; Mesenchy-
mal-to-epithelial transition; CRISPR/Cas9-mediated screening
34
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Poster Abstracts
007
CHARACTERISING EMBRYONIC STEM CELL
DERIVED TENOCYTES AND DETERMINING THE CHANG-
ING ROLE OF SCLERAXIS DURING TENDON DEVELOP-
MENT
Paterson, Yasmin Z.1, Henson, Fran2, Guest, Debbie J.3
1Stem Cell Department, Animal Health Trust, Edmunds, UK,
2Veterinary Medicine, University of Cambridge, UK, 3Stem Cell
Department, Animal Health Trust, Kentford, UK
Tendon injuries are common in human and equine athletes. Adult
tendon injuries heal via the formation of biomechanically inferior
scar tissue resulting in high re-injury rates. However, fetal tendon
injuries undergo total scar-less regeneration. Novel cell therapies
should therefore try to recapitulate this scar-less fetal tendon
regeneration. This study aims to build upon research into the use
of equine embryonic stem cells (ESCs) to aid tendon regeneration
by determining if tenocytes derived from ESCs are more represen-
tative of the fetal or adult stage of tendon development. Using
genome wide transcriptional analysis, we compared fetal, adult
and ESC-derived tenocytes cultured and differentiated in a 3D
collagen matrix. 2,260 genes were differentially expressed
between adult and ESC-derived tenocytes, whereas 1,254 genes
were differentially expressed between fetal and ESC-derived
tenocytes (log fold change ±2; q-value <0.05). Genes which were
signicantly upregulated in adult tenocytes tended to be ex-
pressed at similar levels in fetal and ESC-tenocytes and were
highly associated with immune system processes and the
recognition of foreign external stimulus. Genes which were
signicantly upregulated in fetal tenocytes tended to be even
further upregulated in ESC-derived tenocytes and were highly
associated with cellular adhesions. To further study how adult and
fetal tenocytes differ, scleraxis (SCX), an important transcription
factor involved in tendon formation was knocked down in four
lines of fetal and adult tenocytes. Quantitative PCR determined
that SCX knock-down had differential effects in adult and fetal
cells on the expression of several genes including matrix metallo-
proteinases and collagens. RNA-sequencing is currently being
performed to provide more global information on the effects of
SCX knock-down in fetal and adult tenocytes. In summary, our
initial work towards global gene expression proling in fetal and
adult tenocytes has yielded useful information into key over-repre-
sented pathways and novel genes that are likely to play a role in
regulating tendon development and healing. Future work will now
delve into the developmental differences between ESC-derived,
fetal and adult tenocytes.
Funding Source
Petplan Charitable Trust; BBSRC
Keywords: Equine; ESC-derived Tenocytes; Tendon-injury
008
AIPL1-/- END-STAGE RETINAL DEGENERATION MICE
AS A MODEL FOR CELL TRANSPLANTATION
Claudio, Ribeiro, Joana, Filipa, Procyk, Christopher, Goh, Debbie,
Naeem, Arifa, Jumbo, Neeraj, Sampson, Robert D., Hoke, Justin,
Hare, Aura, Bainbridge, James W.B, Smith, Alexander J., Pearson,
Rachael A., Gonzalez-Cordero, Anai, Ali, Robin R.
Institute of Ophthalmology, University College London, UK
Cell transplantation is a promising therapeutic approach to treat
degenerative retinal diseases, the leading cause for blindness in
the western world. Although hESC-derived photoreceptors have
been used extensively for transplantation studies in various
mouse models of retinal degeneration, further optimization is
still required to achieve substantial improvement of visual
function. We have previously shown that hESC-derived cone
photoreceptors can survive following transplantation into
Aipl1-/- mice. Here we sought to characterize in detail the
degeneration and remodelling of the Aipl1-/- retina to optimize
the transplantation of hESC-derived cone photoreceptors into
this model. Advanced photoreceptor degeneration was already
evident by post-natal stage 18 (P18) with a single row of rod
and cone photoreceptors in the ONL. In the INL rod bipolar cells
were displaced and their axon processes appeared retracted.
Both horizontal and amacrine cells are reduced in number.
Post-synaptic markers are absent in interneurons at 3 months
of age. No difference in the number of ganglion cells was
observed. Following transplantation, no signicant improve-
ment in visual function was detected by the methods used, but
cell maturation and pre-synaptic proteins were identied in the
transplanted hESC-derived cone photoreceptors. This data
shows that Aipl1-/- retina suffers severe remodelling as it
degenerates. hESC-derived cone photoreceptors survive and
mature following transplantation. Our data suggests that by 3
months of age, the Aipl1-/- retina might have undergone
extensive remodelling that prevents the establishment of
functional connections with the transplanted cone photorecep-
tors. Further studies are required to establish the optimal
conditions for achieving functional rescue.
Funding Source
MRC; erc; NIHR; Moorelds eye Charity; Fighting for Blindness
Keywords: Cell Transplantation; Human Stem cells; End-
Degeneration retina
009
THE ENGINEERED ACTIVIN A PROVIDES
PROLONGED AND SUSTAINABLE SIGNALLING
ACTIVITY IN HUMAN STEM CELL CULTURING
Wang, Xuelu, Hyvonen, Marko
Department of Biochemistry, University of Cambridge, UK
Activin A, a member of TGF-beta superfamily, is widely used in
human stem cell culturing to maintain the cell pluripotency
and induce differentiation. Its signalling activity also induces
the cells to produce follistatin, a secreted antagonist of
activin A, which binds to activin A and inhibits its signalling in
a negative-feedback loop. This leads to gradual reduction of
activin A bioactivity during the stem cell culturing. With the
aim to develop an engineered form of activin A with more
sustainable activities, we have designed a number
of activin A mutants that preserve the wild-type signalling
activity, but resist inhibition by follistatin. We have used
activin A-responsive luciferase assay to conrm that these
mutants have more sustained signalling activities during the
culturing of human stem cells. These engineered forms of
activin A could provide a more stable and sustainable
signalling environment for stem cell culturing. Moreover, there
are potentials to reduce the amount of the engineered activin
A added into the culture due to their longer half-life times.
Funding Source
This work is supported by the Biotechnology and Biological
Sciences Research Council (BBSRC) Follow-on funding.
Keywords: Human stem cell culturing; TGF-beta superfamily;
Sustained activity
35
AMSTERDAM NETHERLANDS
Poster Abstracts
010
FUNDAMENTAL ROLE FOR HIF-1A IN INTESTINAL
HOMEOSTASIS AND AUTOIMMUNE DISEASE
Wen, Jinming1, Bittel, Miriam2, Günther, Claudia2,
Schett, Georg1, Bozec, Aline1
1Department of Internal Medicine, Friedrich–Alexander University
Erlangen–Nürnberg, Germany, 2Department of Medicine,
Friedrich-Alexander-University Erlangen-Nürnberg, Germany
HIF transcription factors (HIF-1a and HIF-2a) are central
mediators of cellular adaptation to hypoxia. Because the
resting partial pressure of oxygen is low in the intestinal
lumen, epithelial cells are believed to be mildly hypoxic. Having
recently established a link between HIF and mucosal inamma-
tion and dysbiosis, we hypothesized that HIFs stabilized in the
hypoxic intestinal epithelium, may also play critical roles in
regulating intestinal homeostasis. To explore this idea, we
generated small intestine organoids and cultivated them under
normoxia (21% O2), or Hypoxia (1% O2) conditions. As expected,
hypoxia strongly inuenced the viability of organoids. Organ-
oids maintained for 8 hours under hypoxia conditions dis-
played high levels of HIF1a and elevated numbers of apoptotic
cells. Next we generated conditional knockout mice that
lacked Hif1a specically in the intestinal epithelium (HIF-
1α∆IEC mice). Using in vitro HIF-1α∆IEC organoid culture, we
showed that HIF-1α is essential for organoid cell survive after
hypoxia exposure. Moreover, intestinal inammation and
dysbiosis have been linked to autoimmune diseases such as
rheumatoid arthritis (RA), however, the underlying mechanisms
remain incompletely understood. Using collagen induced
arthritis in HIF-1α∆IEC mice, we demonstrated that decient
mice have severer than littermate controls, which was
correlated with an increase of intestinal IgA+ B cells and
splenic Th1 cells in HIF-1α∆IEC mice.Taking together, these
nding indicated that HIF-1a play an important role in small
intestine homeostasis and autoimmune diseases by controlling
epithelial cell survival.
Funding Source
DFG
Keywords: HIF1a(hypoxia induced factor), organoid;
inammation; epithelial
011
SCALABLE MULTIWELL MICROELECTRODE
ARRAY (MEA) TECHNOLOGY FOR THE EVALUATION
OF CARDIAC AND NEURAL THREE-DIMENSIONAL
CELL CULTURES
De Filippi, Giovanna, Sullivan, Denise, Nicolini, Anthony,
Arrowood, Colin, Millard, Daniel
Axion BioSystems, Inc., Atlanta, GA, USA
Three-dimensional induced pluripotent stem cell
(iPSC)-derived in vitro models, commonly referred to as
spheroids, organoids, or “mini-brains”, more accurately
recreate the multicellular organization and structure of in
vivo tissues when compared to traditional monolayer stem
cell cultures. However, to effectively characterize 3D iPSC
cell cultures, or to extract meaningful and predictive
information from these models, new technology is required
for evaluating functional cellular and network responses. For
electro-active cells, like cardiomyocytes or neurons,
measurements of electrophysiological activity across a
networked population of cells provide a comprehensive view
of function. Microelectrode array (MEA) technology offers
such a platform by directly connecting key biological variables,
such as gene expression or ion channel distributions, to
measures of cellular and network function. Furthermore, the
advent of multiwell MEA platforms has enabled scalable
throughput capacity for 3D cell culture applications including
toxicological and safety screening, disease modeling, and
developmental biology. Raw neural or cardiac electrical
activity can be captured simultaneously from each electrode
on an MEA multiwell plate and in relation to the position of
the spheroid on the array. Here, we present data supporting
the use of multiwell MEA technology as an efcient
non-invasive approach to capture electrophysiological
activity from individual iPSC-derived spheroids. Human
iPSC-derived cardiomyocytes and neural spheroids were
cultured on 6-well MEA plates and monitored throughout
maturation of their network connections. Cardiac
electrophysiological activity, including spike amplitude and
eld potential duration, were recorded in response to
compounds to provide information on the depolarization and
repolarization of the cardiomyocyte action potential. For
neural cultures, functional endpoints, such as network
bursting and synchrony, were measured to dene cellular
activity across neural spheroids within a network. These
results support the continued development and use of
human iPSC-derived cardiomyocyte and neural spheroid
assays on multiwell MEA technology for high throughput
drug toxicity and safety assessment, evaluation of
phenotypic disease-in-a dish models, and cell development.
Keywords: microelectrode array (MEA); spheroids; electro-
physiology
012
PRIMARY HUMAN HEPATOCTYE 3D SPHEROIDS
FOR STUDYING HEPATIC FUNCTION AND DRUG
TOXICITY
Kuninger, David, Kennedy, Mark, Lahiri, Sujoy, Hoover,
Jenni, Tieberg, Deborah, Horton, Robert, Connolly, Michael,
Witek, Rafal, Powers, Mark
Cell Biology, Thermo Fisher Scientic, Frederick, MD, USA
Primary Human Hepatocyte (PHH) culture provides the
closest in vitro model to human liver that can produce a
metabolic prole of a given drug very similar to that found in
vivo. Hence, PHH culture is the gold standard for studying
the in vitro hepatic biology, liver function, and drug induced
hepatotoxicity. The conventional 2-dimensional (2D) PHH
culture is limited by de-differentiation and rapidly loss of
hepatic specic functions. Therefore, there is a need for
more robust in vitro models that reects in vivo liver biology
with better culture longevity. Recently, 3-dimensional (3D) in
vitro models for hepatocytes have gained a lot of attention
for their ability to recapitulate the hepatic function and greater
longevity. Recently we have developed an easy-to-assemble
in vitro PHH 3D-spheroid model. Our initial work shows that
PHH can assemble into spheroids using low attachment
96-well U-bottom plates within 5 days of seeding. Interesting-
ly, we have also found that not every lot of PHH can assem-
36
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Poster Abstracts
ble into 3D-spheroids. We have shown that seeding 1,500
PHH/well resulted in spheroid formation with h
omogenous
morphology and consistent size (~200 μm diameter). These
PHH spheroids can live up to 28 days in culture and can
retain hepatocyte-specic functions. To assess whether
hepatocyte-specic functions were maintained in the PHH
spheroids during prolonged culture, albumin secretion,
CYP3A4 activity and levels of ATP synthesized were ana-
lyzed. These parameters were found to remain stable during
prolonged culture period. Also, gene expression prof
iles at 5,
7, 14 and 21 days showed a relatively higher expression of
hepatocyte specic genes, such as albumin and CYP3A4,
compared to that of the 2D-culture. Finally, we have per-
formed cytotoxicity assay using compounds causing drug
induced liver injury (DILI), such as Chlorpromazine and
Diclofenac, and found comparable IC50 values between the
2D and 3D cultures using PHH. These results indicate that
the PHH 3D-spheroid system developed by us constitutes a
promising in vitro tool to evaluate hepatic function. As part
of our future work, we are investigating the possibility of
introducing nonparenchymal liver cells like Kupffer and
Stellate cells to the spheroid system to assess feasibility of
creating various liver disease models.
Keywords: Organoid; Disease modeling; Hepatic spheroid
013
IN VITRO DRUG EFFICACY EVALUATION IN
CULTURED
HUMAN IPSC-DERIVED NEURONS USING
MEA SYSTEM
Suzuki, Ikuro
Department of Electronics, Tohoku Institute of Technology,
Sendai, Miyagi, Japan
Micro-electrode array (MEA) assay using human iPSC-de-
rived neurons are expected to one of in vitro assays to
predict the toxicity and predict the mechanism of action of
drugs. Human iPSC-derived cortical neurons and astrocytes
were cultured on 24-wells MEA plate for extracellular
recording using MED64 Presto. Twelve compounds (penty-
lenetetrazole, picrotoxin, 4-aminopyrdine, linopyridine,
amoxapine, strychnine, pilocarpine, amoxicillin, chlorproma-
zine, enoxacin, phenytoin, and acetaminophen) and dimethyl
sulfoxide were tested at 5 concentrations for each com-
pound (n>6). In this study, we aimed to develop an analytical
method enabling the detection of toxicity of convulsants and
the prediction of mechanism of action (MoA) using deep
learning. We rstly had articial intelligence (AI) learned the
data of convulsants and the data of non-convulsants. Next
this AI predicted the Toxicity of the data that not used for
learning. The toxicity probability of unlearned sample data
was 90% or more, and the toxicity probability of the un-
learned convulsants was also 80% or more. In addition, the
negative probability of non-convulsants and the prediction
probability of MoA was more than 80%. These results
indicated that this AI analysis method is useful for predicting
the convulsion toxicity and the MoA of new drugs using
hiPSC-derived neurons.
Keywords: MEA; iPSC-derived neurons; drug
014
PRECISE LEVELS OF SNF5 ARE REQUIRED FOR
HUMAN PLURIPOTENT STEM CELLS DEVELOP-
MENTAL FATE REGULATION
Urbach, Achia, Carmel-Gross, Ilana
Faculty of Life Science, Bar Ilan University, Ramat-Gan, Israel
SNF5 is one of the core subunits of the SWI/SNF
chromatin-remodeling complex. Therefore, loss of function
(LOF) or gain of function (GOF) of this protein might have
signicant effects on the epigenetic state of the cells and on
their phenotype. Indeed, SNF5 LOF is the solely genetic lesion
in Rhabdoid tumor (RT) of the kidney and in AT/RT tumor in the
brain. SNF5 is also required for early embryonic development
as it was shown that SNF5-/- mouse blastocysts lose their
hatching capacity and that SNF5 regulates the expression of
Nanog during the differentiation of mouse embryonic stem
cells. Here we aim to study the effect of SNF5 LOF and SNF5
GOF on human pluripotent stem cells (hPSCs). For this
purpose, we targeted the endogenous SNF5 gene and intro-
duced a conditional SNF5 over-expressing cassette into the
AAVS1 locus. Using this system, we show that both SNF5 GOF
and SNF5 LOF lead to rapid changes in cell fate, however in a
different manner. SNF5 GOF results in down-regulation of
pluripotency markers such as Oct4 and in cell differentiation.
SNF5 LOF by contrast, doesn’t show a direct effect on the
expression of pluripotency markers but rather seems to affect
the morphology of the cell colonies which start to grow as 3D
structures instead of at monolayer colonies. Global gene
expression analysis reveals signicant down-regulation in
pathways related to the extra cellular matrix (ECM) and
integrins upon SNF5 LOF. Notably, earlier attempts to downreg-
ulate SNF5 in embryonal carcinoma cells (NCCIT) led to similar
morphological changes while down-regulation of the other
SWI/SNF core subunits in hPSCs not. Taken together, these
results reveal that SNF5, but not other SWI/SNF subunits,
regulates the interactions between hPSCs and the ECM. This
observation is further supported by the fact that re-expression
of SNF5 in mouse RT cells leads to upregulation of cellular
pathways related to cell adhesion, extracellular space, integrin
signaling etc. To conclude, our results reveal that SNF5 levels
have to be tightly regulated in hPSCs. Furthermore, the oppo-
site effect of SNF re-expression in RT cell line and SNF5 LOF in
hPSCs demonstrates the capacity of our system to be used as
a novel tool to study RT formation.
Keywords: SWI/SNF complex; Rhabdoid tumor formation;
Cell-ECM interactions
37
AMSTERDAM NETHERLANDS
Poster Abstracts
015
EVIDENCE FOR NEURONAL PLASTICITY IN
BIOENGINEERED NEURONAL ORGANOIDS
Zafeiriou, Maria Patapia
1
, Bao, Guobin
2
, Halder, Rashi
3
,
Fischer, Andre
3
, Schild, Detlev
2
, Zimmermann, Wolfram
1
1
Institute of Pharmacology and Toxicology, University Medical
Center Goettingen, Germany,
2
Institute of Neurophysiology
and Cellular Biophysics, University of Goettingen, Germany,
3
Research Group for Epigenetics in Neurodegenerative
Diseases, German Center for Neurodegenerative Diseases,
Goettingen, Germany
To study human neuronal network function, we developed a
dened, Matrigel-free 3D cell culture system termed human
bioengineered neuronal organoids (BENOs). Neural differen-
tiation of pluripotent stem cells (iPSCs) embedded in a
collagen matrix was directed under dened serum-free
conditions. Calcium imaging revealed spontaneous tetrado-
toxin (1 µM)-sensitive signals by d25. To test spontaneous
neuronal network activity, BENOs (day 30-60) were subjected
to GABAergic (picrotoxin, 58 µM; saclofen, 330 µM) inhibi-
tion. Spontaneous Ca2+ signals of synchronized neurons
became asynchronous upon GABAR inhibition (2 indepen-
dent experiments). Antagonist washout restored synchronic-
ity suggesting the presence of functional GABAergic
networks. Stimulation (injected current: 20-100 µA)-evoked
Ca2+ inux in remote regions (distance from electrode 0.5
to 1.5 mm) suggested a neuronal network that extends
throughout the organoid. Multi-pulse stimulation demon-
strated a Ca2+ inux pattern similar to paired pulse depres-
sion (PPD). The PPD-like Ca2+ signal pattern was alleviated
by a GABA-A inhibition (picrotoxin 58 µM) and was restored
upon washout (2 independent experiments). Field potential
measurements revealed high frequency stimulation-induced
long-term potentiation (n=3) suggesting neuronal plasticity.
In conclusion, BENOs from human pluripotent stem cells
contain electrically active neuronal networks that exhibit
typical forms of plasticity observed in the human brain.
Keywords: Neuronal netwotk function; Neuronal Plasticity;
Bioengineered neuronal organoids
016
WNT AGONIST ENHANCES THE FUNCTION OF
PROGRANULIN FOR HUMAN DOPAMINERGIC
NEURONAL DIFFERENTIATION
Hao, Hsiao-Nan
1
, Pei, Haitao
2
, Peduzzi, Jean
3
, Tang, Limin
4
,
Hao, Peter
5
, Zhao, Jane
6
, Sun, Xuan
7
1
Department of Orthopeadic Surgery, Wayne State University,
Detroit, MI, USA,
2
Neurology, Qingdao University, Qingdao,
China,
3
Anatomy and Embryology, Wayne State University,
Detroit, MI, USA,
4
Radiology, Qingdao University, Qingdao,
China,
5
Biochemistry, University of Michigan, Ann Arbor, MI,
USA,
6
Neurosurgery, Providence Hospital, Troy, MI, USA,
7
OB/
GYN, Haici Hospital, Qingdao, China
The method to enrich dopaminergic neurons in vitro is
essential for Parkinson’s cell therapy. Our hypothesis is that
maintenance of progranulin (PGRN) expression in neural
precursors may be essential to ensure neuronal differentia-
tion. The reported results from our previous studies indicate
that the dopaminergic neuronal differentiation (DND) occurs
only among the nestin+/CD133+ human fetal brain cells
(HFBC) rather than the cells with same markers isolated
from adult human brain tissue. We also notice that the level
of PGRN expression in fetal neural precursor cultures can be
elevated after the cells treated with Wnt signal enhancer.
The increased PGRN expression is benet to the DND. To
explore the molecular mechanism of PGRN to regulate DND,
both PGRN antisense deoxynucleotides (PGRN-ADON) and
human recombinant PGRN protein were used for this study.
Prior incubation of the nestin+/CD133+ HFBC in dopaminer-
gic differentiation medium, cells were cultured with the
PGRN-ADON medium. After PGRN antisense treatment, the
level of PGRN expression in the nestin+/CD133+ HFBCs was
reduced. The decreased PGRN expression was conrmed
using RT-PCR, Northern blotting and protein assays. No
elevated apoptotic signal was detected after the cultures
treated with PGRN-ADON. Interestingly, inhibition of PGRN
expression declines the potential of dopaminergic differenti-
ation among nestin+/CD133+ HFBCs. Meanwhile, it is likely
reducing of PGRN expression increases GFAP expression
and astrocytic differentiation. In contrast, using exogenous
recombinated-PGRN protein to pretreat the nestin+/CD133+
HFBCs is able to supress the PGRN-ADON induced glio-
differentiation. The results from this study indicate that high
level of PGRN promotes neuronal differentiation. Although
the mechanism of DND may be not limited to the intracellu-
lar PGRN expression level, effect of PGRN on DND deserves
further investigation.
Funding Source
University internal fundings; University hospital internal
fundings.
Keywords: Wnt agonist, active Wnt signals; Progranulin,
differentiation regulator; Dopaminergic neurons, produce
dopamine in CNS
017
ELECTROPHYSIOLOGICAL PHENOTYPE
CHARACTERIZATION OF HUMAN IPSC-DERIVED
DOPAMINERGIC NEURONAL LINES BY MEANS OF
HIGH-RESOLUTION MICROELECTRODE ARRAYS
Zorzi, Giulio A.
1,2
, Fiscella, Michele
2
, Emmenegger, Vishalini
1
,
Leary, Noelle
1
, Ronchi, Silvia
1
, Hierlemann, Andreas
1
1
Department of Biosystems Science and Engineering, ETH
Zurich, Basel, Switzerland,
2
MaxWell Biosystems Basel,
Switzerland
High-resolution-microelectrode-array (HD-MEA) technology
enables to study neuronal dynamics at different scales, rang-
ing from axonal physiology to network connectivity. We have
used this HD-MEA technology to characterize and compare
the electrical phenotypes of commercially available human
dopaminergic neurons. Furthermore, we have studied the
effect of human astrocytes on neural-culture development.
Astrocyte/neuron co-cultures showed higher signal ampli-
tudes and higher ring rates than neural cultures without
astrocytes. Adding astrocytes to neural cultures changed the
whole culture morphology by promoting cell clustering.
Interestingly, astrocyte/neuron co-cultures showed a lower
sample-to-sample variability across multiple preparations
and HD-MEA recordings compared to neural cultures without
astrocytes. We compared action potential propagation
velocities along axons between dopaminergic A53T α-synu-
38
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Poster Abstracts
clein neurons and the wild-type isogenic control cell line. We
found that in both, wild-type and disease-model neurons,
axonal action potential propagation velocities were lower
than those in rat primary cortical neurons. Furthermore, we
found different axonal-action-potential-velocity-development
proles of A53T α-synuclein dopaminergic neurons and the
wild-type counterpart. Finally, we were able to precisely
evoke action potentials in individual single human neurons
by subcellular-resolution electrical stimulation. HD-MEA
systems were found to enable access to novel electrophysi-
ological parameters of iPSC-derived neurons, which can be
potentially used as biomarkers for phenotype screening and
drug testing.
Funding Source
EU, ERC Advanced Grant “neuroXscales”, contract number
694829; EU, ERC-PoC “MwHresEP”, contract number
755383; CH, Project CTI-No. 25933.2 PFLS-LS “Multi-well
electrophysiology platform for high-throughput cell-based
assays”
Keywords: iPSC;
microelectrode,
neurodegeneration
018
SALIVARY GLAND STEM CELL DESMOSOME
DYSREGULATION AND LYMPHOEPITHELIAL
LESIONS IN PRIMARY SJÖGREN’S SYNDROME
Pringle, Sarah
1
, Wang, Xiaoyan
1
, Jara Araya, Daniela
2
,
Terpstra, Janneke H.1
1
, Kuipers, Jeroen
3
, Giepmans, Ben
N.G
3
, Vissink, Arjan
4
, Spijkervet, Fred K.L
4
, Bootsma, Hendri-
ka
3
, Coppes, Robert P.
5
, Kroese, Frans G.M
1
1
Rheumatology and Clinical Immunology, University Medical
Center, Netherlands,
2
Instititute of Biomedical Sciences,
University of Chile, San Tiago, Chile,
3
Biomedical Sciences of
Cells and Systems, University Medical Centrum Groningen,
Netherlands,
4
Oral and Maxillofacial Surgery, University
Medical Centrum Groningen, Netherlands,
5
Biomedical
Sciences of Cells and Systems & Department of Radiotherapy,
University Medical Centrum Groningen, Netherlands
We have previously demonstrated that hyposalivation in the
autoimmune disease Sjogren’s syndrome (pSS) is likely due
in part to replication-induced senescence of salivary gland
stem cells (SGSCs), resident in the ducts of salivary glands
(SGs). In order to establish if SGSCs are capable themselves
of producing cytokines driving their own proliferation, we
stimulated SGSC-derived organoid toll-like receptors using
TLR stimulants Poly(I:C) and Imiquimod (specic for TLR3
and TLR7, respectively). Organoid exposure to IMQ induced
initial cell death (passages 1-3) followed by partial rescue of
proliferation (passage 4). TLR3 stimulation induced similar
but less signicant effects. Surviving organoids at p4 of IMQ
treatment upregulated expression of proinammatory
cytokines (TNFα, IFNα, IFNβ, IL6) and became disorganized
in morphology. In p4 cultures containing these disorganized
structures, desmosomes components were downregulated
at transcript level, and appeared to be scarce when exam-
ined by electron microscopy. Lymphoepithelial lesions
(LELs) are poorly understood facets of pSS SG pathology,
and presumed to be the precursor to MALT lymphomas in
pSS. They develop from the striated ducts (SDs), and involve
proliferation and loss of organization of the epithelial cells,
beginning from the cells in the SD basal layer (BSD). The
BSD layer is also one of the stem cell niches in the human
SG. When immunostained for the BSD marker K14, we
observed proliferation, lack of organization, and aberrant
differentiation of BSD cells into acinar cells, in LELs. Reason-
ing that TLR-induced desmosome dysregulation in BSD
stem cells may lead to this phenotype ad be important in
LELs formation (organoid cultures used contained a majority
of SD cells), we examined LELs from pSS patient tissue,
using a previously established LEL severity grading system.
Desmosomes in all LEL stages (1-3) were difcult to identify
in BSD cells. We hypothesize that TLR-induced desmosome
dysregulation in BSD resident SGSCs, in combination with
their ability to attract B cells through cytokine/chemokine
expression, represents the rst stages of lymphoepithelial
lesion formation. and therefore also potentially of MALT
lymphomas in pSS patients.
Funding Source
This research was funded by the Dutch Arthritis Foundation
Translational Research Grant (T015-052) and Cancer
Research
UK (grant number: C17199/A18246), and unre-
stricted research
grant from Bristol-Myers Squibb.
Keywords: primary Sjogren’s syndrome; Salivary gland stem
cells/organoids; Lymphoepithelial lesion
019
GENERATING DISEASE MODELS USING CRIS-
PR-
CAS9 GENE EDITING OF HUMAN PLURIPOTENT
STEM CELLS AND CEREBRAL ORGANOIDS
Lee, Vivian M.
1
, Anonuevo, Adam
1
, Hirst, Adam J.
1
, Eaves,
Allen
2
, Thomas, Terry
1
, Louis, Sharon
1
, Chew, Leon H.
1
1
Research & Development, STEMCELL Technologies Inc.,
Vancouver, BC, Canada,
2
Corporate Administration, STEM-
CELL Technologies Inc., Vancouver, BC, Canada
Human pluripotent stem cells (hPSCs), in combination with
CRISPR/Cas9 gene editing technologies, are emerging as
important tools to study mechanisms of pathogenesis and
modelling human diseases. Here we used CRISPR-Cas9 to
target the CDK5 regulatory subunit-associated protein 2
(CDK5RAP2), a gene implicated in the development of primary
microcephaly. Briey, a blood-derived hPSC line was dissociat-
ed into a single cell suspension, electroporated with the
ArciTect™ CRISPR-Cas9 ribonucleoprotein complex and
allowed to recover in mTeSR1™ supplemented with CloneR™.
After the initial round of clone selection, heterozygous clones
were subjected to a second round of gene editing to generate
a compound heterozygous clone harbouring frameshift
mutations in both alleles of CDK5RAP2. Clones were expand-
ed in mTeSR1™ and screened for cell quality attributes
including karyotype, pluripotency and hPSC marker expres-
sion. Expression levels of CDK5RAP2 were conrmed by
RT-qPCR and immunostaining, with only the compound
heterozygote exhibiting decreased CDK5RAP2 expression.
Compound heterozygote lines were then used to generate
cerebral organoids using the STEMdiff™ Cerebral Organoid Kit
to investigate effects of CDK5RAP2 on organoid formation,
morphology and size. Cerebral organoids generated from the
compound heterozygote line were approximately 10% - 40%
smaller in diameter compared to the heterozygote and control
at the EB formation (Day 5), neural induction (Day 7), expan-
sion (Day 10), and maturation stages (Day 18); n=4 per clonal
line, 12 - 16 EBs per measurement. Expression of neuronal
39
AMSTERDAM NETHERLANDS
Poster Abstracts
markers (DCX and TuJ-1) were increased while markers for
neural progenitors (SOX2 and PAX6) were slightly decreased in
the compound heterozygote compared to the heterozygote
and control lines. Morphology of the cortical region in the
compound heterozygote also exhibited decreased organiza-
tion of the ventricular zone and cortical plate with an increase
in neuronal marker expression. In summary, we have opti-
mized a gene editing protocol using the ArciTect™ CRIS-
PR-Cas9 system combined with mTeSR1™ supplemented with
CloneR™ to derive high quality clonal edited hPSC lines, which
can be used to generate and study brain development in a
cerebral organoid culture system.
Keywords: Human Cerebral Organoid; Gene editing; Human
pluripotent stem cell
020
DEVELOPMENT OF CORTICAL ORGANOIDS TO
STUDY CACH/VWM SYNDROME DEVELOPMENTAL
ALTERATIONS
Pain, Bertrand
1
, Baquerre, Camille
1
, Huyghes, Aurelia
2
,
Wianny, Florence
1
, Dehay, Colette
1
, Boespug-Tanguy, Odile
3
1
Stem Cell and Brain Research Institute, U1208 INSERM, Bron,
France,
2
Centre Léon Bérard, U1052 INSERM, UMR 1088
CNRS, Lyon, France,
3
Robert Debré Hospital, UMR 1141
INSERM, Paris, France
EIF2B-related disorders are autosomal recessive leukodys-
trophies due to mutations in Eukaryotic Initiation Factor 2B
genes (EIF2B). EIF2B genes are involved in the translation
initiation and protein synthesis regulation and are expressed
ubiquitously in the tissues. But Childhood Ataxia with Central
Hypomyelination/Vanishing White Matter Syndrome (CACH/
VWM) is a neurodegenerative disease and the archetype
form of those eIF2B-related disorders. It affects mainly the
development and full maturation of the brain and is charac-
terized by either a quick or a progressive evolution depend-
ing on the identied point mutation in the various EIF2B
genes. In the present work, we developed an in vitro model
allowing us to mimic the developmental evolution and
alteration resulting from the CACH/VWM Syndrome. We
generated cortical organoids from human induced pluripo-
tent stem cells (hiPSc) derived either from CACH patients
(EIF2B5 R113H/R113H – a moderate phenotype) or from
sex and aged matched control patients using a 3D specic
protocol that starts with cell aggregates. The results of this
approach allowed us i) to validate a robust protocol for
generation of long term cultured cortical organoids, ii) to
start to decipher the molecular mechanisms presiding the
defects observed in this unique developmental genetic
disease and iii) to identify morphological alterations in the
neuroepithelial structures observed during the organoïds
formation. Development of these 3D cortical organoïds
helps us to mimic the physiopathological disorders specic
to this neurodegenerative disease.
Keywords: cortical organoïds; neurodegenerative disease;
leukodystrophy
021
VISUALIZATION OF STEM CELL NICHE METABO-
LISM AND PROLIFERATION IN THE INTESTINAL
ORGANOID MODEL
Dmitriev, Ruslan I., Papkovsky, Dmitri B., Okkelman, Irina A.
School of Biochemistry and Cell Biology, University College
Cork, Ireland
Studies of 3D tissue models reveal dual role of cell metabo-
lism for multicellular organization and development: it plays
both bioenergetic and cell signaling functions. Recent
studies demonstrate that cell metabolism depends on the
cell type and it can directly play an instructive role in regula-
tion of the stem cell niche, balancing proliferation and
differentiation. Growing interest in this area correlates with
the need of new live imaging methods for detailed analysis
of cell composition, proliferation and cellular metabolism in
toto with organoid and related 3D tissue models. Here we
report the application of phosphorescence lifetime imaging
microscopy (PLIM) with the cell-penetrating O2-sensitive
probe for analysis of oxygenation, an important marker of
aerobic metabolism, in mouse intestinal organoid culture.
O2-PLIM method allows for high-resolution quantitative
imaging of O2 in individual organoids in real-time. Our
preliminary experiments with primary adult stem cell-derived
organoids revealed strong inter-cultural heterogeneity of
oxygenation (27-92 μM O2 for individual organoids); in
addition, the oxygenation also varied within the single
organoids and resulted in transmembrane gradients.
However, even with such variability, organoids displayed high
activity of the mitochondria and responded to stimulation
with uncoupling agent (FCCP) and inhibitor of respiration
metformin. We also studied differences in oxygenation,
depending on the glucose content in the growth medium.
We further demonstrated that O2-PLIM method can be
multiplexed with uorescence lifetime imaging microscopy
(FLIM) of DNA-binding probe Hoechst 33342 for detection of
BrdU-accumulating cells in S phase, conventional uores-
cence microscopy of Lgr5-GFP-positive cells and some
other biomarkers. We found that the same live organoid can
be analyzed for presence of stem cells (Lgr5), proliferating
cells (Hoechst FLIM) and oxygenation. This allows investiga-
tion of organoid metabolism and cell fate, by localizing and
identifying new regions with respect to their oxidative
phosphorylation and proliferation status.
Funding Source
This work was supported by the Science Foundation Ireland
(SFI) grants 13/SIRG/2144, 12/RC/2276 and by Agilent
University Research Program (ACT-UR) No. 4225.
Keywords: Hypoxia imaging; Intestinal organoid; Cell
proliferation
40
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Poster Abstracts
022
KIDNEY ORGANOIDS FROM NEPHROTIC DISEASE
DERIVED IPSCS EXHIBIT IMMATURE NEPHRIN
LOCALIZATION AND SLIT DIAPHRAGM FORMA-
TION IN PODOCYTES
Islam, Mazharul
1
, Tanigawa, Shunsuke
1
, Sharmin, Sazia
1
,
Naganuma, Hidekazu
1
, Yoshimura, Yasuhiro
1
, Haque, Fahim
1
,
Era, Takumi
2
, Nakazato, Hitoshi
3
, Nakanishi, Koichi
4
, Sakuma,
Tetsushi
5
, Yamamoto, Takashi
5
, Kurihara, Hidetake
6
, Taguchi,
Atsuhiro
7
, Nishinakamura, Ryuichi
1
1
Department of Kidney Development, Institute of Molecular
Embryology and Genetics, Kumamoto University, Kumamoto,
Japan,
2
Department of Cell Modulation, Institute of Molecular
Embryology and Genetics, Kumamoto University, Kumamoto,
Japan,
3
Department of Pediatrics, Faculty of Life Sciences,
Kumamoto University, Kumamoto, Japan,
4
Department of
Child Health and Welfare (Pediatrics), Graduate School of
Medicine, University of the Ryukyus, Okinawa, Japan,
5
Depart-
ment of Mathematical and Life Sciences, Graduate School of
Science, Hiroshima University, Hiroshima, Japan,
6
Department
of Anatomy and Life Structure, Juntendo University School of
Medicine, Tokyo, Japan,
7
Department of Genome Regulation,
Max Planck Institute for Molecular Genetics, Berlin, Germany
Mutations in the NPHS1 gene, which encodes NEPHRIN,
cause congenital nephrotic syndrome, characterized by mas-
sive proteinurea after birth due to impaired kidney slit
diaphragm (SD). However, methods for SD reconstitution
have been unavailable, thereby limiting studies in the eld. In
the present study, we established human induced pluripotent
stem cells (iPSCs) from a patient with an NPHS1 missense
mutation and reproduced the SD formation process using
iPSC-derived kidney organoids. The mutant NEPHRIN failed
to become localized on the cell surface for pre-SD domain
formation in the induced podocytes. Upon transplantation,
the mutant podocytes developed foot processes, but
exhibited impaired SD formation. Overexpression of mutated
protein in Human embryonic kidney (HEK293) cell line
indicated that impaired translocation might be due to
impaired glycosylation, resulting in retention of mutated
protein in the cytoplasm. Treatment with a combination of
chemicals revealed improved translocation of the mutated
protein in HEK293 cells. However, similar treatment to
patient iPSC exhibited no effect. In contrast, genetic correc-
tion of the single amino acid mutation restored NEPHRIN
localization and phosphorylation, colocalization of other
SD-associated proteins, and SD formation. Thus, these
kidney organoids from patient-derived iPSCs identied SD
abnormalities in the podocytes at the initial phase of
congenital nephrotic disease.
Funding Source
The study was supported by: Japan Society for the Promo-
tion of Science, Japan Agency for Medical Research and
Development (AMED).
Keywords: iPS cells; Nephrotic syndrome; Kidney, Podocyte
023
TUBEROUS SCLEROSIS COMPLEX MUTATIONS
AFFECT HUMAN IPSC DERIVED ASTROCYTES
Dooves, Stephanie
1
, Nadadhur, Aishwarya
2
, Gasparotto,
Lisa
1
, Heine, Vivi
1
1
Department of Pediatrics, Amsterdam UMC location VUmc,
Amsterdam, Netherlands,
2
Department of Functional Genom-
ics, Vrije Universiteit Amsterdam, Netherlands
Tuberous Sclerosis Complex (TSC) is a genetic multisystem
disorder caused by mutations in the TSC1 or TSC2 gene.
Patients show benign tumor formation in multiple organs
including the brain. Additional neurological problems like
epilepsy, autism and intellectual disability are common and
the major cause of disability in TSC patients. Astrocytes, one
of the major cell types in the brain, are responsible for
keeping the homeostasis of the brain and are involved in
most, if not all, brain processes like neuronal signaling,
myelination and maintenance of the blood-brain barrier.
Astrocytes have been implicated in morphological abnormal-
ities (tubers and supependymal giant astrocytoma’s) that are
observed in post-mortem brain tissue from TSC patients.
Glutamate and potassium buffering is affected in TSC-mu-
tated astrocytes, which can promote neuronal excitability
and seizures. Most studies looking at TSC brain pathology
used transgenic mice or post-mortem brain tissue. We have
previously made human induced pluripotent stem cells
(hiPSC) lines from TSC patients and controls and used these
to model neuronal and oligodendrocytic abnormalities. In
the current study we used hiPSC lines to look at astrocytic
pathology. We differentiated hiPSC towards astrocytes using
previously established protocols and looked at proliferation
rates, marker expression and cell morphology. Preliminary
data suggest an increased cell proliferation and increased
expression of GFAP, a marker for astrocyte reactivity. In a
rst pilot study no morphological changes in astrocytes
were observed. We are now planning to analyze differences
between TSC and control hiPSC derived astrocytes with RNA
sequencing and to study the effects of TSC astrocytes on
neuronal functioning and OPC maturation. The rst results
of this study show that human astrocytes are affected by
TSC mutations and may be important targets in therapy
development.
Keywords:Tuberous Sclerosis Complex; Astrocytes; human
iPSCs
024
CTIP2-DEPENDENT PKA ACTIVATION REGULATES
CA2+ SIGNALING AND DARPP32 PHOSPHORYLA-
TION IN HUMAN MEDIUM SPINY NEURONS
Fjodorova, Marija
1
, Noakes, Zoe
1
, Louessard, Morgane
2
, Li,
Zongze
1
, De La Fuente, Daniel
1
, Dyke, Emma
1
, Brooks,
Simon
3
, Errington, Adam
1
, Perrier, Anselme L.
2
, Li, Meng
1
1
Neuroscience and Mental Health Research Institute, Cardiff
University, Cardiff, UK,
2
INSERM, I-Stem, Paris, France,
3
Brain
Repair Group, Cardiff University, Cardiff, UK
The mechanisms underlying the selective degeneration of
medium spiny neurons (MSNs) in Huntington’s disease (HD)
remain largely unknown. CTIP2, a transcription factor
41
AMSTERDAM NETHERLANDS
Poster Abstracts
expressed by all MSNs, is implicated in HD pathogenesis
due to its interactions with mutant huntingtin. We demon-
strate that human ESC-derived MSNs decient in CTIP2
display impaired mitochondrial health, increased vulnerabili-
ty to oxidative stress and abnormal responses to physiologi-
cal stimuli. We uncovered a key role for CTIP2 in Ca2+
transport and protein phosphorylation via governing protein
kinase A (PKA) signaling. This is demonstrated by a PKA-de-
pendent rescue of Ca2+ signaling decits and signicant
reduction in phosphorylation of DARPP32 and GLUR1, two
PKA targets in CTIP2-decient MSNs. Moreover, we show
that CTIP2-dependent dysregulation of protein phosphoryla-
tion is shared by HD hPSC-derived MSNs and striatal tissues
of two HD mouse models. Our study therefore establishes
an essential role for CTIP2 in human MSN homeostasis and
provides mechanistic and potential therapeutic insight into
striatal neurodegeneration.
Funding Source
This work has been funded by MRC UK and European
Commission within its FP7 Programme.
Keywords: CTIP2-decient medium spiny neuron; Neural
differentiation; Huntington’s disease
026
THE LOSS OF FRAGILE X MENTAL RETARDATION
PROTEIN ALTERS THE DEVELOPMENT OF HUMAN
FOREBRAIN ORGANOIDS
Wen, Zhexing
1
, Zhou, Ying
1
, Kang, Yunhee
2
, Zhang, Feiran
2
,
Xu, Jie
3
, Landry, Corey R.
4
, Niu, Weibo
1
, Machleidt, Kara B.
5
,
Forest, Craig R.
4
, Jin, Peng
2
1
Department of Psychiatry and Behavioral Sciences, Emory
University, Atlanta, GA, USA,
2
Human Genetics, Emory
University, Atlanta, GA, USA,
3
Genetics and Molecular Biology
Graduate Program, Emory University, Atlanta, GA, USA,
4
Woodruff School of Mechanical Engineering, Georgia
Institute of Technology, Atlanta, GA, USA,
5
Antibodies and
Immunoassays Systems, Thermosher Scientic, Rockford,
MD, USA
Fragile X syndrome (FXS) is the most common inherited
form of intellectual disability and a leading genetic cause of
autism. FXS is caused by the loss of functional fragile X
mental retardation protein (FMRP). FMRP is an RNA-binding
protein forming a messenger ribonucleoprotein complex
with polyribosomes in the regulation of protein synthesis.
Despite major progress to characterize underlying disease
mechanisms in animal models that has led to several
clinical trials, improvements of behavioral and cognitive
outcomes in patients have unfortunately been unsuccessful,
a strong need for human-specic models of FXS to under-
stand the unique factors that underlie human disease and to
test the efcacy of candidate compounds. Three-dimension-
al (3D) organoid culture of human-induced pluripotent stem
cells (iPSCs) has evolved from embryoid body cultures, quite
faithfully following human organogenesis, and provides a
new platform to investigate human brain development in a
dish, otherwise inaccessible to experimentation. To deter-
mine whether the loss of FMRP could alter the development
of human brain organoids, we have generated forebrain
organoids from three FXS male patients and three healthy
male controls. We observed dysregulated proliferation of
neural progenitor cells and neural differentiation as well as
perturbed cell migration in FXS forebrain organoids. Interest-
ingly these decits were not observed with FXS mouse
model. To compare the differential gene expression caused
by the loss of FMRP between human and mouse, we then
performed RNA-seq to identify the differentially expressed
genes using both mouse embryonic brain cortex and human
forebrain organoids at the comparable developmental
stages. We detected very few genes differentially expressed
in the absence of Fmrp in mouse. However, we identied
200 genes downregulated and 126 genes up-regulated in
human FXS organoids, indicating human-specic impact
caused by the loss of FMRP. These results together suggest
that the loss of FMRP could cause neurodevelopmental
decits specically in human, and fragile X organoids could
provide a unique platform to study the molecular pathogene-
sis of FXS and identify human-specic druggable targets for
FXS and autism in general.
Funding Source
National Institutes of Health, FRAXA Research Foundation,
Emory Woodruff Health Sciences Center.
Keywords:Fragile X syndrome; FMRP; Forebrain organoids
027
MODELLING THE NEURODEVELOPMENTAL
DISORDER FRAGILE X SYNDROME BY DIRECT
CELLULAR
REPROGRAMMING OF ADULT HUMAN
FIBROBLASTS
Connor, Bronwen J., Edwards, Nicole
Department of Pharmacology, Centre for Brain Research,
University of Auckland, New Zealand
Fragile X Syndrome (FXS) is the leading monogenic cause of
intellectual impairment and autism spectrum disorder. FXS
results from epigenetic silencing of the FMR1 gene due to
expansion of the CGG repeat in the 5’UTR of FMR1. Study of
the molecular and cellular mechanisms that link FMR1
inactivation to impaired neuronal development and function
is limited by the inability to access live FXS affected human
neurons. We have established a direct cellular reprogram-
ming strategy using a chemically modied mRNA gene
delivery system to enhance the conversion of adult human
dermal broblasts (HDFs) to induced neural precursor cells
(hiNPs) from which cortical forebrain neurons can be
generated. We propose that by generating hiNPs directly
from HDFs derived from FXS patients, we will improve our
understanding of the molecular, cellular and pathological
basis of FXS, with potential to identify new therapeutic
compounds. This study shows the ability to directly repro-
gram FXS patient-derived HDFs to hiNPs by transient ectopic
expression of the pro-neural transcription factors, SOX2 and
PAX6 using chemical modied mRNA (cmRNA) transfection.
A mixed population of hiNP-derived neurons and astrocytes
were derived after differentiation in dened media, exhibiting
expression of the astrocyte marker S100B as well as
glutamatergic genes TBR2 and VGLUT1 and GABAergic
genes MEIS2 and GAD67. FMR1 was not detected in
independent FXS lines (n = 3) following differentiation. We
observed an increase in neurite length as well an alteration
in the ratio of neurons to astrocytes in FXS hiNP-derived
neurons compared to unaffected controls (n = 3/condition).
42
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Poster Abstracts
These observations were correlated with the CGG-repeat
dynamics and epigenetic prole of independent FXS lines
using genotyping and DNA methylation analyses. Overall,
these ndings provide proof-of-concept for the application
of direct-to-hiNP reprogramming to undertake further
research analyzing the molecular and cellular basis of FXS.
Funding Source
The Neurological Foundation of New Zealand.
Keywords:Cell reprogramming; Fragile X Syndrome; Neural
precursor cells
028
AUTISM SPECTRUM DISORDERS: FROM DYNAMIC
DEVELOPMENTAL DISEASE TRAJECTORIES TO
GENE NETWORK HETEROCHRONICITY
Schafer, Simon T.
1
, Paquola, Apua
1
, Stern, Shani
1
, Gosselin,
David
2
, Ku, Manching
3
, Pena, Monique
1
, Mansour, Abed
AlFatah
1
, Glass, Christopher K.
4
, Mertens, Jerome
1
, Gage,
Fred H.
1
1
Laboratory of Genetics, The Salk Institute for Biological
Studies, La Jolla, CA, USA,
2
Département de Médecine
Moléculaire, Université Laval, Québec, QC, Canada,
3
Center for
Pediatrics and Adolescent Medicine, University of Freiburg,
Germany,
4
Department of Cellular and Molecular Medicine,
University of California, San Diego, La Jolla, CA, USA
Autism is a neurodevelopmental disorder with a complex
genetic etiology. Recent advances in systems biology
approaches have caused a paradigm shift in the eld from a
single gene causation model to pathway perturbation
models and the cellular and molecular events that lead to
autism likely occur very early during human fetal cortical
development. However, one of the current challenges in
understanding autism pathophysiology is to determine
critical neurodevelopmental periods and cellular states that
might provide the ground for disease propensity. This task
requires an in-depth exploration of the temporal dimension
of shifting biological processes by treating the disorder as
an evolving, dynamic system. Using a time series approach
to monitor patient-derived induced pluripotent stem cells
(iPSCs) throughout early stages of neurodevelopment
enabled us to reconstruct the dynamics of transcriptional
gene network programs and led to the identication of an
intrinsic network-specic heterochronicity. Our data show
that these ASD-related changes are likely to be primed
during an earlier stage of development and we propose that
studying molecular disease trajectories could maximize our
chance of capturing relevant mechanistic disease states, as
well as the processes by which these states unfold.
Funding Source
This work was supported by the Engman Foundation, The
Paul G. Allen Family Foundation and The Leona M. and Harry
B. Helmsley Charitable Trust (Grant #2012-PG-MED00) and
the German Research Foundation (DFG).
Keywords: Autism; Development; Gene networks
029
DECIPHERING THE ETIOLOGY OF MICROCEPHALY
USING CEREBRAL ORGANOIDS GENERATED FROM
NIJMEGEN BREAKAGE SYNDROME PATIENT-
DERIVED INDUCED PLURIPOTENT STEM CELLS
Martins, Soraia1, Bohndorf, Martina1, Wruck, Wasco1,
Chrzanowska, Krystyna2, Adjaye, James1
1
Institute for Stem Cell Research and Regenerative Medicine,
University Hospital Düsseldorf, Germany,
2
Department of
Medical Genetics, The Children’s Memorial Health Institute,
Warsaw, Poland
Nijmegen Breakage Syndrome (NBS) is a rare autosomal
recessive genetic disorder caused by a mutation within the
NBN gene and thus resulting in genomic instability, premature
aging, microcephaly, growth retardation, immune deciency,
impaired puberty and infertility in females. The consequence
of these manifestations is a decrease in average life span,
caused by cancer or infection of the respiratory and urinary
tract. We previous reported that broblasts from NBS
patients can be reprogrammed into induced pluripotent stem
cells (iPSCS) and thus by-passing premature senescence.
Global transcriptome analysis comparing NBS broblasts to
healthy cells and NBS-iPSCs to hESCs unveiled de-regulated
cancer related pathways such as p53, cell cycle and glycoly-
sis. Furthermore, molecular analysis of NBS-iPSCs derived
neural progenitor cells (NPCs) revealed de-regulated expres-
sion of neural developmental genes in-part due to NBS-NPCs
inability to maintain normal levels of P53. Recent advances in
in vitro culture of 3D cerebral organoids derived from iPSCs
have illuminated the early mechanisms of mammalian
neuro-development. To decipher the etiology of microcephaly
in NBS patients, we rst generated another iPSC line from a
NBS patient bearing the homozygous 657del5 within NBN.
Next, we used the generated NBS-iPSC line together with the
previously reported NBS-iPSC line bearing the heterozygous
657del5 and a healthy control iPSC line to comparatively
generate cerebral organoids. Here, we show that NBS-derived
organoids are signicantly smaller during the early
differentiation stage, showed decrease proliferation of the
neural progenitor cells (NPCs) and key pathways related to
neurogenesis, DNA damage response and cell cycle are
differentially regulated compared to the healthy control iPSC
line. In conclusion, our observations are (i) successful
generation of a 3D cerebral organoid model of NBS, (ii)
delayed neurogenesis of the NBS organoids probably due to
less proliferation of the NPCs and (iii) de-regulated expression
of neurogenesis-associated genes and P53, which might
account for the development of microcephaly in NBS patients.
Funding Source
Medical faculty of Heinrich Heine University Düsseldorf.
Keywords: Microcephaly; Nijmegen Breakage Syndrome;
Cerebral organoids
43
AMSTERDAM NETHERLANDS
Poster Abstracts
030
GENERATION AND CULTURE OF MURINE
PANCREATIC EXOCRINE ORGANOIDS USING
PANCREACULT™ SERUM-FREE MEDIUM
Micsik, Roxana1, Segeritz-Walko, Charis1, Luu, Yvonne2,
Young, Megan3, Tiriac, Herve3, Stingl, John1, Riedel, Michael J.2,
Tuveson, David3, Thomas, Terry E., Eaves1, Allen, Louis, Sharon1
1Research and Development, STEMCELL Technologies Inc.,
Cambridge, UK, 2Quality Control, STEMCELL Technologies
Inc., Vancouver, BC, Canada, 3RND, Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY, USA
Despite the prevalence and urgency of pancreatic cancer
research throughout the previous decades, a method for the
long-term in vitro maintenance of pancreatic exocrine tissue
as 3-dimensional (3D) organoids has been described only
recently. Pancreatic exocrine organoids are composed of a
polarised monolayer epithelium that retains many of the
features of in vivo exocrine pancreatic tissue, and thus can
serve as a physiological model system to address diverse
research questions related to pancreatic development, stem
cell biology, secretory function, and disease modeling. We
have developed PancreaCult™ Organoid Growth Medium
(Mouse), a dened cell culture medium for the initiation and
long-term maintenance of pancreatic exocrine organoids. To
establish these cultures, resected mouse pancreatic tissue
was enzymatically dissociated to liberate ductal fragments
that contain the putative stem cell niche. When embedded
into Corning® Matrigel® domes and cultured in PancreaCult™,
these ductal fragments formed spherical organoids within 1
week (n = 59 mice). Established organoids were passaged
weekly at an average split ratio of 1:25 and maintained in
continuous culture for > 1 year. Expanded organoids were
also cryopreserved using CryoStor® CS10 for long-term
storage. Pancreatic exocrine organoids cultured in Pancrea-
Cult™ are composed of cells expressing genes specic for
pancreatic stem cells (Lgr5), progenitor cells (Pdx1, Sox9),
and ductal cells (Car2, Muc1, Krt19, Cftr). Additionally, we
observed that primary and metastatic tumor cells isolated
from Kras+/LSL-G12D; Trp53+/LSL-R172H; Pdx-Cre mice
and cultured in PancreaCult™ generate tumor organoids that
recapitulate the features of the original tumor, thus providing
a model system to study ductal pancreatic carcinoma. Due
to the robust growth of pancreatic exocrine organoids in
PancreaCult™ and their close resemblance to the in vivo
pancreatic epithelium, this organoid technology can comple-
ment or replace other experimental methodologies for
studying the exocrine pancreas and could reduce or even
eliminate the need for animal experimentation.
Keywords: Pancreas; Organoids; Cancer
031
EXPLOITING IPSC-DERIVED NEUROSPHEROIDS TO
RECAPITULATE HUMAN BRAIN MICROENVIRON-
MENT IN DISEASE
Terrasso, Ana P.1, Simão, Daniel1, Silva, Marta M.1,
Arez, Francisca1, Painho, Beatriz1, Sousa, Marcos F.1,
Gomes-Alves, Patricia1, Raimundo, Nuno2, Kremer, Eric J.3,
Alves, Paula M.1, Brito, Catarina1
1ACTU, iBET, Instituto de Biologia Experimental e Tecnológica
and Instituto de Tecnologia Química e Biológica António
Xavier, Universidade Nova de Lisboa, Oeiras, Portugal,
2Institut für Zellbiochemie, University of Goettingen, Germany,
3IGMM, Universite de Montpellier, France
Brain microenvironment plays important roles in
neurodevelopment and pathology. Neural cell culture
typically relies on the use of heterologous matrices that
poorly resemble brain ECM or reect its pathological features.
We have shown that perfusion bioreactor-based 3D differenti-
ation of iPSC-derived human neural stem cells (iPSC-NSC)
sustains the concomitant differentiation of the three neural
cell lineages. We hypothesized that if the neurospheroid
strategy would also allow deposition of native neural ECM, it
would be possible to (i) mimic cellular and microenvironment
remodeling occurring during neural differentiation, without the
confounding effects of exogenous matrices and (ii) recapitulate
pathological phenotypic features of diseases in which alter-
ations in homotypic/heterotypic cell-cell interactions and ECM
are relevant. Quantitative transcriptome (NGS) and proteome
(SWATH-MS) analysis showed that neurogenic developmental
pathways were recapitulated in our system, with signicant
changes in cell membrane and ECM composition. We
observed a signicant enrichment in structural proteoglycans
typical of brain ECM, a downregulation of basement mem-
brane constituents and higher expression of synaptic and ion
transport machinery. Neurospheroids were generated using
iPSC-NSC derived from Mucopolysaccharidosis type VII (MPS
VII) patient. MPS VII is a neuronopathic lysosomal storage
disease caused by decient β-glucuronidase (β-gluc) activity,
leading to glycosaminoglycan (GAGs) accumulation in the
brain. The main MPS VII molecular hallmarks were recapitu-
lated, e.g., accumulation of GAGs. MPS VII neurospheroids
showed reduced neuronal activity and disturbance in network
functionality, with alterations in connectivity and synchroniza-
tion. The proteome of MPS VII neurospheroids is being
analysed to further elucidate molecular alterations related
with the neuronal dysfunction observed in the disease. These
data provide insight into the interplay between reduced β-gluc
activity, GAG accumulation, alterations in the neural network,
and its impact on MPS VII-associated cognitive defects.
Further, our model provides a platform to unveil the cellular
alterations responsible for brain dysfunction in neurological
diseases and to test and optimize new therapies.
Funding Source
BD /78308/2011; /52202/2013; /52473/2014 PhD
fellowships from FCT, Portugal and iNOVA4Health-UID
Multi/04462/2013 supported by FCT/ MEC, Portugal through
national funds and co-funded by FEDER under PT2020
Partnership Agreement.
Keywords:Neurospheroids; Mucopolysaccharidosis type VII;
Brain microenvironment
44
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Poster Abstracts
032
DEVELOPMENT AND CHARACTERIZATION OF
BRAIN REGION-SPECIFIC ORGANOIDS FROM
HUMAN INDUCED PLURIPOTENT STEM CELLS
FOR MODELLING OF RETT SYNDROME
Gomes, Ana R.1, Fernandes, Tiago2, Vaz, Sandra3, Xapelli,
Sara3, Sebastião, Ana Maria3, Cabral, Joaquim Manuel
Sampaio2, Diogo, Margarida2
1Stem Cell Engineering Research Group, Instituto Superior
Técnico, Lisboa, Portugal, 2Department of Bioengineering and
Institute for Bioengineering and Biosciences and The Discov-
eries Centre for Regenerative Precision Medicine, Instituto
Superior Técnico, Universidade de Lisboa, Instituto Superior
Técnico, Lisboa, Portugal, 3Instituto de Medicina Molecular -
João Lobo Antunes and Instituto de Farmacologia e Neurociên-
cias, Faculdade de Medicina da Universidade de Lisboa,
Lisbon, Portugal
Organoid biology and process engineering strategies provide a
novel platform to model brain development and neurological
related disorders, by providing a more reliable recapitulation
of the neurodevelopment process when compared to 2D
culture systems. Among these diseases, Rett Syndrome (RTT),
a rare neurodevelopmental disorder that affects multiple
neuronal subtypes originated from forebrain sub-regions, can
greatly benet from the merging of these technologies. The
main objective of this work is the development and
characterization of standardized ventral and dorsal forebrain
organoids from both RTT patient-specic and wild-type human
induced pluripotent stem cells (hiPSC). We demonstrated
that these organoids successfully recapitulate ventral and
dorsal forebrain progenitor zone organization, as well as the
process of neurogenesis. Importantly, both regions contain
the two major populations of cortical neurons, glutamatergic
pyramidal neurons and GABAergic interneurons, as
determined by qRT-PCR, immunocytochemistry and
spontaneous currents recorded by whole-cell path clamp.
However, while ventral organoids from wild-type hiPSC
displayed a predominant neuronal-like prole, revealed by an
increase in Ca2+ levels in response to KCl but not to hista-
mine stimulation, ventral organoids obtained from RTT
hiPSC lines exhibited a more immature-like prole. Moreover,
it was found that along the maturation process of ventral
organoids, DCX+ immature neurons are present mostly in
RTT cell lines, while MAP2+ neurons are enhanced in
organoids differentiated from wild-type cell lines. These
neurons are currently being characterized in terms of their
electrophysiology prole. Preliminary results from these
studies suggest that wild-type hiPSCs generated neurons
have faster rate of maturation and excitability, ring more
action potential in response to depolarizing current
injection, when compared with RTT hiPSCs generated
neurons. These results will contribute to the characterization
and understanding of RTT during the early stages of
development, before disease inception, by recapitulating the
complex human neurodevelopmental defects. Moreover, this
platform may also constitute a promising tool for disease
modeling as well as for testing potential therapeutic drugs.
Funding Source
H2020-WIDESPREAD-01-2016-2017 – Teaming Phase2,
DISCOVERIES CTR: The Discoveries Centre for Regenerative
and Precision Medicine.
Keywords: Human induced pluripotent stem cells; Rett
Syndrome; Forebrain organoids
033
MODELING FAMILIAL CEREBRAL CAVERNOUS
MALFORMATIONS USING PLURIPOTENT STEM
CELLS
Ameneiro, Cristina1, Moreira, Tiago2, Fuentes-Iglesias,
Alejandro1, Souto, Yara1, Fiel, Manuel2, Pombo, Celia1,
Zalvide, Juan1, Guallar, Diana3, Fidalgo, Miguel1
1Physiology, Center of Research in Molecular Medicine
and Chronic Diseases, Universidade de Santiago de
Compostela, Spain, 2CiMUS, Universidade de Santiago de
Compostela, Spain, 3Biochemestry, Universidade de Santiago
de Compostela, Spain
Familial cerebral cavernous malformation (FCCM) is a rare
disease characterized by vascular malformations, mainly
localized within the brain, that cause cerebral haemorrhage,
seizures and strokes. Despite the severity of the lesions
caused by loss-of-function mutations in any of three Ccm
genes, the molecular mechanisms behind this dysregulated
endothelial cell (EC) behaviour have not been elucidated.
Here, we develop an improved model in vitro using pluripotent
cells to decipher the role of each Ccm gene during endothelial
cell-fate specication. Although we found that Ccm genes
are dispensable for self-renewal of embryonic stem cells,
their function is required for early differentiation towards
endothelial lineage and vessel formation in 3D cell cultures.
Our data indicates that absence of Ccm genes alters a
conserved epigenetic landscape required for the transcrip-
tional transition during mammalian EC development. These
ndings will allow us to identify novel druggable targets and
therapeutic approaches for this severe, and so far, incurable,
human disease.
Funding Source
This research was funded by the Fundación Ramón Areces
(2016-PO025). M.F. is a recipient of a Ramón y Cajal Award
(RYC-2014-16779) from the Ministerio de Economía y
Competitividad of Spain.
Keywords: FCCM disease; Endothelial Cell; Epigenetic
landscape
45
AMSTERDAM NETHERLANDS
Poster Abstracts
034
COMPARISON OF LARGE 1Q21.1 AND TARGETED
NOTCH2NL DELETIONS IN CEREBRAL ORGANOIDS
SUGGEST A MAJOR ROLE FOR NOTCH2NL IN
1Q21.1 DISTAL DELETION PATHOLOGY
Mantalas, Gary L., Bosworth, Colleen M., Mooring, Meghan M.,
Fiddes, Ian, Haussler, David, Salama, Soe
Genomics Institute, University of California, Santa Cruz, CA,
USA
Cerebral cortex organoids (cerebral organoids) derived from
human pluripotent stem cell lines provide a model system
for studying how genetics can affect human brain
development. We recently demonstrated that deletion of
human-specic NOTCH2NL genes in human embryonic
stem cell (hESC)-derived cortical organoids accelerates
differentiation of neural stem cells resulting in smaller
organoids with a higher proportion of cells expressing
markers of excitatory projection neurons at early time points.
This led to the hypothesis that NOTCH2NL expression in the
human cortex increases the self-renewal capacity of neural
stem cells. We also found that NOTCH2NLA and
NOTCH2NLB ank a genomic locus (Chromosome 1q21.1)
that is highly repetitive and is recurrently deleted or
duplicated in patients with a variety of neurodevelopmental
disorders including autism, schizophrenia, microcephaly and
macrocephaly. Here we present our ndings further explor-
ing the phenotypes of NOTCH2NL deletions and larger
deletion events that mimic those observed in patients with
1q21.1 distal deletions by comparing hESC-derived cerebral
organoids with a variety of altered genotypes by single cell
RNA-seq to determine how these alterations effect early
brain development with respect to the proportions of cell
types present and the transcriptional proles of these
resulting cell types. These studies will reveal the relative
contributions of NOTCH2NL genes and the other genes in
this genomic locus on cerebral organoid development and
could provide a model system for exploring ways to mitigate
the developmental effects of pathogenic genomic
rearrangements at this locus.
Funding Source
Howard Hughes Medical Institute, Schmidt Family Founda-
tion, NIH/NIGMS, CIRM.
Keywords: Neurodevelopmental disorders; NOTCH signaling;
Cerebral cortex organoids
035
KIDNEY ORGANOIDS: DISEASE MODELING AND A
PATH TO PERSONALIZED MEDICINE
Emani, Maheswarareddy1, Subramanian, Ayshwarya1,
Sidhom, Eriene-Heidi1, Vernon, Katherine1, Sahakian, Nareh1,
Kost-Alimova, Maria2, Marshall, Jamie1, Rosenblatt-Rosen,
Orit3, Regev, Aviv3, Greka, Anna1
1
Kidney Disease Initiative, Broad Institute of MIT and Harvard,
Boston, MA, USA,
2
CDOT, Broad Institute of MIT and Harvard,
Boston, MA, USA,
3
Computational and Systems Biology, Broad
Institute of MIT and Harvard, Boston, MA, USA
Human pluripotent stem cell (hPSC)-derived three-dimen-
sional kidney organoid is an important model system to
study kidney development, disease and a pre-clinical model
for personalized therapeutics. However, human kidney
organoid model requires a substantial improvement in terms
of maturation of nephrons with functional vascularization.
To achieve this objective, we generated renal subcapsular
transplantation of hPSC derived kidney organoids or human
kidney Organoid Transplants (HKOTs). Following 26 days
post renal subcapsular transplantation, we performed
comprehensive single cell atlas of HKOTs and demonstrated
vascularization and maturation of nephrons. Our results
showed that host-derived vascularization in kidney organ-
oids signicantly improved tubular and podocyte maturity at
single cell resolution.
Keywords:Kidney Orgnaoids; Human kidney Organoid
Transplants (HKOTs); Personalized therapeutics
036
MODELING CANCER STEM CELL AND DIFFERENTI-
ATED CANCER CELL PHENOTYPES IN LIVER
CANCER ORGANOIDS
Lieshout, Ruby, Verstegen, Monique M.A, Jorna, Terry, van
Gilst, Demy, Kan, Yik Y., Ijzermans, Jan N.M, van der Laan,
Luc J.W
Department of Surgery, Erasmus Medical Center, Rotterdam,
Netherlands
Cholangiocarcinoma (CCA) is a biliary-type liver tumor
with a dismal prognosis, due to late diagnosis, high
chemo-resistance and tumor heterogeneity. Recently, we
established long-term 3D CCA organoids which retain the
histological architecture, gene expression prole and genomic
landscape of the original tumor and are amenable to drug
screening approaches. CCA organoids represent a cancer
stem cell (CSC)-like phenotype. CSC’s are known to be
resistant to chemo- and radiation therapy. However, the bulk
of cancer cells in a tumor have a differentiated (non-stem
cell) phenotype and are more therapy-sensitive. The aim of
our study is to establish a model to study both the CSC and
differentiated cancer cell phenotypes in CCA organoids and
apply this in drug sensitivity screening. Organoids from CCA,
non-tumorous adjacent liver and healthy liver tissue
(all n=3-6) were differentiated by blocking cancer stem cell
signaling pathways. At day 5, cell viability, proliferation, cell
death and differentiation potential was tested on gene
expression (qPCR) and protein (immunohistochemistry) level
and compared to CSC-like organoids. Drug sensitivity of
differentiated and CSC-like CCA organoids was tested with
sorafenib. Live/dead staining revealed that the level of cell
death in differentiated organoids was similar to
CSC-type cultures. Differentiated CCA organoids had a
reduced proliferative rate, as demonstrated by reduced EdU
incorporation and downregulation of Ki67 gene expression
(p<0.001). Even though proliferation is inhibited, metabolic
activity was stable, indicating a higher metabolic activity per
differentiated cell. Gene expression analysis showed that
known CSC markers LGR5 (p<0.001), CD44 (p=0.01) and
46
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Poster Abstracts
CD133 (p<0.01) were downregulated upon differentiation.
Preliminary results from a drug sensitivity assay suggested
that differentiation of CCA organoids increased their sensitivity
to sorafenib. This study shows that it is feasible to differentiate
liver tumor-derived organoids from a CSC-like phenotype
towards a differentiated cancer cell phenotype. Differentiated
CCA organoids are less proliferative, downregulate CSC
markers and are more sensitive to sorafenib. More elaborate
drug screenings are ongoing in order to more accurately
identify effective compounds to treat CCA.
Funding Source
This project is nanced with help of ZonMW - InnoSysTox
projectnumber 40-42600-98-14017.
Keywords: Primary liver cancer; Cholangiocarcinoma
organoids; Tumor stem cells
037
HUMAN INTESTINAL AND AIRWAY ORGANOIDS
TO MODEL AND STUDY VIRAL INFECTIONS
Zhou, Jie1, Li, Cun1, Chiu, Man Chun1, Wong, Bosco1, Chu,
Hin1, Chen, Honglin1, Clevers, Hans C.2, Yuen, Kwok-yung1
1Microbiology, The University of Hong Kong, Hong Kong,
2Hubrecht Institute, Utrecht, Netherlands,
Human intestinal organoids, the rst human organoid model
derived from adult stem cells, can faithfully simulate the
multi-cellular complexity and functionality of human
intestinal epithelium. MERS-CoV has caused human
respiratory infection since 2012, with a fatality rate over 30%.
Airway exposure is intuitively assumed to be the infection
route. However, various evidence implicates human may
acquire MERS via gastrointestinal tract. We demonstrate
that MERS-CoV can robustly replicate in human intestinal
organoids. After direct intragastric gavage, the MERS-CoV
permissive hDPP4 transgenic mice developed progressive
intestinal infection. With the progression of enteric infection,
inammation, virus-infected cells and live viruses emerged
in the lung tissues, indicating the development of sequential
respiratory infection. Collectively, the results suggest that
the human intestinal tract may serve as an alternative
infection route for MERS-CoV. In order to study respiratory
viral infection, we established long-term expanding human
airway organoids which accommodate four types of airway
epithelial cells: ciliated, goblet, club, and basal cells. We
report differentiation conditions which increase ciliated cell
numbers to a nearly physiological level with synchronously
beating cilia readily discernible in every organoid. We also
established improved 2D monolayer culture conditions for
the differentiated airway organoids. To demonstrate the ability
of differentiated airway organoids to identify human-infective
virus, 3D and 2D differentiated airway organoids are applied
to evaluate two pairs of viruses with known distinct infectivity
in humans, H7N9/Ah versus H7N2 and H1N1pdm versus an
H1N1 strain isolated from swine (H1N1sw). The human
infective H7N9/Ah virus replicated more robustly than the
poorly human-infective H7N2 virus; the highly
human-infective H1N1pdm virus replicated to a higher
titer than the counterpart H1N1sw. Collectively, we
developed differentiated human airway organoids which can
morphologically and functionally simulate human airway
epithelium. These differentiated airway organoids can be
applied for rapid assessment of the infectivity of emerging
respiratory viruses to humans.
Keywords: Human intestinal organoids; Human airway
organoids; Virus infection
038
USING INTESTINAL ORGANOIDS TO STUDY THE
ROLE OF GROUP 3 INNATE LYMPHOID CELLS
(ILC3S) IN INFLAMMATORY BOWEL DISEASE (IBD)
PATHOGENESIS
Tsakmaki, Anastasia1, Pantazi, Eirini2, Polychronis, Pavlidis2,
Powell, Nick2, Bewick, Gavin1
1School of Life Course Sciences, King’s College London,
London, UK, 2School of Immunology and Microbial Sciences,
King’s College London, UK
IBD is characterised by chronic mucosal inammation.
There are two types of IBD: Crohn’s disease, where any part
of the gastrointestinal tract can be inamed and ulcerative
colitis, which usually only affects the colon. In patients with
IBD, mucosal T cells and ILCs respond to microbes by
uncontrollably producing cytokines that can promote chronic
inammation. ILCs are divided into 3 groups based on
transcription factor expression and cytokine production.
ILC3s are further subdivided according to their expression of
natural cytotoxicity receptors (NCRs). NCR+ ILC3s contribute
to the maintenance of intestinal homeostasis. However, less
is known about the role of NCR- ILC3s. To investigate the
functional role of NCR- ILC3s the Tbx21-/- Rag2-/- Ulcerative
Colitis (TRUC) model of disease was used, as T-bet deciency
results in failure of NCR+ ILC3 development and presence
only of NCR- ILC3s. Interestingly, we found that in TRUC
disease IL22 produced by colonic NCR- ILC3s had a
pathogenic role. The IL22 receptor (IL22R1) is expressed
in the epithelial cells of the colon, so to understand the
mechanisms by which IL22 mediates colitis, we treated
mouse colonoids with recombinant IL22 and conducted
transcriptomic analysis. Comparison of microarray data
from IL22-treated colonoids with data from the colon of
TRUC mice revealed that IL22 responsive transcripts, as
identied in IL22-treated colonoids, were signicantly
enriched in the colon of TRUC mice. Pathway analysis
showed, as expected, changes in the expression of transcripts
involved in microbial sensing, anti-microbial responses and
barrier function. However, it also showed changes in the
transcript levels of endoplasmic reticulum (ER) stress
response genes, a pathway not previously linked to ILCs.
Upregulation of ER stress response genes was conrmed by
real time PCR. This effect was enhanced in the presence of
IL17, another cytokine secreted by NCR- ILC3s. IL22-induced
ER stress affected all colonic epithelial cells, not only stem
cells, and caused ER stress-induced apoptosis. To conclude,
our ndings indicate that IL22 and IL17 co-produce by
NCR- ILC3s can drive chronic colitis through induction of
epithelial ER stress.
Keywords: IBD; IL22; ER stress
47
AMSTERDAM NETHERLANDS
Poster Abstracts
039
SYNAPTIC DEFECTS AND IMPAIRED AUTOPHAGY
IN A HUMAN IPSC-BASED MODEL OF FAMILIAL
PARKINSON’S DISEASE
Kouroupi, Georgia1, Taouk, Era1, Kloukina, Ismini2, Xilouri,
Maria2, Wrasidlo, Wolf3, Masliah, Eliezer4, Stefanis, Leonidas2,
Matsas, Rebecca1
1Neurobiology, Hellenic Pasteur Institute, Athens, Greece,
2Center of Clinical Research, Experimental Surgery and
Translational Research, Biomedical Research Foundation
of the Academy of Athens, Greece, 3Department of
Neurosciences, University of California, San Diego, CA, USA,
4Division of Neuroscience, National Institute on Aging,
National Institute of Health, Bethesda, MD, USA
Parkinson’s disease (PD) remains an incurable neurodegenera-
tive disorder with variable clinical characteristics, age of onset
and course of progression. The hallmark of PD, whether
sporadic or familial, is the deposition of protein aggregates,
which are composed mainly of alpha-synuclein (αSyn). αSyn is
a pre-synaptic protein with N-terminal binding to acidic lipids
that can sense and generate changes in membrane curvature,
suggesting its participation in presynaptic events, including
endocytosis and exocytosis whilst its involvement in pre-synap-
tic organization has been postulated in mice. However, the
mechanisms through which mutant αSyn affects synaptic
organization in a human setting remain unknown. αSyn is the
major gene linked to sporadic Parkinson’s disease, while the
G209A (p.A53T) αSyn mutation causes a familial form
characterized by early onset and a generally severe phenotype,
including non-motor manifestations. In this study, using cell
reprogramming technologies, we have developed a robust
induced pluripotent stem cell (iPSC)-based model of PD from
patients harboring the p.A53T-αSyn mutation that faithfully
simulates disease pathogenesis and uncovers novel disease-
relevant phenotypes at basal conditions, including protein
aggregation, compromised neuritic outgrowth and contorted
axons with swollen varicosities containing αSyn and tau, as
well as reduced synaptic connectivity. Global transcriptome
analysis suggested defects in synapse formation and function.
Electron microscopy of p.A53T neurons indicated impaired
organization of synaptic vesicle pools, microtubule disorganiza-
tion and a striking accumulation of autophagic vacuoles. In
agreement, impaired autophagic activity and lysosomal protein
degradation was shown by immunouorescence and biochemi-
cal analysis. Finally, articial synapse formation assay was
used to study synaptogenesis of p. A53T neurons and
monosynaptic rabies virus tracing to assess p.A53T neuronal
circuitry. We aim to complement our investigations in 2D
cultures with the development of a novel 3D model for
synucleinopathy that will address how p.A53T affects the
spatiotemporal organization of the neuronal network. The
p.A53T-model can be used to answer fundamental questions
for PD pathogenesis and serve as a new drug-testing platform.
Funding Source
Stavros Niarchos Foundation Grant to the Hellenic Pasteur
Institute, as part of the Foundation’s initiative to support the
Greek research center ecosystem. G.K. is funded by the
Hellenic Foundation for Research and Innovation.
Keywords: Parkinson’s disease; α-synuclein; Synaptopathy
040
HIGH-FIDELITY DISEASE MODELLING OF
SKELETAL MUSCLE LAMINOPATHIES USING
THREE-DIMENSIONAL HUMAN IPS CELL-DERIVED
BIOENGINEERED MUSCLES
Tedesco, Francesco Saverio1,2, Steele-Stallard, Heather B.1,2,
Pinton, Luca1,3, Sarcar, Shilpita1, Ozdemir, Tanel3, Mafoletti,
Sara M.1, Zammit, Peter3
1Department of Cell and Developmental Biology, University
College London, UK, 2 Great Ormond Street Institute of Child
Health, University College London, UK, 3Randall Centre for Cell
and Molecular Biophysics, King’s College London, UK
Laminopathies are severe heterogeneous genetic diseases
caused by mutations in A-type lamins, which are encoded
by the LMNA gene. These proteins together with Lamin B1
and B2 form the nuclear lamina: a mesh-like structure
located underneath the nuclear membrane which helps
maintaining nuclear shape and regulating gene expression.
Laminopathies affect multiple cell types and can be tis-
sue-specic or systemic, with some subtypes affecting
striated muscle, peripheral nerve and adipose tissue, while
others cause multisystem disease with accelerated aging.
Although several mechanisms have been proposed, the exact
pathophysiology of laminopathies remains unknown; addition-
ally, the rarity of the disorder and lack of easily accessible cell
types for ex vivo studies negatively impact on therapy develop-
ment. To overcome these hurdles, here we used induced
pluripotent stem (iPS) cells from patients with skeletal muscle
laminopathies such as LMNA-related congenital muscular
dystrophy and limb-girdle muscular dystrophy 1B, to model
disease phenotypes in vitro. iPSC lines from three skeletal
muscle laminopathy patients were differentiated into skeletal
myogenic cells and myotubes. Disease-associated pheno-
types were observed in all genotypes, including abnormal
nuclear shape and mislocalisation of nuclear lamina proteins.
Notably, complex modelling in three-dimensional (3D) articial
muscle constructs resulted in recapitulation of nuclear abnor-
malities with higher delity than standard bi-dimensional
cultures and identied nuclear length as a robust and objec-
tive outcome measure. Finally, we will present and discuss
current efforts and future applications of this novel 3D
organoid-like platform for therapy development and drug
screening for skeletal muscle laminopathies. These results
demonstrate that patient-specic iPS cells can model phenotyp-
ic readouts of skeletal muscle laminopathies with high delity
upon 3D differentiation in vitro, laying the foundation for future
therapy screening platforms for skeletal muscle laminopathies
and other severe muscle disorders.
Funding Source
BBSRC, European Research Council, EU FP7 projects PluriMes
& EBiSC, AFM, MDUK and National Institute for Health Re-
search (the views expressed are those of the authors and not
necessarily those of the NHS, NIHR or Department of Health).
Keywords:Articial skeletal muscle; Disease modelling; iPS
cells
48
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Poster Abstracts
041
MODELLING IDIOPATHIC PARKINSONS DISEASE
IN HUMAN INDUCED PLURIPOTENT STEM
CELL-DERIVED MIDBRAIN ORGANOIDS
Monzel, Anna S.1, Cardoso Pereira, Sandro L.1, Badanjak,
Katja1, Wasner, Kobi1, Antony, Paul1, Bolognin, Silvia1, Ghel,
Jenny1, Ouzren, Nassima1, Treff, Antoine1, Zagare, Alise1,
Krüger, Rejko1, Diederich, Nico2, Seibler, Philip3, Klein,
Christine3, Grünewald, Anne1, Schwamborn, Jens C.1
1Luxembourg Centre for Systems Biomedicine, University
of Luxembourg, Belvaux, Luxembourg, 2Neurology, Centre
Hospitalier de Luxembourg, Luxembourg, 3Institute of
Neurogenetics, University of Lübeck, Germany
Parkinson’s disease (PD) is the most common age-related
motoric neurodegenerative disorder. The motor decits,
including tremor, rigidity and bradykinesia, result from the
loss of dopaminergic neurons (DNs) in the substantia nigra
and the lack of the neurotransmitter dopamine in the
striatum. Over the last decades, several genetic variants
have been suggested to either cause or increase the risk of
PD. Nevertheless, most cases are idiopathic and the
underlying environmental triggers remain elusive. Our
current understanding of PD suggests a multifactorial nature
of the disease. Research on PD and other neurological
disorders is limited by the lack of advanced experimental in
vitro models that truly capture the complex nature of the
human brain. Although animal models recapitulate some
aspects of human physiology, they often fail to recreate
specic pathogenic events, with low success rates in
clinical trials. Thus, classical 2D cell culture approaches with
isolated cell types poorly model human physiology due to
distinct cellular microenvironments. In contrast, advanced
human induced pluripotent stem cell (hiPSC)-derived 3D
models represent patient-specic in vitro systems, which
mimic certain aspects of the physiological situation and
can reproduce specic features of human diseases. Here
we present the usage of hiPSC-derived neuroepithelial stem
cells (hNESCs), thereof derived midbrain dopaminergic
neurons (mDNs) and complex 3D human midbrain
organoids (hMOs) as idiopathic PD (IPD) in vitro models.
Using these cell culture models of the different developmental
stages, we investigate cellular hallmarks of PD such as
mitochondrial dysfunction, oxidative stress and DN
degeneration in a longitudinal fashion. Our studies include
the evaluation of distinct mitochondrial DNA parameters,
such as deletions, copy number and replication/transcription
rate as well as proteomic and transcriptomic data and
high-content image analysis. In the hMOs in particular we
explore differential dynamics of neuronal dopaminergic
specication in IPD patients. Overall, this project aims to
capture the complex nature of PD and to clarify molecular
mechanisms underlying the degeneration of DNs in IPD.
Funding Source
Fonds National de la Recherche Luxembourg.
Keywords: Parkinson’s disease; Human midbrain organoids;
Dopaminergic neurons
042
GENERATION OF DISEASE MODEL OF NEONATAL
DIABETES MELLITUS WITH INS GENE MUTATION
IN HUMAN
Panova, Alexandra V.1, Klemetieva, Natalia1, Goliusova,
Daria2, Kiselev, Sergey2
1Institute of Personalized Medicine, Endocrinology Research
Center, Moscow, Russia, 2Stem Cell Laboratory, Vavilov
Institute of General Genetics, Moscow, Russia
One of the causes of diabetes in infants is the defect
of the insulin gene (INS). The mutations that lead to
hypoinsulinemia are usually heterozygous. The cause of the
absolute deciency of insulin in patients with heterozygous
mutations is still not clear. Despite the presence of one
normal allele of the insulin gene, the disease is manifested
with the absolute deciency in insulin in blood. We aimed to
generate an isogenic system, i.e. a pair of insulin-producing cell
lines with the same genome with the presence and the
absence of a mutation in the INS gene. Patient specic iPS
cell lines were generated from primary broblasts by
non-integrative method of reprogramming from a patient
with an intronic mutation of INS gene. The mutation leads to
emergence of an ectopic splice site within the INS gene and
clinically manifested at 6 months as a rare disease:
permanent neonatal diabetes mellitus (PNMD). This
mutation was edited by CRISP-Cas9 system by deletion of
the mutant splice site. The editing led to the restoration of
the RNA sequence of INS gene. IPSC lines with mutant INS
and with edited INS were differentiated to the pancreatic
beta cells to conrm the restored function in the edited cell
line and to study the pathogenesis at the molecular and
cellular level in an isogenic model.
Keywords: CRISPR-Cas9; iPSC; Permanent neonatal diabe-
tes mellitus
043
EFFICIENT DIFFERENTIATION OF MACROPHAGES
FROM HUMAN PLURIPOTENT STEM CELLS AND
THEIR APPLICATION IN MODELING INFLAMMATORY
DISEASES IN 3D ORGANOIDS
Na, Jie
School of Medicine, Tsinghua University, Beijing, China
Macrophages derived from human pluripotent stem cells
(hPSCs) can be used to model inammatory or autoimmune
diseases, and may be genetically engineered for immune
therapy. We established a monolayer, chemically dened
culture system to induce hematopoietic differentiation and
macrophage formation from hPSCs. We found that insu-
lin-free medium allowed hPSC to leave pluripotency prompt-
ly and preferably enter the vascular lineage. Addition of
insulin during later stage of differentiation was essential for
the efcient induction of hemogenic endothelium and the
emergence of large numbers of CD34+CD43+ hematopoietic
stem/progenitor cells (HSPCs), while no insulin condition
preferably permit endothelial differentiation. HSPCs
generated from our protocol can be subsequently induced to
form macrophages (iMacs) at high efciency. Global
49
AMSTERDAM NETHERLANDS
Poster Abstracts
transcriptome proling revealed that iMacs differentiated using
our protocol was similar to macrophages developed in vivo.
IMacs demonstrated robust phagocytic ability and upregulated
inammatory cytokines upon recombinant model pathogen
stimulation. These iMacs are included in a range of 3D
organoids as tissue residential immune cells to study inam-
matory responses that implicated in many human diseases.
Funding Source
the National Key R&D Program of China Grant
2017YFA0102802; the National Natural Science Foundation of
China (NSFC) grants 91740115.
Keywords:
Human pluripotent stem cells; Differentiation; Macro-
phages
045
A BREAST CANCER ORGANOID INVASION MODEL
IN A SYNTHETIC BIOELASTIC MATRIX
De Jong, Menno R.1, Khalil, Antoine2, Smits, Daan1, Bruekers,
Stéphanie1, Van Den Boomen, Onno1, Van Den Dolder,
Juliette1, De Rooij, Johan2
1Noviocell BV, Oss, Netherlands, 2Department of Molecular
Cancer Research, University Medical Center Utrecht, Nether-
lands
Breast cancer invasion is a mechanically regulated process
that critically depends on a subset of tumor cells with basal
properties. During the malignant progression of breast cancer,
these basal cells, expressing basal cell markers such as
cytokeratin (CK) 14 are found at the invasive front of a
tumor. Mammary tumor organoids derived from MMTV-PyMT
mice grow with benign morphology under standard condi-
tions in Basement Membrane Extract (Laminin 111 and
Collagen IV) but transform into invasive tumors when
transferred to a collagen I matrix. The transformation from
benign to invasive form is accompanied by the appearance
of CK14 positive basal cells in the invasive parts of the
tumor. It is clear that adhesion-controlled changes in tumor
stiffness are crucial in malignant transformation. However,
which specic adhesive interactions are involved and how
they could be targeted to inhibit tumor metastasis is not yet
known. In this study, we set out to increase our understand-
ing of such adhesive interactions, by using a synthetic, fully
dened matrix based on polyisocyanopeptide (PIC) hydro-
gels. PIC hydrogels were chosen for this study since they,
uniquely among synthetic hydrogels, exhibit the same
strain-stiffening mechanical properties as collagen and
other ECM proteins. In addition, they can be functionalized
with various cell-binding peptides derived from ECM pro-
teins. MMTV-PyMT mouse tumor organoids exhibited a
benign morphology in most PIC hydrogels but transitioned
to an invasive morphology with CK14 expressing cells at the
tips of invasive outgrowths when grown in PIC matrices
modied with collagen peptides and tuned to specic
stiffnesses. Thus, we developed a tumor invasion model with
increased control over adhesive interactions and mechanical
properties for targeting adhesive interactions responsible for
invasive behavior. The outcomes of our research will increase
our understanding on the control over cellular mechanics,
may drive or prevent malignant transformation, and ultimate-
ly help in guiding more effective therapeutic strategies.
Keywords: Invasion; Tumor Organoids; Hydrogels
046
ORGANOID MODELING OF THE REPRODUCTION-
CRUCIAL ENDOMETRIUM IN BOTH HEALTHY AND
DISEASED STATE
Vankelecom, Hugo, Boretto, Matteo
Department of Development and Regeneration, KU Leuven,
Belgium
The endometrium is of crucial importance for mammalian
reproduction and undergoes dynamic reiterative tissue
remodeling during the menstrual cycle. Knowledge on the
cellular and molecular underpinnings of endometrium
biological remodeling is poor, as well as on the processes
that go awry in endometrium pathogenesis. This limited
understanding is primarily due to the lack of research
models reliably recapitulating endometrium biology and
disease in nature and heterogeneity. Therefore, we embarked
on the development of organoid models from human healthy
endometrium as well as from a wide spectrum of endometri-
al diseases. We established organoids from healthy endo-
metrium biopsies which reproduced molecular, histological
and cellular phenotype of the tissue’s epithelium. The organ-
oids phenocopied the physiological responses to reproduc-
tive hormones thereby mimicking the menstrual cycle at both
morpho-histological and molecular level. Furthermore, the
organoids showed long-term expansion capacity while
remaining genomically, transcriptomically and functionally
stable. Transcriptomic proling advanced candidate markers
of the still undened endometrium epithelial stem cells.
Long-term expandable organoids were also established from
a broad range of endometrium pathologies, ranging from
endometriosis and endometrium hyperplasia to low and high
grade endometrial cancer. The organoids obtained recapitu-
lated characteristics of the patients’ diseased tissue and
faithfully captured the clinical heterogeneity of the different
pathologies regarding histological markers, differentiation
status, genomic alterations and drug responses. Finally,
endometrial disease organoids reproduced the original lesion
when transplanted in vivo. Taken together, we established
new organoid models for endometrium and a wide spectrum
of endometrial diseases, thereby providing powerful tools to
decipher the mechanisms underlying the biology (including
embryo receptivity) and pathology of this key reproductive
organ. The eventually generated organoid biobank will allow
to in-depth explore genetics and pathway alterations in
diverse forms of endometrium pathology, and at the same
time serve as screening platform to test (new) drugs, even in
a patient-personalized manner.
Keywords: Endometrium; Reproduction; Endometrial cancer
50
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Poster Abstracts
047
IDENTIFYING THERAPEUTIC TARGETS IN THE
INTRINSIC TUMOR IMMUNITY GENETIC PROGRAM
USING PATIENT-DERIVED GLIOBLASTOMA
ORGANOIDS
Imitola, Jaime1, Banasavadi-Siddegowda, Yeshavanth2,
Hollingsworth, Ethan W.3, Hester, Mark4, Quiñones-Hinojosa,
Alfredo5, Watanabe, Fumihiro3
1Neurology and Neuroscience, The Ohio State University
Wexner Medical Center, Columbus, OH, USA, 2Cancer Biology,
UT Houston, TX, USA, 3Neurology, The Ohio State University,
Columbus, OH, USA, 4Pediatrics , Nationwide Children’s
Hospital, Columbus, OH, USA, 5Neurosurgery, Mayo Clinic,
Jacksonsville, FL, USA
Glioma research currently lacks patient-derived platforms
that can effectively recapitulate gliomagenesis in vitro to
guide the search for molecular vulnerabilities and screen
for therapies. Tumor organoid cultures have been
generated from several cancers; however, it is unknown
whether organoids possess the tissue organization and
genetic programs of glioblastoma multiforme (GBM), or if
they can be leveraged to determine translational targets.
Here, we describe a multi-level cellular, molecular discovery
platform using cultures of patient-derived GBM organoids
(GBMO) to recapitulate the unique histologic and genetic
architecture of GBM and further understand GBM intrinsic
immunity. We characterized GBMOs by comparing their
cytoarchitecture with iPSC-derived organoids and molecular
landscape with NSC-derived organoids. GBMO mimics
stereotypic tumor architecture with distinct proliferative and
apoptotic microenvironments but lacks the developmental
self-organization recapitulated in iPSC organoids, despite
hosting basal and outer radial glia. Targeted deep-parallel
sequencing of GBMO validated a mutational load consistent
with GBM, including ATRX and KDM6A mutations, while
transcriptome data showed robust activation of cancer
hallmarks, like the Warburg Effect. Notably, we devised a
computational approach for identifying drivers of tumor
immunity from transcriptome data to nd an innate immunity
landscape in GBMO enriched for aryl hydrocarbon receptor
(AHR) genes, leading to the identication of an
AHR-dependent program. Pharmacological and genetic
inactivation of AHR revealed this program to be critical for
glioblastoma stem cells (GSCs) and organoid evolution.
Enhanced survival of mice intracranially injected with
AHR-inactivated GSCs reinforces the in vivo relevance of our
GBMO paradigm. Finally, we highlight the value of our
organoid platform for discovering 41 prognostic candidate
GBM biomarkers, 19 of which are novel, and expression
signatures important for population-level survival by using
advanced statistical models. Our work demonstrates, for the
rst time, the utility of patient-derived GBMO as a
personalized-medicine approach toward in vitro pre-clinical
trials for identifying molecular vulnerabilities and advancing
therapy for this devastating tumor.
Keywords: Glioblastoma; Organoids; Radial glia
048
FRAGILITY OF DEVELOPMENTAL TRAJECTORIES
OF HUMAN CORTICOGENESIS REVEALED BY
SINGLE INFLAMMATORY PERTURBATION OF
CEREBRAL ORGANOIDS
Hollingsworth, Ethan W.1, Julian, Katie2, Watanabe, Fumihiro2,
Julian, Dominic2, Hartlaub, Annalisa M.3, Hester, Mark3,
Imitola, Jaime2
1Medical Scientist Training Program, University of California,
Irvine, CA, USA, 2Neurology and Neuroscience, The Ohio State
University, Columbus, OH, USA, 3Center for Perinatal
Research, Nationwide Children’s Hospital, Columbus, OH, USA
Despite strong impetus from the recent Zika outbreak,
resurgence of neurotoxic viral infections, and clear association
to neurodevelopmental disorders, the fundamental principles
by which maternal infection and prenatal inammation
impact human brain development remain unclear. Here we
adopted a reductionist, systems neurobiology approach and
perturbed developing human cerebral organoids derived
from healthy donor iPSCs with proinammatory cytokine
interferon-γ (IFN-γ), the topmost hierarchal molecule of the
antiviral response cascade in radial glia (RG), to unravel the
downstream developmental consequences of a single
perturbagen on a complex biological system. IFN-γ exposure
restricts organoid growth across several concentrations and
timepoints. Utilizing uorescent DiD labeling, we visualized
the RG scaffold in organoids to nd marked underdevelopment
of progenitor zones following IFN-γ administration. Subse-
quent immunostaining showed that IFN-γ exposure alters
cell cycling of RG, disrupts adherens junctions of ventricular
RG, and diminishes outer RG proliferation, leading to
alteration of the prototypical radial unit. Likewise, RNA-
sequencing revealed IFN-γ dysregulates the normal
spatiotemporal expression pattern of the developing brain,
activating postnatal programs while downregulating early
neurogenic pathways. Importantly, inammatory induction
by IFN-γ also reshapes developmental hierarchies of organoid
corticogenesis toward a ventral cell fate with increased
GABAergic lineage output. This GABA-glutamatergic
neuronal imbalance is accompanied by dysregulation of
protein and ribosomal synthesis, both of which are implicated
in autism spectrum disorder (ASD). Bioinformatic analyses
further suggest the IFN-γ organoid transcriptome signature
to be aligned with ASD and maternal infections, opposed to
congenital neurodevelopmental disorders. Finally, mathe-
matical modeling indicates IFN-γ perturbation affects
nonlinear cell dynamics in a multilineage, parallel manner.
Altogether, our results highlight the feasibility of a single
perturbation organoid paradigm to model environmental,
non-genetic determinants of human brain development and
reveal a novel molecular and cellular link between
neuroinammation and ASD-like phenotypes.
Funding Source
OSU NRI, Nationwide Children’s Hospital.
Keywords:Neuroinammation; Infection; Neurogenesis
51
AMSTERDAM NETHERLANDS
Poster Abstracts
049
GENE CORRECTION REVERSES PHAGOCYTOSIS IN
IHPSC-DERIVED RETINAL PIGMENT EPITHELIAL
CELLS FROM RETINITIS PIGMENTOSA PATIENT
Erceg, Slaven, Artero-Castro, Ana, Machuca Arellano,
Candela, Lukovic, Dunja
Stem Cell Therapies in Neurodegenerative Diseases, Centro
de Investigación Príncipe Felipe, Valencia, Spain
Hereditary retinal dystrophies (HRD) are an important cause
of blindness af
fecting mostly retinal pigment epithelium
(RPE) and photoreceptors (PRs), with no effective treatments.
Both cell types are highly specialized, interact mutually in
retinal function and it is thought that the majority of the HRD
are primary in one cell type leading to a secondary dystrophy
in the other one. One of the monogenic primary RPE
dystrophies includes mutations MERTK gene that has
essential role in phagocytosis, one of the major functions of
the RPE. One of the strategies to treat this rare disease is to
replace diseased RPE with healthy autologous RPE tissue to
prevent PR cell degeneration. Patient-derived induced
pluripotent stem cells (ihPSCs) provide an unprecedented
opportunity to create autologous stem cell source that is able
to differentiate to virtually any human cell type including RPE
and PRs. The major drawback of ihPSC cell therapy is that
affected cells derived from ihPSC will carry the same MERTK
gene mutation. Recently we reported the generation of a
cellular model of MERTK-associated RP, which recapitulates
the disease phenotype and efciently genetically corrected
MERTK. In order to determine whether gene corrected ihPSC is
a faithful source for MERTK-associated RP in this study we
efciently differentiated ihPSC from corrected and patient´s
ihPSC and showed that gene corrected ihPSC-RPE reverse
the full expression of MERTK protein as well as reinstate
phagocytosis of labelled photoreceptor outer segments.
These ndings represent a proof-of-principle the usefulness of
gene correction in ihPSC to provide unlimited cell source for
potential personalized cell therapy for these rare blinding
disorders.
Keywords:
Retinitis pigmentosa; Gene correction; Phagocyto-
sis
051
MODELLING MICROENVIRONMENTAL MECHANI-
CAL PROPERTIES IN DUCHENNE MUSCULAR
DYSTROPHY IPSC-DERIVED CARDIOMYOCYTES
Pardon, Gaspard1, Chang, Alex C.Y.1, Pruitt, Beth L.2,
Blau, Helen H.1
1Baxter Laboratory for Stem Cell Biology, Department of
Microbiology and Immunology, Stanford University School of
Medicine, Stanford, CA, USA, 2Bioengineering & Systems
Biology, Department of Mechanical Engineering, University of
California, Santa Barbara, CA, USA
Duchenne Muscular Dystrophy (DMD) is an X-linked disease
affecting ~1:3500 boys per year that culminates in heart
failure in early adulthood. DMD results from >200 possible
genetic mutations on dystrophin. The lack of dystrophin
disrupts the anchoring of the cell sarcomere to the
extracellular matrix (ECM), affecting cardiomyocyte
contraction. With disease progression, tissue increases in stiff-
ness due to brosis and changes in ECM composition in
accordance with a dilated cardiomyopathy phenotype. We
hypothesize that this entails a positive feedback loop involving
multiple mechanosensing pathways. Here, we use a single-cell
platform to model the brotic remodeling in DMD. We measure
the force production of single human induced pluripotent stem
cell derived cardiomyocytes (hiPSC-CMs) on hydrogel sub-
strates with a stiffness matching that of healthy (~10kPa) or
brotic (~35kPa) tissue. In addition, single iPSC-CMs are
patterned in an elongated 1:7 aspect ratio by using microcon-
tact printing of ECM proteins. This enhances their intracellular
structural maturity and enables standardized measurements
on consistently oriented elongated contractile cardiomyocytes.
We use traction force microscopy to computes the contractile
strength as a function of bead displacement in the hydrogel
substrate using Digital Image Correlation (DIC) and Fourier
Transform Traction Cytometry (FTTC). We show that DMD
hiPSC-CMs have a dramatically reduced ability to produce force
on stiffer substrates compared to their isogenic controls. This
loss of contractile function correlates with an increase in
reactive oxygen species (ROS) and mitochondrial dysfunction.
The effect of stiffness in this difference in contractile function
uncovers a potent role of mechanosignaling mediated by the
dystroglycan complex. This platform will increase our under-
standing of the biophysics underlying cardiomyocyte mechano-
sensing.
Funding Source
This work was partially funded by the Swiss National Science
Foundation (SNF) through the Postdoc.Mobility fellowship
grant number P400PM_180825/1.
Keywords:
Human induced pluripotent stem cells; Duchenne
Muscular Dystrophy cardiomyopathy; Mechanosensing
052
MODELLING CANCER IMMUNOMODULATION USING
EPITHELIAL ORGANOID CULTURES
Kretzschmar, Kai1,2, Bar-Ephraim, Yotam E.1,2, Asra, Priyanca1,2,
de Jongh, Evelien1,2, Boonekamp, Kim E.1,2, Drost, Jarno3, van
Gorp, Joost4, Pronk, Apollo5, Smakman, Niels5, Gan, Inez J.6,
Sebestyen, Zsolt6, Kuball, Jürgen H. E.6, Vries, Robert G. J7,
Clevers, Hans C.1,2
1Hubrecht Institute, Royal Netherlands Academy of Arts and
Sciences, Utrecht, Netherlands, 2Oncode Institute, Utrecht,
Netherlands, 3Princess Maxima Center for Pediatric Oncology,
Utrecht, Netherlands, 4Pathology, Diakonessenhuis, Utrecht,
Netherlands, 5Surgery, Diakonessenhuis, Utrecht, Netherlands,
6Laboratory of Translational Immunology, University Medical
Center Utrecht, Netherlands, 7N.A., Foundation Hubrecht
Organoid Technology, Utrecht, Netherlands
Immune escape has been recognised as one of the hallmarks of
cancer. Overcoming this immunomodulatory process by tumour
cells has become a major therapeutic target. Suitable model
systems are missing to study this process in culture and test
possible drugs. Organoid technology using epithelial cells has
emerged as a powerful tool for the ex vivo modelling of tissue
morphogenesis, cell differentiation and cancer biology. Yet,
epithelial (cancer) organoid cultures lack all non-epithelial compo-
nents of the tissue microenvironment such as immune cells.
However, using transcriptional proling and ow cytometry, we
found that human colorectal cancer (CRC) organoids maintain
differential expression of immunomodulatory molecules present
52
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Poster Abstracts
in primary tumours. We hypothesised that this may allow us
to study immune escape using CRC organoid cultures. To
assess anti-tumour immunity and possible cancer immuno-
modulation, we developed a method of co-culturing CRC
organoids with antigen-specic cytotoxic T cells. In this
proof-of-principle assay, CRC organoids presenting specic
peptides were readily killed by these cytotoxic T cells
expressing matched T-cell receptors (TCRs). We did not
observe signicant CRC organoid cytotoxicity with a pep-
tide-TCR mismatch. By applying the cytokine interferon
gamma, we found that upregulation of immune checkpoints
by CRC organoids suppressed T-cell mediated killing in the
co-culture. By application of immune checkpoint inhibitors,
we could reinstate T-cell mediated killing of CRC organoids,
demonstrating that this method can be used to model cancer
immunodulation and test cancer immunotherapy drugs. Our
co-culture system may further serve as a rst step towards
rebuilding the tumour microenvironment in vitro.
Keywords: Organoids; Colorectal cancer; Cancer immunotherapy
053
EMERGENCE OF CELLULAR HETEROGENEITY IN
LIVER METASTASES OF HUMAN COLORECTAL
CANCERS IS ESSENTIAL FOR SUSTAINED GROWTH
Heinz, Maria C.1, Oost, Koen C.1, Fumagalli, Arianna2,
Peters, Niek3, Quintas Glasner de Medeiros, Gustavo4,
van Voorthuijsen, Lisa5, Lindeboom, Rik G.H5,
Verlaan-Klink, Ingrid1, Vermeulen, Michiel5, Liberali, Prisca4,
Kranenburg, Onno3, van Rheenen, Jacco2, Snippert, Hugo J.G1
1Molecular Cancer Research, Center for Molecular Medicine,
University Medical Center Utrecht, Netherlands, 2Molecular
Pathology, The Netherlands Cancer Institute, Netherlands,
3Cancer Center, University Medical Center Utrecht, Netherlands,
4Quantitative Biology, Friedrich Miescher Institute for
Biomedical Research, Switzerland, 5Department of Molecular
Biology, Faculty of Science, Radboud Institute for Molecular
Life Sciences, Radboud University Nijmegen, Netherlands
Accumulating evidence suggests that tumors are caricatures of
normal tissues, where cancer stem cells are believed to be
the driving forces of tumor growth. To identify intestinal stem
cells (SCs) in the organoid system, the Snippert group has
developed the SC reporter STAR, a minigene that reports
activity of the transcription factor ASCL2, the master
regulator of intestinal stem cell fate. Previous experiments in
the Snippert group have shown that single colorectal cancer
cells grow into organoids regardless of their STAR positivity.
Intriguingly, while monitoring stem cell activity over time, we
noticed that emergence of cellular heterogeneity is essential
for tumor growth since complete stem cell organoids
remained small and unsuccessful. Strikingly, the strict
interdependency between cancer stem cells and neighboring
support cells that underlies tumor growth seems to be
independent of the mutational background of the organoids. In
particular, this also holds true for highly mutated organoids
with ‘so-called’ metastatic niche-independency. Importantly,
these ‘symmetry breaking’ phenotypes are in agreement with
the initial growth of liver metastases of a murine CRC model
(VillinCre-ERT2; APC/; KRASLSL-G12D/+; P53KO/KO;
R26R-Confetti; Lgr5DTR-eGFP), where emergence of
heterogeneity between stem and non-stem cells correlates
with the transition between micro- and macrometastases.
In conclusion, we identied the existence of a universal
signalling mechanism between cancer stem and non-stem
cells – a cross-talk that is essential for metastatic tumor
growth. We will combine our expertise in human organoid
technology, high-resolution live imaging of tumor organoids
and in vivo mouse models for in-depth characterization of the
signalling crosstalk that support cancer stem cell
function, especially in metastatic situations.
Keywords:
heterogeneity; cancer stem cells; metastases
054
TOWARDS AN IN VITRO MODEL OF WILMS TUMOR:
GENERATING TUMOR-ASSOCIATED MUTATIONS IN
HUMAN INDUCED PLURIPOTENT STEM CELL-
DERIVED KIDNEY ORGANOIDS
Stevenson, Matthew, O’Brien, Lori
Cell Biology and Physiology, University of North Carolina at
Chapel Hill, NC, USA
Wilms tumor (WT) is the most common pediatric kidney
cancer. Blastemal-predominant tumors represent the most
malignant and chemotherapy-resistant form of WT. This
tumor subtype expresses markers found only within the
nephrogenic niche of the fetal kidney, suggesting it arises
from a malignant transformation of these cells. Sequencing
of blastemal-predominant tumors has identied a recurring
single amino acid mutation, Q177R, within the conserved DNA
binding domain of either of the closely related transcription
factors SIX1 and SIX2. These factors are critical for the
maintenance of nephron progenitors during mammalian
kidney development. Therefore, we hypothesize that this
recurring mutation disrupts the regulatory networks controlled
by SIX1/2 resulting in aberrant gene expression, an inability of
nephron progenitors to fully differentiate, and their malignant
transformation. Efforts to model WT in mice have proven
challenging and differences in gene expression dynamics
between mouse and human nephrogenesis, including the
temporal expression of SIX1, necessitates a novel system in
which to investigate the tumorigenic potential of the Q177R
mutation. HiPSC-derived kidney organoids recapitulate key
stages of human kidney development in vitro, resulting in the
generation of complex nephron-like structures. Based on
previously developed protocols, we have established our own
minimal, modied 3D protocol to reproducibly generate kidney
organoids for use as a model system. With a focus on SIX1,
ChIP-qPCR revealed proper targeting of SIX1 to its canonical
DNA targets beginning by day 5 with signicantly increased
binding through day 8 of the protocol, recapitulating the in
vivo activities of SIX1 during human nephrogenesis. Utilizing
the Sleeping Beauty transposon system, we have exploited
the dened differentiation timeline of our kidney organoid
protocol to express the mutant SIX1 protein at critical
developmental time points to elucidate the tumorigenic
program initiated by the Q177R mutation. Our results will shed
light onto the etiology of WT, opening up the possibility of
new, targeted therapies for this disease.
Funding Source
Supported in part by a grant from the National Institute of
General Medical Sciences under award 5T32 GM007092
Startup funds from UNC-Chapel Hill.
Keywords: Organoids; Tumorigenesis; Transcription
53
AMSTERDAM NETHERLANDS
Poster Abstracts
055
AN ALPHA-ACTININ REPORTER CELL LINE TO
MODEL SARCOMERIC DYNAMICS IN HUMAN
HESC-DERIVED CARDIOMYOCYTES RELATIVE
TO SUBSTRATE STIFFNESS
Slaats, Rolf1, Catarino Ribeiro, Marcelo1, Schwach, Verena1,
Tertoolen, Leon G.J.2, van Meer, Berend J.2, Molenaar,
Robert3, Mummery, Christine L.2, Claessens, Mireille M.A.E.3,
Passier, Robert C.J.J.1
1Applied Stem Cell Technologies, University of Twente,
Enschede, Netherlands, 2Department of Anatomy and
Embryology, Leiden University Medical Centre, Leiden,
Netherlands, 3Nanobiophysics Group, University of Twente,
Enschede, Netherlands
Cardiovascular disease is a major cause of morbidity and
mortality in modern society, being responsible for 37% of all
deaths in the EU in 2017 and an overall estimated cost to the
EU economy of 210 billion euro annually. Cardiac disease is
often accompanied by remodeling of the heart wall (for
example following myocardial infarction), resulting in cardio-
myocyte hypertrophy and increased extracellular brosis in
response to stress or injury. Although cardiac remodeling may
be an adaptive response at rst, cardiomyocyte hypertrophy
and cardiac brosis, resulting in an increased wall stiffness,
may ultimately lead to end-stage heart failure. Previous work
demonstrated that cardiomyocytes are less effective in
applying strain on stiff substrates than on physiologically
elastic substrates, leading, in combination with the increased
stiffness itself, to diastolic heart failure. Understanding the
underlying mechanisms of cardiomyocyte adjustment to
increased wall stiffness may provide a better insight in how to
preserve or restore heart function. We generated an α-acti-
nin-mRubyII (fusion protein) uorescent reporter line in a
previously documented cardiac NKX2.5-GFP human embryon-
ic stem cell (hESC) reporter line using CRISPR-Cas9 technolo-
gy. This α-actinin-mRubyII-NKX2.5-GFP-hESC double uorescent
reporter line allows visualization and live monitoring of z-disks
during cardiomyocyte contraction. Using this reporter cell line,
we analyzed sarcomeric dynamics in cardiomyocytes cultured
on uorescent bead-laden polyacrylamide gels with a stiffness
ranging from below physiological (4 kPa) heart conditions to
pathologically high (>100 kPa) levels. We found that absolute
sarcomeric shortening, but not contraction time, depends on
substrate stiffness. In addition, sarcomeric elongation during
relaxation is more rapid on stiff substrates rather than compli-
ant substrates. Furthermore, we will compare sarcomeric
contraction and relaxation to contractile strain applied on the
substrate by the cardiomyocyte. For this, we will determine
the applied strain by quantifying bead displacement in the
acrylamide gel, as previously described. With this data, we aim
to demonstrate the validity of an α-actinin uorescent cell line
in a model for myocardial stiffness.
Keywords: heart failure; sarcomere dynamics; reporter cell
line
056
HEMATOPOIETIC DIFFERENTIATION POTENTIAL
OF INDUCED PLURIPOTENT STEM CELLS DERIVED
FROM OSTEOPETROSIS PATIENTS’ PERIPHERAL
BLOOD MONONUCLEAR CELLS
Cevher, İnci1, Alkan, Berna1, Özdemir Saka, Cansu1,
Uçkan Çetinkaya, Duygu2, Visal Okur, Fatma2
1PEDI-STEM, Health Science Institute, Hacettepe University
Center for Stem Cell Research and Development, Ankara,
Turkey, 2Faculty of Medicine, Pediatric Hematology/Bone
Marrow Transplantation Unit and Center for Stem Cell
Research and Development, PEDI-STEM, Health Science
Institute, Hacettepe University, Ankara, Turkey,
Malignant infantile osteopetrosis (MIOP) is a rare inherited
disease characterized by increased bone mass and density
resulting from defects in osteoclast formation and/or
function. Only available therapeutic option is allogeneic
stem cell transplantaion. Disease modeling with patient-
derived IPS cells for better understanding of disease biology
could pave the way for development of new therapies. Aim of
this study is to evaluate hematopoietic differentiation potential
of osteopetrotic-iPSC carrying TCIRG1 mutation, one of the
most common mutations identied in autosomal recessive
form, and their functionality in in vitro culture system. Banked
peripheral blood MNC samples were used as a cell source
because of ease of obtaining cells with a minimally invasive
method. Erythroid progenitor cells (EPC) were expanded using
erythroid expansion medium. Expression of CD45, CD34, CD36,
CD235a and CD3 surface marker was checked prior to
reprogramming. Enriched PB-EPC of three patients and one
healthy donor were reprogrammed by using CytoTune-IPS 2.0
Sendai (SeV) kit. iPSC colonies which started to appear
around day 12-14 were picked, manually passaged and
expanded in matrigel-coated plates. Colonies were character-
ized (≥ 3 clones/line) by colony morphology, ow cytometry,
alkaline phosphatase (AP) and immunouorescence (IF)
staining. Expression of pluripotency genes, detection of SeV
were evaluated with PCR. In vitro pluripotency was assessed
by embryoid body (EB) assay. iPSC lines exhibiting typical ESC-
like colony morphology were shown to express pluripotency
markers (OCT4, SSEA-4, SOX2, TRA-1- 60) by IF staining.
85-99% of the cells were found positive for SSEA-4 and Oct3/4
and negative for CD36 and CD235a with ow cytometry. All
iPSC lines expressed pluripotency related genes (OCT4, SOX2,
c-Myc, Klf4, Nanog, DNMT3B, CDH1, UTF1, REX1, TERT). Loss
of SeV genome was observed at later passages. Fluorescence
staining and lineage specic gene expressions (α-SMA, MAP-2
and SOX17) of EB and EB-based hematopoietic differentiation
experiments of iPSC are in progress. In conclusion, results of
this study would provide valuable information regarding the
impact of osteoclast defect on the cellular components of
hematopoietic niche specically hematopoietic stem compart-
ment in patients with MIOP.
Keywords: Osteopetrosis; induced pluripotent stem cells;
hematopoietic differentiation
54
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Poster Abstracts
057
CRISPR-INDUCED ‘SECOND HIT’ MUTATIONS IN
APC CAUSE DIFFERENTIATION OF HESCS DERIVED
FOLLOWING PREIMPLANTATION GENETIC DIAG-
NOSIS FOR FAMILIAL ADENOMATOUS POLYPOSIS
Preisler, Livia1, Mayshar, Yoav2, Ben-Yosef, Dalit3
1Cell and Developmental Biology, Tel Aviv University, Tel-Aviv,
Israel, 2Racine IVF Unit, Tel-Aviv Sourasky Medical Center,
Tel-Aviv, Israel, 3Cell and Developmental Biology, Tel-Aviv
Sourasky Medical Center, Tel-Aviv, Israel
Inactivation of adenomatous polyposis coli (APC), a major
controller of Wnt signaling, is one of the most important
genetic alterations in the development of colorectal cancer
(CRC). Familial adenomatous polyposis (FAP) patients carry
a single mutated APC allele, which invariably leads to CRC at
a relatively young age due to sporadic loss of the remaining
functional allele. Our goal in this study is to establish the
role of APC in human embryonic stem cells (hESCs) and
their derived colon organoids in order to understand its
contribution to CRC carcinogenesis. Three FAP-hESC lines
carrying different germline mutations in the APC gene were
derived in our lab from blastocysts following preimplantation
genetic diagnosis (PGD) from three unrelated FAP-affected
families. We used CRISPR with specic gRNAs to target the
APC gene in these FAP-hESC lines in order to induce the
somatic mutation in two sites in the second (wild-type) allele
of the gene. A total of 59 clones were isolated, and 11 (19%)
of them were identied by NGS as carrying CRISPR-induced
mutations. Interestingly, all clones were found by western
blot analysis to express the full-length APC protein despite
harboring frameshift mutations in one allele indicating that
the targeted allele was the germline mutant in all cases. A
follow-up time course FACS analysis of FAP-hESCs carrying
a β-catenin GFP reporter that were subjected to CRISPR
mutations in the APC gene revealed that the proportion of
APC double-mutated cells (GFP+) declined signicantly with
time, a result that was also conrmed by NGS. RNA extract-
ed from double mutant cells isolated by FACS sorting
demonstrated relatively high expression of the endodermal
markers, CDX2 and FOXA2, and low expression of the
pluripotent genes, Nanog and Oct4, indicating that their loss
during long-term culture is due to differentiation. In conclusion,
APC knockout in hESCs results in Wnt signaling activation
and loss of pluripotency, highlighting the central role of this
pathway for hESC self-renewal. We are now generating colon
organoids from these hESCs in order to further understand
the role of APC in CRC. Inducing the APC mutations in the
differentiated cells of the organoids will allow further
clarication of the roles of APC in more clinically relevant cells.
Funding Source
Israel Cancer Research Foundation (ICRF), The Dahlia
Greidinger Anti Cancer Fund, Or Shapira Fund.
Keywords: Adenomatous polyposis coli (APC); Familial
adenomatous polyposis (FAP); Human embryonic stem cells
(hESCs)
058
DIGEORGE SYNDROME; MODELING A COMPLEX
DISORDER
Apáti, Ágota1, Berecz, Tünde1, Szabó, Brigitta1, Szabó, Eszter 1,
Molnár, Andrea Á2, Haltrich, Irén3, Homolya, László1,
Földes, Gábor4, Réthelyi, János5
1Institute of Enzymology, Research Centre for Natural
Sciences, HAS, Budapest, Hungary, 2Heart and Vascular
Center, Semmelweis University, Budapest, Hungary, 32nd
Department of Pediatrics, Semmelweis University, Budapest,
Hungary, 4Heart and Vascular Center, Semmelweis University,
Budapest, Hungary, 5Department of Psychiatry and
Psychotherapy, Semmelweis University, Budapest, Hungary
Results describing molecular mechanisms underlying
complex disorders are limited by currently available
model systems. Human iPSC-derived cell cultures are new,
promising models for studying disease related phenotypes
in vitro especially in cell types which cannot be investigated
directly and maintained in long-term cultures (such as neural
or cardiovascular cells) or when appropriate animal models
are not available (such as psychiatric diseases). Studying
the entire differentiation process may provide insights to the
pathomechanism and disease development. Our aim is to
investigate in vitro cellular phenotypes of DiGeorge
syndrome, a genetically well-determined, multiorgan
(cardiovascular, nervous and immune systems) disease
caused by a microdeletion in 22q11.2. For our investigations
we have chosen a family with inherited form of the syndrome
and blood was taken from the index individual, a severely
affected child, her mother and grandfather (all carriers of
the deletion) and from father and grandmother as healthy
controls. Reprograming of peripheral blood mononuclear
cells to pluripotent state was performed by forced expression
of four transcription factors – Oct3/4, Sox2, Klf4 and c Myc
– using Sendai virus vectors. Stabilized iPSCs lines were
genetically analyzed and subsequently differentiated into
cell types relevant to the diseases by mimicking the in vivo
developmental program. We performed cardiac and neural
differentiation and disease-affected cells were compared to
the control cells. Our data show impaired neural development
and altered beating activity in patient-derived cells.
Funding Source
Supported by the National Brain Research Program of
Hungary (NAP 2017-1.2.1-NKP-2017-00002 and KTIA_
NAP_13-2014-0011), and by the National Research, Develop-
ment and Innovation Ofce (NVKP_16-1-2016-0017 and
OTKA- K128369).
Keywords: induced pluripotent stem cells; DiGeorge syn-
drome; directed differentiation
55
AMSTERDAM NETHERLANDS
Poster Abstracts
059
CEREBRAL ORGANOIDS AS A NEW WAY TO MODEL
ADHD PATHOPHYSIOLOGY: A LOOK AT MOLECULAR,
CELLULAR AND CONNECTIVITY DEFICITS
Saraiva Lopes, Claudia A.1, Ko, Sanghyeok1, Jang, Yongwoo1,
Kim, Yeahan1, Jonathan, Copetta2, Cohen, Bruce1,
Teicher, Martin1, Kim, Kwang-Soo1
1Psychiatry, McLean Hospital, Harvard Medical School,
Belmont, MA, USA, 2Draper Laboratories, Cambridge, MA, USA
Attention decit hyperactivity disorder (ADHD) is a
heterogeneous neurodevelopmental disorder with a
devastating impact on the quality of life of millions of
children, adolescents and adults. While ADHD is thought to
be highly heritable with unknown etiology, it is is likely to
involve a combination of environmental factors and the
contribution of multiple genes defects. To understand the
molecular underpinnings of ADHD, we hypothesize that 3D
neuralized structures (organoids) derived from patient-specic
induced pluripotent stem cells (iPSCs) can be used as a
potential platform. In particular, the Prefrontal Cortex (PFC)
is emerging to be of central relevance to the neural pathways
of ADHD, as it connects extensively to sensory and motor
cortices, as well as to the basal ganglia and cerebellum.
These areas are intricately interconnected and modulated by
a mesh of neurons that in ADHD display heavy decits in
dopaminergic and noradrenergic transmission. Thus, it is
critical to understand the molecular inuences modulating
PFC’s function in order to develop novel medications for
patients aficted with the disorder. We have started to
generate and characterize iPSC-derived cortical organoids
from ADHD patients and healthy sibling controls to study the
molecular and cellular differences in corticogenesis between
diseased and control brains. Particularly, we propose that
the root cause of the PFC’s smaller structure involves a limited
progenitor pool and impaired radial migration. To achieve
these long-term goals, we attempted to use our novel and
non-viral reprogramming methods to generate high quality
control and ADHD-iPSC lines, to optimize in vitro organoid
generation. Our approach will facilitate examination of how
disease risk is translated at the cellular and tissue levels
through comparative studies of processes such as progenitor
cell proliferation, migration and connectivity during
development.
Funding Source
Narsard Award for Young Investigator
Keywords: ADHD; Corticogenesis; Progenitor
060
DYNAMIC HYDROGELS TO MODEL FIBROSIS IN VITRO
Madl, Christopher M., Blau, Helen M.
Baxter Laboratory for Stem Cell Biology, Stanford University,
Stanford, CA, USA
Despite the ubiquitous role of brosis in tissue dysfunction
arising from aging and disease, no adequate in vitro model of
the brotic microenvironment exists. Fibrosis is characterized
by excess extracellular matrix (ECM) deposition that leads to a
progressive stiffening of the cellular microenvironment. Thus,
to model brosis in vitro, cell culture substrates that permit
dynamic tuning of matrix stiffness and composition would be
ideal. To date, existing hydrogel culture platforms do not
readily enable real-time, cytocompatible manipulation of these
properties wherein a single cell can be visualized as matrix
properties change. Instead, cell populations are compared after
culture on either elastic or stiff hydrogels. To address this need,
we have developed a system that employs bioorthogonal
chemical reactions to increase hydrogel crosslink density and
stiffen the matrix in situ. Hydrogels are synthesized from
chemically-functionalized multi-arm poly(ethylene glycol)
(PEG) macromers and characterized by rheometry. Control
static materials are designed to possess mechanical
properties approximating both healthy tissue (soft, elastic
modulus (E) ~ 1-10 kPa) and brotic tissue (stiff, E ~ 20-50
kPa). Dynamic materials are prepared that allow user-con-
trolled stiffening to mimic brosis. Cell adhesion to the
materials is controlled by chemically-coupling extracellular
matrix (ECM) components (such as laminin and bronectin) to
the hydrogels. The hydrogel platforms are amenable to
characterization techniques such as live-cell time lapse
microscopy and immunostaining. These materials enable
tracking of real-time, single-cell responses to alterations in
substrate stiffness to probe the temporal changes in mecha-
notransduction during brosis.
Keywords: brosis; extracellular matrix; biomaterials
061
PHENOTYPIC EVALUATION OF FRESHLY-ISOLATED
AND CRYOPRESERVED HPSC-CMS
van den Brink, Lettine1, Brandao, Karina O.1, Mol, Mervyn P.H1,
Mummery, Christine L.1, Verkerk, Arie O.2, Davis, Richard P.1
1Anatomy and Embryology, Leiden University Medical Centre,
Leiden, Netherlands, 2Department of Medical Biology, UMC
Amsterdam, Netherlands
Human induced pluripotent stem cell-derived cardiomyocytes
(hiPSC-CMs) have emerged as a powerful platform for in
vitro modelling of cardiac diseases, safety pharmacology and
drug screening, and in vivo cardiac regenerative therapies. All
these applications require large quantities of hiPSC-CMs. To
facilitate this, cryopreservation of hiPSC-CMs without altering
their biochemical and functional phenotype is essential. Not
only does the ability to cryopreserve hPSC-CMs make the
generation more cost and time effective, but is also enables
the same batch of hPSC-CMs to be evaluated in multiple
functional assays to investigate a disease phenotype. For this
56
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Poster Abstracts
reason, this study aimed to functionally and molecularly
compare freshly-derived and cryopreserved hiPSC-CMs
generated from two different hiPSC lines. While the cryopre-
served hiPSC-CMs exhibited poorer recovery than their
freshly-derived counterparts, no signicant differences in
terms of cardiac-specic marker expression and contractility
were observed. Similarly, one hiPSC line showed no differ-
ence in action potential parameters, although a second
hiPSC line did exhibit a prolongation in action potential
duration. These results provide evidence that cryopreserva-
tion does not compromise the molecular, physiological and
mechanical properties of hiPSC-CMs, thereby enabling large
quantities of hiPSC-CMs to be stored for prolonged periods
and allowing the same batch of hiPSC-CMs to be used for
multiple applications and evaluations.
Funding Source
This work was funded by an ERC-stG and a VIDI fellowship
from the Netherlands Organisation for Scientic Research
(NWO).
Keywords: hPSC-CMs; Cryopreservation; Electrophysiology
062
GENERATION OF ASTROCYTE SUBTYPES FROM
HUMAN AND MOUSE INDUCED PLURIPOTENT STEM
CELLS TO MODEL VANISHING WHITE MATTER
Hillen, Anne E.J.1, Dooves, Stephanie1, Leferink, Prisca1,
Watanabe, Kyoko2, Jacobs, Gerbren1, Gasparotto, Lisa1,
Cornelissen-Steijger, Paulien1, van der Knaap, Marjo1,
Heine, Vivi M.1
1Child Neurology, Amsterdam UMC, Amsterdam, Netherlands,
2Complex Trait Genetics, Amsterdam UMC, Amsterdam,
Netherlands
Astrocytes have become an important player in research
into various neurological diseases, such as demyelinating
disorders. Drug screening experiments and regenerative
studies on rare leukodystrophies would be greatly facilitated
by the generation of astrocytes from induced pluripotent
stem cells. As such, it has become the focus of various
studies. Whereas human models are likely to provide
especially useful insight, high variability and low sample size
impede mouse-based models to a lesser degree. We have
developed two astrocyte differentiation protocols for
derivation from both mouse and human induced pluripotent
stem cells to model the leukodystrophy Vanishing White
Matter. By using fetal bovine serum or ciliary neurotrophic
factor, associated with grey and white matter respectively,
we obtained two different astrocyte subtypes. Both expressed
astrocyte-associated markers, showed appropriate
morphology, and displayed reactivity when challenged. The
obtained white matter-like and grey matter-like astrocytes
differed in size, process arborization, and expression prole.
White matter-like astrocytes were found to be selectively
vulnerable to Vanishing White Matter mutations. Human
astrocytes of both subtypes derived from Vanishing White
Matter iPSC lines and control iPSC lines were subjected to
whole cell RNA sequencing. The analysis indicated white
matter-like astrocytes were more affected than grey
matter-like astrocytes. To compare human and mouse data,
whole cell RNA sequencing of white matter-like astrocytes
derived from Vanishing White Matter or control mouse iPSC
lines was performed. In mouse, differentially expressed
genes were related to the extracellular matrix and the
immune system (based on Gene Ontology). Human but not
mouse Vanishing White Matter astrocytes additionally
showed differentially expressed genes involved in neuronal
functioning, vasculature, and cell development. The
stimulatory effect of hyaluronidase-treated astrocyte
conditioned medium on oligodendrocyte progenitor
maturation was also specic to human Vanishing White
Matter astrocytes. These human- and mouse-based models
of astrocyte subtype differentiations can underscore disease
mechanisms when combined, and are useful in disease
modeling in vitro and regenerative applications in vivo.
Funding Source
This research is funded by a ZonMw VIDI research grant
(91712343; VMH), a European Leukodystrophy Association
(ELA) Research Grant (2014-012L1; VMH), an E-Rare Joint
Call project (9003037601; VMH), and an NWO Spinoza grant
(MSvdK).
Keywords: iPSC differentiation; astrocyte subtypes; Vanish-
ing White Matter
063
SCREENING CHEMICALS FOR RECOVERING
IMPAIRED
OSTEOGENESIS IN CFC SYNDROME-IPSCS
Kim, Bumsoo1, Choi, Jung-Yun2, Hong, Beom-Jin1, Lee,
Beom-Hee3, Yoo, Han-Wook3, Kim, Mi Young1, Han, Yong-Mahn1
1Department of Biological Sciences, Korea Advanced
Institute of Science and Technology (KAIST), Daejeon, Korea,
2Graduate School of Medical Science and Engineering, Korea
Advanced Institute of Science and Technology (KAIST),
Daejeon, Korea, 3Department of Pediatrics, Asan Medical
Center Children’s Hospital, University of Ulsan College of
Medicine, Seoul, Korea
Cardiofaciocutaneous (CFC) syndrome is a type of
RASophathy, which is mainly caused by the germline
mutations in BRAF gene. Among various symptoms of CFC
syndrome, skeletal phenotypes, including short stature, bone
growth delay, and low bone mineral density are characterized
in the majority of CFC patients. We previously reported that
CFC-induced pluripotent stem cells (CFC-iPSCs) showed
impaired osteogenesis. In fact, CFC-mesenchymal stem
cells (CFC-MSCs) differentiated from CFC-iPSCs represented
aberrant alkaline phosphatase activity and mineralization
during osteogenic differentiation in vitro. In addition,
TGF-beta signaling was activated and BMP signaling was
downregulated in CFC-MSCs compared to wild-type (WT)-
MSCs. Based on these results, we screened potential
chemicals to rescue defective osteogenic differentiation of
CFC-MSCs using para-Nitrophenylphosphate (pNPP) assay on
MSCs-plated 384-wells. Among 2261 clinical compounds
(provided by Korea Chemical Bank), 10 potential compounds
such as signaling inhibitors and nucleoside analogues were
screened. Those chemicals also revealed pharmacological
effects on the recovery of defective osteogenesis in CFC-
MSCs. Our ndings provide novel insights on the pathologi-
cal mechanism and therapeutic targets in CFC syndrome.
Keywords: Drug screening; CFC syndrome; Osteogenesis
57
AMSTERDAM NETHERLANDS
Poster Abstracts
064
DISEASE MODELING FOR FABRY DISEASE USING
PATIENTS-DERIVED INDUCED PLURIPOTENT STEM
CELLS
Do, Hyosang1, Park, Sang-Wook1, Lee, Beom-Hee2,
Yoo, Han-Wook2, Han, Yong-Mahn1
1Department of Biological Sciences, Korea Advanced
Institute of Science and Technology (KAIST), Daejeon, Korea,
2College of Medicine, Asan Medical Center, Seoul, South Korea
Fabry disease (FD) is an X-linked inherited lysosomal storage
disorder which is caused by α-galactosidase(GLA) deciency.
Mutated GLA results in immoderate globotriaosylceramide
(Gb3) accumulation in various cell types, thereby causing to
progressive complications with age. In particular, accumulation
of Gb3 in endothelium causes life-threatening complications
such as ischemic stroke, hypertrophic cardiomyopathy, and renal
failure at the terminal stage of FD patients. However, cellular
mechanisms whether GB3 accumulation leads to these vascu-
lopathies in FD are poorly understood. To study cellular model-
ing in vitro, we generated induced pluripotent stem cells (iPSCs)
from four Fabry patients. Although FD-iPSCs exhibited low
a-galactosidase activity and excessive Gb3 accumulation in
undifferentiated state, they could differentiate vascular cells
such as endothelial cells (ECs) and smooth muscle cells.
Accumulated Gb3 was transiently cleared by treatment with
alpha-galactosidase recombinant protein (Fabrazyme
®
) during
endothelial differentiation of FD-iPSCs. Nonetheless, FD-ECs
showed a lower expression of angiogenic factors such as ANG2,
VEGF than normal ECs. These results demonstrate that endothe-
lial dysfunction is associated with low activity of angiogenic
factors in Fabry disease.
Keywords:
human iPSCs; Fabry disease; disease modeling
065
CEREBRAL ORGANOIDS REVEAL AN IMPAIRED
DEVELOPMENT OF CORTICAL NEURONS IN
MONOZYGOTIC TWINS DISCORDANT FOR
SCHIZOPHRENIA
Koskuvi, Marja1, Lehtonen, Sarka1, Plotnikova, Lidiia1,
Puttonen, Katja1, Hyötyläinen, Ida1, Gao, Yanyan1, Ojansuu,
Ilkka2, Vaurio, Olli2, Cannon, Tyrone3, Lönnqvist, Jouko4,
Therman, Sebastian4, Suvisaari, Jaana4, Kaprio, Jaakko5,
Tiihonen, Jari2, Koistinaho, Jari1
1A. I. Virtanen Institute for Molecular Sciences, University of Eastern
Finland, Kuopio, Finland, 2Department of Forensic
Psychiatry, University of Eastern Finland, Niuvanniemi Hospital,
Kuopio, Finland, 3Department of Psychology and Psychiatry, Yale
University, New Haven, CT, USA, 4Department of Public Health
Solutions, National Institute for Health and Welfare, Helsinki, Finland,
5Department of Public Health, University of Helsinki, Finland
According to the neurodevelopmental theory of schizophrenia
(SZ), pathologic changes contributed by genetic and environ-
mental factors start taking place during the rst trimester of
pregnancy, even though the onset of SZ with drastic symptoms
(hallucination, delusion, social dysfunction) appear typically only
in young adulthood. If one of the identical twins has SZ, the risk
of illness for the other one is about 50% despite of the shared
genome and childhood environment. We have generated human
induced pluripotent stem cell (hiPSC) derived cerebral organoids
from monozygotic twin pairs discordant for SZ and unrelated
healthy controls to examine gene expression and morphological
changes in cortical development. On the rst 60 days of cerebral
organoid development, RT-qPCR showed gradually increased
expression in DCX and MAP2 gene markers for neuron progenitor
cells and mature neurons, respectively. Interestingly, the expres-
sions of both genes were increased in affected twins at all
measured time points compared to non-affected twins and
controls. Furthermore, we detected an increased expression in
markers of various neuronal subtypes: glutamatergic (SLC17A7,
FC>20), dopaminergic (TH, FC>1000) and GABAergic neurons
(GABRA1 and GAD2, FC>5), all known to be implicated in SZ. The
developing organoids derived from discordant pairs were smaller
than organoids derived from control patients. Morphologically,
the organoids of affected twins lacked ventricle-like structures
and displayed disorganized rosette formation at 20 days of
organoid differentiation, indicating malformation of the progeni-
tor zone. In summary, the disruption of cortical neuronal program-
ming suggests network dysfunction and contribution of several
neuronal subtypes to SZ phenotype already at early stage
development. Further investigation studies will focus on neuronal
connectivity and signaling.
Keywords:
schizophrenia; induced pluripotent stem cell; cerebral
organoid
066
FUNCTIONAL EVALUATION OF CRISPR/CAS9
EDITED KCNQ1 MUTATIONS IN CARDIOMYOCYTES
DERIVED FROM IPS CELLS OF LONG QT SYNDROME
PATIENTS
Cagavi, Esra1, Sahoglu Goktas, Sevilay2, Torun, Tugce3, Kazci,
Yusuf Enes2, Durdu, Zeynep B.3, Ors, Gizem3, Akgul Caglar, Tuba2
1Department of Medical Biology, Regenerative and Restorative
Medicine Research Center, Medipol University, Istanbul, Turkey,
2Neuroscience Programme, Regenerative and Restorative Medicine
Research Center, Medipol University, Istanbul, Turkey, 3Medical
Biology and Genetics Programme, Regenerative and Restorative
Medicine Research Center, Medipol University, Istanbul, Turkey
Congenital Long QT Syndrome Type-1 (LQTS-1) is a common
form of cardiac arrhythmia that is characterized by a prolonga-
tion of the QT interval on an electrocardiogram. LQTS-1 is
associated with mutations in the KCNQ1 gene encoding a
voltage-gated potassium channel. Here, we reported to recapitu-
late the LQTS-1 disease phenotype in vitro by using cardiomyo-
cytes derived from patient-specic iPSCs, to correct KCNQ1 gene
mutation by CRISPR/Cas9 and to evaluate the electrophysiologi-
cal phenotype of cardiac tissue following genome editing. First,
we have reprogrammed venous blood cells of a LQTS-1 patient
and his healthy family member to iPSCs via Sendai virus encod-
ing pluripotency markers. After reprogramming, iPSC identity was
conrmed by expression of pluripotency genes by qRT-PCR,
immunocytochemical staining and teratoma assay. To correct the
heterozygous point mutation at pore region of KCNQ1 gene,
patient-specic iPSCs were co-electroporated with guide RNA,
Cas9 enzyme and GFP encoding plasmid together with ssDNA
carrying wild-type sequences. GFP expressing iPSCs were
single-cell sorted by FACS. From the screened 476 individual iPSC
colonies, 26 colonies showed CRISPR/Cas9 genome editing
revealed by Sanger sequencing. Using a similar strategy, the
disease causing mutation was generated in KCNQ1 gene of
healthy control iPSCs with 3% efciency. To electrophysiological-
ly evaluate the cardiac phenotype, cardiac tissue generated from
t
he healthy, patient-derived and gene-edited iPSCs were analysed
58
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Poster Abstracts
for Ca2+ currents by Fluo-4 analysis and eld potential by the
multielectrode system. While we have observed a signicant
prolongation of QT interval in cardiomyocytes derived from
LQTS-1 patient compared to healthy control, KCNQ1 gene
correction shorten the QT interval and functionally alleviated the
in vitro disease symptoms. Collectively, we have showed a direct
correlation of electrophysiological cardiac phenotype with KCNQ1
gene sequence, sheding light on the disease mechanism and the
potential corrective use of gene-editing in therapy. We are in the
process of generating healthy, patient-derived and genome edited
iPSC-derived cardiac organoids for drug screens in the future.
Funding Source
This study is supported by The Scientic and Technological
Research Council of Turkey (TUBITAK) under 1003 Scientic
and Technological Research Projects Funding program with
Project number 213S192.
Keywords:
Induced pluripotend stem cells; cardiac disease
modeling; genome editing
067
SIMULTANEOUS MULTI-PARAMETER FUNCTIONAL
ANALYSIS OF HUMAN PLURIPOTENT STEM CELL-
CARDIOMYOCYTES USING FLUORESCENCE-BASED
IMAGING
Davis, Richard, Miller, Duncan C., Meer, Berend J., Brandão, Ka-
rina O., Krotenberg Garcia, Ana A., Mol, Mervyn P.H., Mummery,
Christine L.
Department of Anatomy and Embryology, Leiden University Medi-
cal Centre, Leiden, Netherlands
Pharmacological compounds and genetic variants can affect
different and sometimes multiple aspects of cardiac physiology,
such as the cardiomyocyte (CM) action potential (AP), intracellu-
lar calcium transients, and contractility. Simultaneous and higher
throughput modelling of these effects in human pluripotent stem
cell (hPSC)-derived CMs can be achieved optically, using com-
plementary voltage- and calcium-sensitive uorescent indicators
together with membrane labelling. Using an imaging platform
capable of fast simultaneous recording in three separate uores-
cence channels, we have determined the effects of a KCNH2
mutation, known to affect IKr current, on these three functional
parameters using organic dyes in the mutant hPSC-CMs com-
pared to the isogenic control. As an alternative to organic dyes,
we are investigating genetically encoded voltage and calcium
indicators (GEVIs and GECIs), since they may mitigate potential
toxicity issues associated with the use of organic dyes. We nd
transfection of in vitro transcribed (IVT) mRNA to be efcient
and gentle in hPSC-CMs. Comparison of two GEVIs, ASAP2f and
ArcLight1, indicate that although ASAP2f has a weaker signal,
faster kinetics mean that AP morphology is more similar to that
of organic voltage sensitive dyes (VSDs). Two GECIs, GCaMP6f
and jRCaMP1b, also exhibit a robust signal for Ca2+ transients.
Together with an advanced imaging platform, combinations of
uorescent organic dyes or GEVIs and GECIs can facilitate bona
de and multi-parameter modelling of disease mutations or drug
effects in hPSC-CMs.
Funding Source
This work was supported by an ERC-Starting Grant (StemCardio-
Risk; #638030) and the Netherlands Organisation for Scientic
Research grant VIDI-917.15.303 (Illuminate).
Keywords:
cardiac disease modelling; optical measurements;
optogenetics
068
MODELLING SUCCINIC SEMIALDEHYDE DEHYDROGE-
NASE DEFICIENCY USING PATIENT IPSC-DERIVED
CEREBRAL ORGANOIDS
Brennenstuhl, Heiko
1
, Ebrahimi-Fakhari, Darius
2
, Sahin, Mustafa
2
,
Pearl, Phillip
2
, Lohrer, Benjamin
3
, Thome, Christian
4
, Opladen,
Thomas
5
, Karow, Marisa
3
, Jung-Klawitter, Sabine
1
1
Division for Neuropediatrics and Metabolic Medicine, Heidelberg
University Hospital, Heidelberg, Germany,
2
Department of Neurolo-
gy, Children’s Hospital Boston, Harvard Medical School, Boston,
MA, USA,
3
Biomedical Center, Ludwig Maximilian University of
Munich, Munich, Germany,
4
Institute of Physiology and Pathophysi-
ology, Medical Faculty of Heidelberg University, Heidelberg,
Germany,
5
Division for Neuropediatrics and Metabolic Medicine,
Heidelberg University, Heidelberg, Germany
Succinic semialdehyde dehydrogenase deciency (SSADHD) is a
monogenetic autosomal-recessive neurological disorder caused
by variants of the ALDH5A1 gene. Clinical symptoms develop
during early childhood and include intellectual disability, develop-
mental delay, behavioral problems, impaired motor coordination
and seizures. Malfunction of the SSADH enzyme results in high
concentrations of the neurotransmitter gamma-amino butyric
acid (GABA) and its degradation product gamma-hydroxybutyrate
(GHB). To date, it is unclear how altered concentrations of GABA
and GHB could contribute to the disease etiology of SSADHD.
Furthermore, little is known about the extra-synaptic role of these
neurotransmitters in early brain development. Using patient-de-
rived induced pluripotent stem cells (iPSCs) and isogenic control
lines from two separate families harboring different genetic
alterations in ALDH5A1 we generated a cerebral organoid model
to study the underlying pathophysiology of SSADHD. Our rst
immunohistochemical analyses of the organoids at early stages
revealed an increased signal and diffuse spreading of the
neuronal marker MAP2 in patient-derived organoids with simulta-
neous reduction of SSADHD neuronal progenitor cells. Moreover,
we investigated mitotic activity of organoid-resident progenitors,
showing a strong reduction of M-Phase cells in SSADHD organ-
oids, which may point to an overall decreased stem cell pool and/
or premature cell cycle exit of symmetrically dividing stem cells.
Currently, we are further studying the cellular composition of the
SSADH organoids at later stages following aggregation. More-
over, the concentration of GABA and GHB in the supernatant of
SSADHD and control organoids are determined using HPLC and
additional analysis also include electrophysiological activity of
SSADHD organoid-resident neurons. We propose an unprece-
dented role of GABA and GHB as extra-synaptic, non-neurotrans-
mitter related trophic factors, contributing to the pathophysiology
of SSADHD.
Funding Source
This research was supported by the SSADH-Dezit e.v. parental
association and the Dietmar Hopp Foundation (St. Leon-Rot,
Germany).
Keywords:
neurotransmitters; SSADHD; neuronal development
59
AMSTERDAM NETHERLANDS
Poster Abstracts
069
ZOOMING IN ON CRYOPRESERVATION OF HIPSCS
AND NEURAL DERIVATIVES: A DUAL-CENTER
STUDY USING ADHERENT VITRIFICATION
Kaindl, Johanna1, Meiser, Ina2, Majer, Julia2, Sommer, Annika1,
Krach, Florian1, Katsen-Globa, Alisa2, Winkler, Jürgen3,
Zimmermann, Heiko2, Neubauer, Julia C.2, Winner, Beate1
1Department of Stem Cell Biology, Friedrich–Alexander
University Erlangen–Nürnberg, Erlangen, Germany,
2Department of Cryo- and Stem Cell Technology, Fraunhofer
Institute for Biomedical Engineering, Sulzbach, Germany,
3Department of Molecular Neurology, Friedrich–Alexander
University Erlangen–Nürnberg, Erlangen, Germany
Human induced pluripotent stem cells (hiPSCs) are an
important research tool and efcient cryopreservation is a
major challenge. The current gold standard for hiPSCs is
slow-rate freezing in suspension, but low recovery rates are
limiting immediate post-thawing applicability. We tested
whether the switch from slow-rate freezing to ultra-fast
cooling by vitrication improves post-thawing survival in a
selection of hiPSCs and small molecular neural progenitor
cells (smNPCs) from Parkinson’s disease and controls.
In a dual-center study, we compared the results by
immunocytochemistry (ICC) and uorescence-activated
cell sorting analysis. Moreover, RNA-sequencing (RNA-seq)
before and after freezing was performed. Adherent vitrication
was achieved in the so-called TWIST substrate, a device
combining cultivation, vitrication, storage, and post-thawing
cultivation. Vitrication resulted in preserved conuency and
signicantly higher post-thawing cell numbers and viability
at day one after thawing, while results were not signicantly
different at day four after thawing. RNA-seq and ICC of hiPSCs
revealed no change in gene expression and pluripotency
markers after cryopreservation, indicating that physical
damage after slow-rate freezing disrupts the cellular
membranes. Scanning electron microscopy revealed
preserved colony integrity and intact cell-cell adhesions by
adherent vitrication. Experiments in smNPCs demonstrated
that adherent vitrication is also applicable to neural
derivatives of hiPSCs. Our data suggest that, compared to
the state-of-the-art slow-rate freezing in suspension,
adherent vitrication is an improved cryopreservation
technique for hiPSCs and derivatives.
Keywords: Cryopreservation; Adherent vitrication; Neural
derivatives
070
GENERATION OF A MIDBRAIN DOPAMINERGIC
REPORTER LINE FOR MODELLING DOPAMINERGIC
DEVELOPMENT AND PARKINSON’S DISEASE
Cardo, Lucia F., Webber, Caleb, Li, Meng
DPMCN, Cardiff University, Cardiff, UK
iPSC-derived midbrain dopaminergic (mDA) neurons are an
invaluable tool for studying mDA development and neurode-
generative disorders as Parkinson’s disease (PD). However,
cellular heterogeneity is signicant confound in these cultures
and a human mDA lineage reporter line that enabled cell type
sorting would signicantly aid cell-type purity. LMX1A is a
transcription factor with a fundamental role in mDA develop-
ment and provides an early marker for this neuronal lineage.
We designed a strategy to generate an LMX1A Blue Fluores-
cent Protein (BFP) reporter line for tracking and purifying early
mDA progenitors and their neuronal derivatives. A silent BFP
expression unit is rstly targeted into the AAVS1 safe harbour
by CRISPR/Cas9 assisted homologous recombination in the
KOLF2 human iPSC line and then Cre will be knocked in to the
LMX1A locus of the derived AAVS1-BFP cells. A pilot study
carried out in HEK293 demonstrated strong BFP expression fol-
lowing co-transfection of AAVS1-BFP and a Cre expression vector.
We can expect strong expression of BFP in all LMX1A expressing
cells and their derivatives (ie. committed mDA neural progeni-
tors and their differentiated progeny) upon excision of the oxed
stop signal upstream of BFP by the LMX1A driven Cre in our
reporter cells. This reporter cell line will serve as a powerful tool
both for in vitro modelling of PD and associated risk genes, as
well as for developing PD cell therapies.
Funding Source
UK Dementia Research Institute.
Keywords: midbrain dopaminergic neurons; Parkinson’s dis-
ease; reporter line
071
CARDIOMYOCYTES FROM ISOGENIC HUMAN
IPSC LINES HARBORING MUTATIONS IN KCNH2
EXHIBIT PROLONGATIONS IN ACTION – AND
FIELD-POTENTIAL DURATION
Brandao, Karina1, van de Brink, Lettine1, Miller, Duncan1,
Martins Grandela, Catarina1, Mol, Mervyn1, Mummery,
Christine1, Sala, Luca2, Verkerk, Arie3, Davis, Richard1
1Anatomy and Embryology, Leiden University Medical Centre,
Leiden, Netherlands 2Center for Cardiac Arrhythmias of
Genetic Origin, Istituto Auxologico Italiano, Milan, Italy,
3Department of Experimental Cardiology, Academic Medical
Center, Amsterdam, Netherlands
Congenital long QT syndrome type 2 (LQT2) is one of the
most common genetic channelopathies in the heart leading
to life-threatening cardiac arrhythmias and sudden cardiac
death (SCD). A broad range of phenotypes are associated with
this cardiac channelopathy and nearly 500 variants identied
in the gene KCNH2 have been associated with this disease.
This phenotype variability impacts the management strategy
of patients as it complicates the task of identifying individuals
at risk. Patient-derived human induced pluripotent stem cells
(hiPSCs) can be used to investigate the pathogenicity of these
mutations, however interline variability complicates the use of
these models to reect genotype-phenotype relationships. To
overcome this issue, we have created isogenic hiPSC by intro-
ducing KCNH2 mutations into a well-established and validated
wild-type hiPSC line using Crispr/Cas9 technology. Preliminary
analysis of cardiomyocytes derived from these isogenic lines
show a prolongation of both action potential and eld potential
duration when compared to wild type. The response of these
hiPSC-CM lines to different pharmacological compounds is
currently being examined.
Funding Source
ERC – European Research Council; NWO – Netherlands
Organization for Scientic Research
Keywords: Long QT syndrome; hiPSC; disease model
60
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Poster Abstracts
072
STEM CELL DERIVED HUMAN BRAIN ORGANOIDS,
A PROMISING MODEL TO STUDY ENTEROVIRUS
INFECTION AND DISEASE PATHOGENESIS
Depla, Josse1, Sridhar, Adithya2, Evers, Melvin3, Wolthers,
Katja2, Pajkrt, Dasja4
1Experimental Virology, Academic Medical Center, Utrecht,
Netherlands, 2Medical Microbiology, University Medical Centre
Amsterdam, Amsterdam, Netherlands, 3Research, UniQure,
Amsterdam, Netherlands, 4Pediatric Infectious Diseases,
Amsterdam UMC, University of Amsterdam, Netherlands
Picornaviruses commonly cause mild disease but can lead
to severe CNS infection in children. Mainly due to lack of
suitable model systems, their pathogenesis is poorly
understood. New models to study picornavirus infection in
the brain are needed to understand the underlying
mechanisms. Brain organoids have increasingly proven to be
useful to study human specic disease. Brain organoids are
self-organizing 3D cultures derived from human stem cells
that recapitulate the organ microenvironment. These brain
organoids have the advantage of complex morphology over
transformed cell lines and the advantage of its human origin
over animal models. From induced pluripotent stem cells,
over the course of 40 days, brain organoids were formed.
Organoids were characterized by immunostaining. Beta
tubulin 3 (Tuj1) and paired box protein 6 (PAX6) were stained
to respectively visualize neurons and neural progenitors.
Besides the presence of neural cell types, the organoid mor-
phology was studied. After characterization, brain organoids
were transduced with an adeno associated virus expressing
GFP. In the future, the organoids will be infected with HPeV3,
EV71 and E68, members from picornavirus family known
to cause CNS infection. We can establish the iPSC derived
human brain organoid model. Organoids expressed GFP,
which allowed for tracking infection in brain organoids. We
are able to establish brain organoids to study viral infection
of the brain. This model will enable us to our knowledge on
picornavirus infection leading to CNS damage.
Keywords: Brain Organoids; Virology; Picorna viruses
073
BUILDING AN ORGANOID-BASED MODEL FOR
OVARIAN CANCER
Lohmussaar, Kadi, Kopper, Oded, Clevers, Hans C.
Hubrecht Institute, Utrecht, Netherlands
Over the last few years several studies led to a signicant
change in the eld of ovarian cancer and it is currently
believed that the fallopian tube and not the ovary surface
epithelium (OSE) is the main origin of high-grade serous
ovarian cancer (HG-SOC). Nevertheless, due to the lack of
unique markers and adequate model systems the relative
contribution of each tissue is not yet clear and the notion
that OSE has a role in HG-SOC development was not cast
aside altogether. In this work we have established novel
organoid systems derived from both mouse OSE and oviduct
(Ovi, the equivalent of human fallopian tube). These systems
recapitulate their tissue of origin and demonstrate differences
in medium requirements as well as gene expression. To
establish comparable tumor progression models for both
OSE and Ovi we used CRISPR-Cas9 technique and targeted
commonly mutated genes in ovarian cancer (Trp53, Brca1,
Nf1 and Pten). Thus, we were able to establish clones with
different combinations of mutations. Histological, metaphase
spread and gene expression analysis of the mutated
organoid clones from both OSE and Ovi demonstrated
different degrees of deviation from their wild type counter-
part. This deviation became more evident as the amount of
introduced mutations increased. Preliminary transplantation
experiments showed that Ovi triple mutants (Trp53, Brca1
and Pten or Trp53, Brca1 and Nf1) are able to give rise to
solid tumors. Further analysis will reveal what are the differ-
ences of ovarian tumors derived from distinct origins. Taken
together, in this study we present the rst comparable Ovi/
OSE research platform that enables addressing questions
related to origin and early stages of HG-SOC development.
Keywords: ovarian cancer modeling; organoids; CRIS-
PR-Cas9
074
CARDIAC FIBROSIS IN A DISH – A PLATFORM FOR
IDENTIFICATION OF ANTI-FIBROTIC COMPOUNDS
Palano, Giorgia1, Jansson, Märta2, Backmark, Anna3,
Sabirsh, Alan3, Müllers, Erik3, Åkerblad, Peter2, Hansson, Emil1
1Department of Medicine Huddinge, Karolinska Institutet,
Stockholm, Sweden, 2CVRM IMED Bioscience Heart Failure,
AstraZeneca, Mölndal, Sweden, 3CVMR IMED, AstraZeneca,
Mölndal, Sweden
Heart failure is a major public health problem, being described
as an epidemic with a worldwide prevalence of 23 million
affected individuals. A key feature in the pathogenesis of
heart failure is cardiac brosis, but currently treatments
specically targeting cardiac brosis are not available. This
is to a large extent due to the fact that informative in vitro
models permitting high-throughput screening of chemi-
cal libraries for anti-brotic effects are lacking. Here, we
describe a platform of cell-based assays aimed at identify-
ing compounds that interfere with extracellular deposition
of collagen from human cardiac broblasts. We provide
evidence that these assays correctly identify chemical
compounds with known anti-brotic effects, and further de-
scribe compound cultures, composed of human pluripotent
stem cell-derived cardiomyocytes and cardiac broblasts,
for further validation of results in a more complex organoid
system. Taken together, we envision that these assays will
provide a powerful tool for drug screening of anti-brotic
chemical compounds and disease modelling of cardiac
brosis.
Keywords: Cardiac brosis; Disease modelling; Organoid
culture system
61
AMSTERDAM NETHERLANDS
Poster Abstracts
075
DNAJB6, A KEY FACTOR IN NEURAL STEM CELL
RESISTANCE TO POLYGLUTAMINE PROTEIN
AGGREGATION
Thiruvalluvan, Arun1, de Mattos, Eduardo2, Brunsting,
Jeanette F.2, Bakels, Rob 2, Barazzuol, Lara2, Conforti, Paola3,
Fatima, Azra4, Cattaneo, Elena3, Vilchez, David4, Bergink,
Steven2, Boddeke, Erik2, Copray, Sjef2, Kampinga, Harm H.2
1European Research Institute for the Biology of Aging,
University Medical Center Groningen, Netherlands, 2Department
of Biomedical Sciences of Cells and Systems, University
Medical Center Groningen, Netherlands, 3Department of
Biosciences, University of Milan, Italy, 4Cologne Excellence
Cluster for Cellular Stress Responses in Aging-Associated
Diseases , University of Cologne, Germany
Spinocerebellar ataxia type 3 (SCA3) is a neurodegener-
ative disorder caused by the expansion of polyglutamine
(polyQ)-encoding CAG-repeats in the ATXN3 gene. The
CAG-repeat length is proportionally related to the aggre-
gation propensity of the ataxin-3polyQ protein. Although the
protein is ubiquitously expressed, it only causes toxicity to
neurons. To better understand this neuronal hypersensitivity,
we generated iPSC-lines from three SCA3 patients. iPSC
generation and neuronal differentiation is unaffected by the
expression of the ataxin-3polyQ. No spontaneous aggregate
formation is observed in the SCA3 neurons. However, upon
glutamate treatment, aggregates form in SCA3 neurons but
not in SCA3-derived iPSCs or iPSC-derived neural stem cells
(NSCs). Analysis of chaperone proteins expression reveals
a drastic reorganization of the chaperone network during
differentiation, including an almost complete loss of
expression of the anti-amyloidogenic chaperone DNAJB6 in
neurons. Knockdown of DNAJB6 in iPSC and NSC derived
from patients leads to spontaneous aggregation of the
polyQ proteins. Moreover, DNAJB6-knockout cells are
hypersensitive to polyQ aggregation, which is prevented by
DNAJB6 re-expression. Our data show that downregulation
of DNAJB6, which occurs upon neuronal differentiation, is
directly linked to neuronal toxicity of polyQ aggregation.
Keywords: IPSCs; HTT and SCA3; Protein aggregation
076
MODELLING GENETICALLY COMPLEX DISEASES
USING IPSCS
Hoekstra, Stephanie D.1, Watanabe, Kyoko2, Heine, Vivi3,
Posthuma, Danielle2
1Complex Trait Genetics, Amsterdam UMC, Amsterdam,
Netherlands, 2Complex Trait Genetics, Vrije Universiteit
Amsterdam Netherlands, 3Emma Children’s Hospital,
Amsterdam UMC, Amsterdam, Netherlands
The discovery of induced pluripotent stem cells (iPSCs)
has provided researchers with a new tool to study
neurodevelopmental disorders in previously sparsely
accessible human cells, such as neurons. These iPSCs
are of particular interest for studies concerning genetically
complex diseases with a strong genetic component, such as
schizophrenia (SCZ) and autism. Since iPSCs are genetically
identical to the donor, the need for gene editing is eliminated
and the complex genetic architecture such diseases is
recapitulated. Past research has focused on the role of
neurons in the pathology of SCZ. However, recent evidence
shows an important glial component in this disease of both
astrocytes and oligodendrocytes. We would like to investigate
the impact of such glial components on neuronal functioning.
In the current research we rst investigated how we could
obtain the best statistical power to investigate this glial
component in schizophrenia. Schizophrenia is a genetically
complex disorder, involving single nucleotide polymorphisms
(SNPs) and copy number variations (CNVs), which makes
this disease very heterogeneous. This heterogeneity in turn
creates variability, lowering the statistical power of iPSC
studies because participants will have varying causal variants
and varying genetic backgrounds. In order to designs a study
that has enough power and allows us to claim causality, we
have strategically selected patients using genetic information.
We based our selection on polygenic risk scores (SNPs) and
the penetrance of CNVs. Furthermore, considering the
limitations in sample sizes, we have validated our model
in order to determine the best research pool using iPSCs:
should we use multiple clones from a smaller number of
individuals or should we use one clone and opt for a large
number of individuals? In order to answer this question we
performed parallel differentiation of nine clones from three
individuals (3 clones each) in order to determine the
variability at different level.
Funding Source
This work was funded by The Netherlands Organization
for Scientic Research (NWO VICI 453-14-005).
Keywords: schizophrenia; variability; modelling
077
A 3D HUMAN INDUCED PLURIPOTENT STEM
CELL-DERIVED NEURAL CULTURE MODEL TO
STUDY CELLULAR AND BIOCHEMICAL ASPECTS
OF ALZHEIMER’S DISEASE IN VITRO
Preeti Prasannan1, Siney, Elodie2, Chaterjee, Shreyasi3,
Kurbatskaya, Ksenia3, Caballero, Mariana V.3, Mudher,
Amrit3, Morawek, Sandrine W.1
1Faculty of Medicine, University of Southampton, UK, 2Cancer
Sciences, University of Southampton, UK, 3Biological
Sciences, University of Southampton, UK
Alzheimer’s disease (AD) is the most prevalent form of
dementia with no cure yet. The pathophysiology is charac-
terized by accumulation of Abeta oligomers forming plaques
and hyperphosphorylation of Tau forming tangles. The major
advantage of using Induced pluripotent stem cell (iPSC) is
that mature neurons could be generated in vitro from AD pa-
tients with inherited mutations thereby allowing patient-spe-
cic insight into biochemical and pathophysiological nature
of AD. 3D culture models provide a physiologically relevant
spatial microenvironment aiding better differentiation and
maturation of cells in vitro. In this study, we aim to develop a
3D neural model derived from AD patients and control iPSC
62
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Poster Abstracts
wherein cells can differentiate, self-organize and mature
to replicate the pathophysiological mechanisms of AD. For
this study, neural progenitor cells derived from AD-iPSCs
with Presenilin1 mutation (L286V, M146L, and A246E) and
age-matched controls were differentiated in matrigel for 18
weeks in vitro. Characterization of cell morphology, protein
prole and synaptic functions were performed using im-
munouorescence and electrophysiology studies. Western
blotting was used to determine tau isoform expression and
disease associated changes. In this study, we found iP-
SCs-derived NPCs differentiate into neurons and astrocytes,
which self-organize into 3D structures by 3 weeks of differ-
entiation in vitro. These heterogeneous 3D cultures express
astrocytic (GFAP), neuronal (beta-3-tubulin, MAP2), pre-syn-
aptic (Synapsin 1) and glutamatergic neuronal (VGLUT1)
markers after differentiation. The foetal 3Rtau and adult
4Rtau isoforms were detected at 6 and 18 weeks respective-
ly. There is evidence of action potential in these cultures at
12 weeks. At 6 weeks, 3D cultures express Abeta oligomers
as well as hyperphosphorylated tau (PHF1) in AD-iPSC
derived 3D cultures. In conclusion, we have developed an in
vitro human 3D iPSCs derived neural model with mature neu-
rons. In the AD-derived cells, we have also shown presence
of hyperphosphorylated 4R tau and Abeta oligomers. This
model recapitulates the early disease biochemical features
and can be a relevant platform for studying early cellular and
biochemical changes for identication of drug targets.
Funding Source
ARUK south coast network to SWM, Commonwealth
Scholarship to PP, Marie Curie to SC.
Keywords: iPSCs; 3D cultures; Alzheimer’s disease
078
INVESTIGATING THE CELLULAR ORIGIN
OF GLIOBLASTOMA USING DROSOPHILA
MELANOGASTER
Hakes, Anna E., Brand, Andrea H.
Gurdon Institute, University of Cambridge, UK
Glioblastoma is the most common malignant glioma in
adults. Patient survival has not improved since the 1980s
and is limited to approximately one year. Developing effective
diagnostic tools and treatments for glioblastoma is reliant
upon understanding the early molecular events of tumour
initiation. Central to this quest is determining the cell of
origin for distinct tumour types. Differentiated glial cells are
one possible cell of origin of glioblastomas, but the
identication of adult neural stem cells (NSCs) with glial
identity also implicated NSC lineages as alternative
potential cells of origin. Studies in mice have shown that
neural lineages can give rise to glioblastomas but it is not
known from which cells tumours arise. Drosophila
melanogaster has proved a useful model for investigating
the diverse molecular mechanisms that underlie
tumourigenesis in vivo. Importantly, 60 % of genes associated
with human cancers are found in the Drosophila genome,
many with extremely high conservation. Current Drosophila
glioblastoma models have focussed on the glial cell of origin
only and have not considered NSCs as a potential route to
tumourigenesis. We are investigating the role of NSC lineages
in glioblastoma initiation. We have analysed the effect of
expressing oncogenes associated with glioblastoma in
Drosophila NSC lineages. To identify the tumour cell of
origin, we restricted expression to specic cells within NSC
lineages and assessed tumourigenic transformation.
Strikingly, we found that the Drosophila and human
counterparts of glioblastoma genes had highly comparable
tumourigenic capacities in NSC lineages and appear to act
through conserved mechanisms. We used Targeted DamID
to map the genome-wide binding sites of transcriptional
regulators involved in tumour initiation in NSCs in vivo to
determine their direct target genes and downstream effectors.
Our results show that tumours resulting from expression of
Drosophila or human genes have common cellular origins
and the molecular pathways involved are consistent with
mammalian studies.
Keywords: Tumourigenesis; Neural stem cell; Brain develop-
ment
079
DISEASE MODELING OF ARRHYTHMOGENIC
RIGHT VENTRICULAR CARDIOMYOPATHY IN
HUMAN INDUCED PLURIPOTENT STEM CELLS
Meier, Anna1, Meier, Anna1, Kornherr, Jessica1,
Dorn, Tatjana1, Parrotta, Elvira2, Zawada, Dorota1, Moretti,
Alessandra1, Laugwitz, Karl-Ludwig1
1Klinikum rechts der Isar, Technical University of Munich,
Germany, 2Department of Experimental and Clinical Medicine,
University “Magna Graecia” of Catanzaro, Italy
Developmental commitment of cells and tissues is governed
by combinations of lineage-specic transcription factors
that act as master switch genes dening and reinforcing
cell type-specic gene expression patterns. While it is
widely accepted that mechanical cues contribute to cell fate
decisions during differentiation of stem cells, little is known
about how mechanical inputs at cell–cell or cell–matrix
adhesions translate into intracellular signals to maintain cell
identity or direct fate switch. Using an induced pluripotent
stem cell (iPSC) model of arrhythmogenic right ventricular
cardiomyopathy (ARVC), we show that in human developing
cardiomyocytes, cell-cell contacts at the intercalated disk
connect to remodeling of the actin cytoskeleton by regulating
the RhoA-ROCK signaling to maintain an active MRTF/SRF
transcriptional program essential for cardiomyocyte identity.
Genetic perturbation of this mechanosensory pathway in
ARVC leads to progressive loss of myocytic identity and
acquisition of an adipocyte phenotype in response to adi-
pogenesis-inducing signals. We also demonstrate by clonal
analysis of cardiac progenitors that cardiac fat and a subset
of cardiac muscle arise from a common precursor expressing
Isl1 and Wt1 during heart development, suggesting related
mechanisms of determination between the two lineages.
Keywords: ARVC; Cardiac progenitors; Lineage conversion
63
AMSTERDAM NETHERLANDS
Poster Abstracts
healthy controls, the MAOA mutant neurons showed higher
neuronal activity, especially at the network level. And the
increased neuronal network activity could be attenuated by
correcting the mutation of MAOA in mutant neurons. No sig-
nicant difference in terms of synaptic strength and synapse
density were found at the single neuron level. Morphological-
ly, the neurons from one of the MAOA mutant lines had a sig-
nicantly more complex morphology than the controls, but
there seems to be heterogeneity of the consequences of the
different mutations in terms of morphology. At the molecu-
lar levels, rst results suggest differences regarding to the
NMDA but not AMPA receptors of the neurons (suggesting
alterations in glutamatergic signalling), but these need to be
further investigated in detail. Our ndings provide a human
cellular model for investigating pathogenic mechanisms un-
derlying BS and identifying the potential therapeutic targets.
Keywords: iPSC Model; Brunner syndrome; CRISPR-cas9
082
DISEASE MODELING OF CARPENTER SYNDROME
IN HUMAN IPSC AND RAB23 MOUSE MUTANTS
REVEALS CILIOPATHY
Hor, Catherine H. H, Leong, Wan Ying, Goh, Eyleen Lay Keow
Neuroscience Academic Clinical Program, Duke-NUS Medical
School, Singapore
The non-motile primary cilium is an organelle found on the
surface of nearly every cell. It serves as a signaling hub
to transduce extracellular growth signals such as Sonic
Hedgehog (Shh), Wnt and several GPCR signaling pathways.
Defective cilia lead to hereditary disorders collectively called
ciliopathy, which is an expanding spectrum of at least 35
diseases involving multiple genes mutations. Resolving the
intricate regulatory networks for cilia biogenesis and signaling
is critical for the understanding of disease pathogenesis.
Rab23 belongs to the family of small GTPases, which genetic
loss-of-function mutations underlie Carpenter syndrome
(CS). CS patients exhibit profound clinical features that
overlap with other ciliopathies. This suggests that RAB23
mutation may cause dysfunctional primary cilia, and
consequently contributes to the pathogenesis of CS. Given
this hypothesis, we aim to elucidate the disease mechanisms
of CS, and its potential relationship with ciliopathy disorder.
A human disease model of CS has been established from
CS patient-derived induced-pluripotent stem cells (iPSC).
In line with our hypothesis, patient iPSCs-derived neurons
showed reduced ciliation, strongly suggesting the clinical
relationship between CS and ciliopathies. Moreover, mouse
models of Carpenter syndrome were established by global
deletion and conditionally knock-out Rab23 in the central
nervous system (CNS) using Actin-cre (Actin-CKO) and
Nestin-cre (Nes-CKO) driver lines respectively. Indeed, our
data show that Rab23-null and brain-specic knockout
mouse mutants of Rab23 exhibited perturbed cilia formation
and neurological impairments bearing some similarities with
CS and ciliopathies. This is the rst in vivo evidence revealing
novel roles of Rab23 in ciliogenesis and neurophysiology.
Taken together, our data uncover novel in vivo function of
Rab23 in ciliogenesis. This suggest RAB23 as a causative
080
COMMON DEVELOPMENTAL ORIGIN OF
CARDIOMYOCYTES AND CARDIAC FAT
Kornherr, Jessica, Meier, Anna, Dorn, Tatjana, Laugwitz,
Karl-Ludwig, Moretti, Alessandra
Regenerative Medicine in Cardiovascular Diseases, Klinikum
rechts der Isar, Technical University of Munich, Germany
Arrhythmogenic right ventricular cardiomyopathy (ARVC) is
a disease characterized by bro-fatty replacement of car-
diomyocytes predominantly in the right ventricle. Mutations
in genes encoding desmosomal proteins account for the ma-
jority of ARVC cases. To date, it remains still not completely
understood which cell type and which molecular mechanisms
are responsible for the bro-adipose substitution. Using
induced pluripotent stem cells from ARVC patients our group
could recently show that a spontaneous conversion of
a subset of cardiomyocytes into fat is the underlying
pathomechanism of the disease and uncover the signaling
linking cell-cell contacts to changes of transcriptional
programs driving this pathological cell fate switch. Based on
these ndings we hypothesized that myocyte-to-adipocyte
transdifferentiation could occur if both lineages are closely
developmentally related. Indeed, by means of in vivo fate
mapping we identied a common Isl1+/Wt1+ expressing
precursor giving rise to cardiac muscle and cardiac fat.
Furthermore, we demonstrated that a single Isl1+/Wt1+
progenitor can differentiate into both cardiomyocytes and
adipocytes in vitro, which ultimately suggest related
mechanisms of determination between the two lineages.
Keywords: ARVC; Cardiac Progenitors; Lineage Conversion
081
AGGRESSION IN A DISH: A HUMAN MODEL FOR
BRUNNER SYNDROME REVEALS INCREASED NEU-
RONAL NETWORK ACTIVITY OF DOPAMINERGIC
NEURONS
Shi, Yan1, Van Rhijn, Jon-Ruben2, Bomann, Maren2, Mossink,
Britt1, Frega, Monica1, Schubert, Dirk2, Brunner, Han1, Franke,
Barbara1, Nadif Kasri, Nael1
1Human Genetics, Radbound University, Nijmegen, Netherlands,
2Cognitive Neuroscience, Radboud UMC, Nijmegen, Netherlands
Brunner syndrome (BS) is a very rare genetic psychiatric
disorder caused by mutations of MAOA at X-chromosome and
featured as mild mental retardation and impulsive aggression
in the male patients. MAOA encodes monoamine oxidase A
which is an important enzyme to catalyze the degradation
of dopamine, noradrenaline and serotonin that are neu-
rotransmitters in the brain modulating movement, emotion
and cognitive functions. However, how the MAOA mutation
contribute to the abnormal behavior of BS patients is largely
unknown. Using the induced pluripotent stem cell (iPSC)-de-
rived dopaminergic neurons from three BS patients and two
unaffected independent controls along with the isogenic
lines generated by CRISPR-Cas9, we investigated how the
MAOA mutation affected the neuronal electrophysiology,
morphology, and the expression prole of neurotransmitter
receptors. Compared to the neurons differentiated from
64
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Poster Abstracts
gene for ciliopathy, further indicating Carpenter syndrome
as a ciliopathy disorder.
Funding Source
This project is supported by National Medical Research
Council, Ministry of Health, Singapore. Young Individual
Research Grant (OFYIRG17nov019).
Keywords: Carpenter Syndrome; Primary cilia; Central Ner-
vous System Development
083
EFFECT OF MESENCHYMAL STEM CELLS THERAPY
ON THE LEVELS OF SERUM BIOMARKERS IN A RAT
MODEL OF OSTEOARTHRITIS
Noorwali, Abdulwahab A.1, AlGhamdi, Amani1, Sattami, Noora1,
Bashah, Taghreed1, Kalamegam, Gauthaman2
1Stem Cells Unit, King Fahd Medical Research Center, King
Abdulaziz University, Jeddah, Saudi Arabia, 2Stem Cell Unit,
Center of Excellence in Genomic Medicine Research, King
Abdulaziz University, Jeddah, Saudi Arabia
Osteoarthritis (OA) is a chronic disease of the joints
characterized by progressive degeneration of connective
tissues. OA treatment are mainly aimed to alleviate pain and
limitations in movement. Mesenchymal stem cells (MSCs)
therapy appear to have a great therapeutic potential, given
their differentiation potential and immunomodulatory
properties. The present study evaluated the effects of
intra-articular injection of BM-MSCs in a rat model of knee
OA and its relation to serum biomarkers. Ethical approval
was obtained from the Bioethics Committee of King
Abdulaziz University for this study. R-BM-MSCs were derived,
established in culture and characterized. Sixty Sprague
Dawley rats were divided into four groups (n=15/group) as
follows: Group I -normal saline controls, Group II – Normal
rats that were given MSCs alone; Group III- Knee OA with
no treatment; and Group IV- Knee OA + MSCs (1x106 cells
in 25ul PBS). Knee OA was induced by injection of sodium-
monoiodo acetate (MIA, 2mg in 25ul vehicle) into the femo-
rotibial joint space of the left hind limb. Five rats from each
group were sacriced at 10, 20 and 30 days for histological
assessment following rBM-MSCs therapy and evaluation
of the serum biomarkers namely, hyaluronic acid (HA),
N-terminal telopeptide of type I collagen (NTX-1), nerve
growth factor (NGF) and calcitonin gene-related peptide
(CGRP) by ELISA. FACS analysis showed that the rBM-MSCs
were positive for MSC related CD markers. Histological
assessment showed near normal restoration of cartilage
structure in the BM-MSC treated group compared to the
normal. All serum biomarkers analyzed showed signicantly
decreased levels in the rBM-MSC treated group compared
to the untreated controls. The mean levels of HA were 4.66,
7.49, 37.37, 15.81 ng/ml; NTX-1 were 31.56, 40.89, 90.78,
64.97 ng/ml; NGF were 568.54, 32.81, 1644.13, 1351.68
pg/ml and CGRP were 106.26, 124.36, 256.34, 189.92 pg/ml
for the Groups I-IV respectively. In conclusion, treatment of
induced knee OA in rats with rBM-MSCs helped in structural
recovery of the damaged cartilage and the levels of the
biomarkers correlate with the disease status. Therefore,
these biomarkers can be useful to determine the burden of
the disease and/or the disease prognosis.
Keywords: Osteoarthritis; in vivo; Biomarkers;
084
MODELING HEREDITARY RETINAL DYSTROPIES BY
PATIENT SPECIFIC IPSC-3D RETINAL ORGANOIDS
Lukovic, Dunja1, Artero Castro, Ana1, Cortón, Marta3,
Cuenca, Nicolas4, Ayuso, Carmen3, Erceg, Slaven2
1National Stem Cell Bank, Centro de Investigación Príncipe
Felipe, Valencia, Spain, 2Department of Genetics and
Genomics, Fundacion Jiménez Díaz, Madrid, Spain,
3Department of Physiology, Genetics and Microbiology,
University of Alicant, Spain,
Hereditary retinal dystrophies (HRD) are clinically diverse
group of disorders which are characterized by progressive
degeneration of light sensing cells, photoreceptors. Here
have set out to develop unique human model of HRD by
generating retinal organoids via patient-specic iPSCs.
These native retina mimics display inner and outer retinal
structure with the presence of correctly layered major retinal
cell type repertoire including photoreceptor cells, featuring
highly differentiated rods and cones. We evaluated the
differentiation of healthy and disease 3D organoids by
immunouorescence detection of molecules associated with
differentiation and function of retinal cell types. Differentiation
and maturation was further evaluated at the ultrastructural
level by transmission electron microscopy. We show that
this patient specic disease model recapitulates molecular
aspects of the disease phenotype.
Keywords: Inhereted retinal dystrophies; Retinal organoids;
Patients’ iPSCs
085
DISEASE MODELING IN A DISH USING HUMAN
INDUCED PLURIPOTENT STEM CELLS (HIPSCS):
PROCESSES AND CONSIDERATIONS FOR SCALE UP
AND QUALITY CONTROL
Ng, Carrie1, Jesseman, Kimberly M.1, Graef, John D.1,
Wallace, Owen B.2
1Cell Biology, Fulcrum Therapeutics, Cambridge, MA, USA,
2Research and Development, Fulcrum Therapeutics,
Cambridge, MA, USA
Technological advancements in human iPSC cell culture
and reprogramming methods have resulted in widespread
adoption of disease modeling in a dish using iPSCs and their
derivatives. By using patient-derived, disease-specic cells,
iPSC models have the potential to recapitulate important
aspects of disease states that present in vivo. Although iPSC
models have led to many breakthroughs, current processes
and methodologies lack consistent metrics of quality control
and reproducibility. Here we describe a process to generate
iPSCs at high throughput without compromising
differentiation capabilities or inherent pluripotency. We
use several genetically distinct iPSC lines to highlight the
importance of monitoring genetic drift and karyotype
instability, genetic identity, and pluripotency.
Keywords: disease modeling; quality control; human pluripo-
tent stem cell
65
AMSTERDAM NETHERLANDS
Poster Abstracts
086
ATRX SPECIFIES STEM CELL IDENTITY AND
NEUROGENIC POTENTIAL IN HUMAN NEURAL
STEM CELLS
Sanosaka, Tsukasa Tomooka, Ryo Chai, MuhChyi Andoh,
Tomoko Okano, Hideyuki Kohyama, Jun
Department of Physiology, Keio University School of
Medicine, Shinjuku-ku, Japan
The prevalence of human diseases caused by mutations in
chromatin remodeling genes underpins the importance of
chromatin structure in gene regulation. One such disease,
Alpha-thalassemia X-linked intellectual disability (ATR-X)
syndrome, is caused by mutations of ATRX gene. This
syndrome is characterized by mental retardation with
severe developmental delay, craniofacial and urogenital
abnormalities, as well as mild anemia. Previous studies have
reported the role of ATRX in α-thalassaemia and cancer;
however, the function of ATRX during human brain develop-
ment remains unknown. In addition, multiple organ defects
observed in ATR-X syndrome patients, and the identication
of ATRX mutation as the sole genetic cause of ATR-X syn-
drome has raised the question of how disruption of single
factor can lead to multiple phenotypes. In the present study,
we found that ATRX regulates stemness and neurogenic
property of human neuroepithelial (NE) cells. Dysregulation
of ATRX results in the loss of NE identity and the aberrant
acquisition of neural crest properties. We also generated
chromatin landscapes of ATRX-bound genomic regions
and found that ATRX displayed correlation with repressive
heterochromatin (H3K9me3), as reported previously. In
addition to its well-established repressive role, we found
that a small but signicant fraction of ATRX-bound regions
was associated with active histone marks. Such an associ-
ation was undetectable in other somatic cells, indicating the
dual function of ATRX is exclusive to neural lineage. Thus, the
function of ATRX as a transcription activator in the regulation
of cell identity during human brain development certainly
warrant further study.
Keywords: ATRX syndrome; chromatin remodeling;
epigenetics
087
GENERATION OF MICE CARRYING HUMAN
NEURONS BY EMBRYONIC INJECTIONS TO MODEL
HUMAN NEUROLOGICAL DISORDERS
Krzisch, Marine A.1, Wu, Hao2, Fu, Dongdong1, Omer, Attya1,
Wert, Katherine J.3, Ma, Haiting1, Jaenisch, Rudolf1
1Whitehead Institute for Biomedical Research, Cambridge,
MA, USA, 2Fulcrum Therapeutics, Cambridge, MA, USA,
3Ophtalmology, Stanford University, Stanford, CA, USA
Animal models are limited for understanding human neuro-
logical disorders. Modeling neurological disease with cultured
human induced pluripotent stem cells (hiPSC) presents
several biases, including high inter-batch variability and the
fact that cell culture conditions do not recapitulate an in vivo
environment. We made the hypothesis that placing neurons
derived from hiPSC in in vivo conditions, i.e. culturing them in a
living organism, might represent a model closer to physiolog-
ical conditions and allow to study neuronal properties such as
neuronal morphology that are hard to study in in vitro models.
Published studies and our results show that transplantation of
human neural precursor cells (hNPCs) in the neonatal mouse
brain leads to the migration of the cells away from the injection
site into different brain regions of the brain. These cells differ-
entiate into several neural cell types including neurons. Howev-
er, neural progenitor cells mostly differentiate into excitatory
neurons and fail to generate a signicant amount of inhibitory
neurons. Also, in our hands, the survival of neurons generated
via neonatal injections decreases over time, limiting analysis at
later timepoints. Here, we hypothesized that transplanting hNPC
prenatally, within the time window of neurogenesis, at embry-
onic day 13.5, might provide more adequate cues for the devel-
opment and integration of neurons in the mouse brain circuitry,
and lead to more robust integration of the neurons and increased
survival. Our data suggests that embryonic engraftment increas-
es neuronal integration into the network compared to neonatal
engraftment and represents a viable in vivo approach to model
human neurological disorders.
Keywords: human-mouse chimera; neuron; neurological
disorders
088
PHENOTYPIC FUNCTIONAL IN VITRO SCREENING OF
PATIENT IPSC-DERIVED MOTOR NEURONS USED FOR
IN VITRO HTS DISEASE MODELING WITH AI-BASED
ANALYSIS OF MICRO ELECTRODE ARRAY DATA
Schroeder, Olaf1, Segura-Castell, Monica1, Juegelt, Konstantin1,
Hendrickson, Michael1, Schultz, Luise1, Bader, Benjamin1
1NeuroProof GmbH, Rostock, Germany, 2BrainXell, Inc.,
Madison, WI, USA,
Patient-derived iPSC models have been designed for various
indications promising higher physiological relevance and
thus, better translation to the in vivo situation. Their application
eventually may decrease attrition rates in drug discovery and
development. We focused on investigating motor neuron dis-
eases (MND) such as amyotrophic lateral sclerosis (ALS) and
spinal muscular atrophy (SMA), both causing loss of motor
neurons and associated symptoms. Here, we phenotypically
describe the consequence of the genetic variation present in
ALS and SMA patient iPSC-derived motor neurons on the func-
tional activity and network connectivity. We further elucidated
how functional ALS and SMA phenotypes separated from
controls during network establishment to enable compound
testing to rescue the disease phenotypes. We cultured patient
iPSC-derived motor neurons (BrainXell) and controls on multi-
well micro-electrode arrays (MEA, Axion Biosystems) for several
weeks to analyze their functional network activity patterns by
multi-parametric analysis (NeuroProof). Our results showed
reproducible spontaneously active motor neuron networks with
synchronized activity. We identied disease-specic functional
phenotypes and showed how reference compounds can affect
them. In conclusion, we show that hiPSC-derived motor neurons
are able to produce functional in vitro phenotypes which can
be associated with known motor neuron diseases. By using
articial intelligence-based multivariate MEA data analyses
combined with reproducible physiologically relevant iPSC neuron
models we provide a functional phenotypic assay platform for
high throughput compound screening.
Keywords: In Vitro Screening; iPSC-Derived Neurons; motor
neuron disease
66
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Poster Abstracts
089
GENERATION OF HUMAN INDUCED PLURIPOTENT
STEM CELL-DERIVED CORTICAL ASTROCYTES IN
FRAGILE X SYNDROME
Peteri, Ulla-Kaisa1, Roybon, Laurent2, Castren, Maija L.1
1Faculty of Medicine, University of Helsinki, Finland,
2Department of Experimental Medical Science, Lund
University, Lund, Sweden
Fragile X syndrome (FXS) is the most common form of
familial intellectual disability and a variant of autism. The
syndrome is caused by the lack of functional fragile X mental
retardation protein (FMRP) due to epigenetic silencing of the
Fmr1 gene. FMRP is crucial for the early neural development
and its absence leads to changes in the synaptic plasticity and
imbalance of excitatory and inhibitory networks. Recently, an
increasing number of studies have shown that astrocytes
also play a role in the FXS pathology. In this study, we
generated frontal cortex astrocytes from induced pluripotent
stem (iPS) cells to model FXS in human astrocytes. The
induction of neural differentiation was accomplished using
dual SMAD inhibition combined with Dickkopf-Related
Protein 1- and cyclopamine treatments to inhibit hindbrain
and midbrain patterning, respectively. Generated progenitors
were directed towards astrocyte lineage with ciliary neuro-
trophic factor. During the differentiation, the cells were
monitored for the expression of neuronal progenitor and
astrocyte markers. Generated astrocytes were positive for
the selected astrocyte markers and were responsive to
environmental stimuli. The differentiation protocol, therefore,
allows the generation of functional patient-derived astrocyte
cultures that can be used to study the mechanisms
underlying impaired astrocyte function in FXS.
Funding Source
The Arvo and Lea Ylppö Foundation and the Academy of
Finland
Keywords: Neurodevelopmental disease; astrocyte;
induced pluripotent stem cells
090
THE CAUDATE NUCLEUS IN SCHIZOPHRENIA,
FROM COMPUTATIONAL FINDINGS TO POSTMOR-
TEM BRAIN TISSUE AND IN VITRO FUNCTION
van de Leemput, Joyce1, Sawada, Tomoyo1,
Benjamin, Kynon J.M1, Jaffe, Andrew E.2, Shin, Joo Heon1,
Consortium, BrainSEQ1, Hyde, Thomas M.1, Kleinman, Joel E.1,
Weinberger, Daniel R.1, Paquola, Apua1, Erwin, Jennifer A.1
1Johns Hopkins School of Medicine, Lieber Institute for Brain
Development, Baltimore, MD, USA, 2Johns Hopkins School of
Medicine; Johns Hopkins Bloomberg School of Public Health,
Lieber Institute for Brain Development, Baltimore, MD, USA
Schizophrenia (SZ) is a devastating neuropsychiatric
disorder unique to humans that affects ~1% of our
population. The disorder is characterized by distortions in
thinking, perception, emotion, and behavior. Decades of
research have offered many insights, including evidence for a
strong genetic component, but we have yet to identify the
disease causing molecular pathways. Advancing human
genetic ndings to reveal the mechanisms of SZ pathogenesis
can identify potential therapeutic targets. A recent ge-
nome-wide association study (GWAS), which tests for
associations between common genetic variants and traits,
identied 108 loci with modest contribution to SZ risk;
however, the association reveals little about the role of these
loci in SZ. Gene expression studies to elucidate the function
of GWAS risk alleles have focused on cortical areas, even
though other regions have been implicated. For example,
dopamine was the rst neurotransmitter implicated in SZ,
and dopaminergic receptors in the caudate nucleus are the
primary targets of antipsychotic drugs. Therefore, we carried
out a comprehensive analysis of the genetic and transcription
landscape of SZ in postmortem caudate nucleus samples
from the Lieber Institute for Brain Development (LIBD) brain
repository for neuropsychiatric disorders; the largest of its
kind. The BrainSEQ™ Consortium led by LIBD has used the
repository to generate and analyze vast amounts of genomic
data including RNA-seq data from SZ and health controls for
dorsolateral prefrontal cortex (N = 500), hippocampus
(N = 450), and caudate nucleus (N = 438) - and genotypes.
We identied expression quantitative trait loci (eQTL) in
caudate nucleus and differentially expressed genes in
schizophrenia. We nd strong evidence that the caudate
nucleus is a primary site of SZ pathogenesis. Within the
108 loci risk SNPs, 6 of 6 gene-SNP pairs have concordant
expression changes in SZ and a heritable genic risk that
alters expression in the same direction. These genes are likely
involved in SZ pathogenesis. Therefore, we are currently
investigating RNA and protein levels of these genes in
postmortem brain tissue, as well as, by studying the func-
tional implications of their altered expression in cerebral and
striatal organoids from dura-derived human induced
pluripotent stem cells (iPSCs).
Funding Source
Funding for this project supported by the Lieber Institute for
Brain Development, the BrainSEQ™ Consortium, and NAR-
SAD Young Investigator Grant from the Brain & Behavior
Research Foundation (JAE).
Keywords: schizophrenia; caudate nucleus; bio-computation
67
AMSTERDAM NETHERLANDS
Poster Abstracts
091
MODELLING HUMAN NEPHROGENESIS USING
HUMAN PSC-DERIVED PATTERNABLE AND
FUNCTIONAL KIDNEY ORGANOIDS
Low, Jian Hui1, Li, Pin2, Zhang, Tian2, Zheng, Nan2, Izpisua
Belmonte, Juan Carlos3, Xia, Yun2
1Nanyang Technological University, Lee Kong Chian School
of Medicine, Singapore, Singapore, 2Lee Kong Chian School of
Medicine, Nanyang Technological University, Singapore,
Singapore, 3Gene Expression Laboratory, The Salk Institute
for Biological Studies, La Jolla, CA, USA
The incidence of end-stage renal disease (ESRD) is increasing
at an alarming rate, but treatment options remain unchanged
over the past 70 years. Directed differentiation of human
pluripotent stem cells (hPSCs) into kidney organoids offers
an unprecedented opportunity to study human kidney
development and pathogenesis, for performing drug
screening, and ultimately to generate functional kidney
tissue for replacement therapy. Despite recent advances in
generating kidney organoids form hPSCs, the ability to
modulate various cellular components within kidney organoids
has not yet been illustrated. Here, we report the establishment
of a highly efcient protocol for differentiating hPSCs into
kidney organoids comprised of segmented nephron
epithelium, vascular endothelium and interstitium. Through
stringent temporal modulation of canonical WNT-signalling
pathway, we could preferentially modulate the choice of
proximal versus distal lineage, as well as the vascular
compartment in the kidney organoids. Within hPSC-derived
kidney organoids, we identied that KDR+ vascular progenitors
originated from a subpopulation of SIX2+SALL1+ cells that
further differentiate and mature into CD31+ endothelial cells
in response to VEGF-A secreted by podocytes. Following
renal capsule implantation into an immunodecient host
mouse, kidney organoids acquired signicant structural
maturation, as represented by the formation of Bowman’s
capsule space surrounding glomerular capillary turfs of a
human origin. The implanted kidney organoids exhibited
size-selective dextran ltration and reabsorption; and the
accumulation of putative ltrate within tubules, demonstrating
functional maturation. Our work represents an advanced
version of kidney organoids that promise to offer extensive
utility in both basic science and clinical practice.
Funding Source
Y.X. is supported by Nanyang Assistant Professorship Grant,
LKCMed-NTU; Ministry of Education grant (2017-T1-001-
086); National Medical Research Council grant (NMRC/
OFIRG/0076/2018) and NTU internal grant (JNT-14/2016-CG)
.
Keywords: Kidney organoid; Patterning; Pluripotent stem
cells
092
CHARACTERIZATION AND TRANSPLANTATION
OF CD73-POSITIVE PHOTORECEPTORS ISOLATED
FROM HUMAN IPSC-DERIVED RETINAL ORGAN-
OIDS
Gagliardi, Giuliana1, Ben M’Barek, Karim2, Slembrouck-Brec,
Amelie1, Nanteau, Celine1, Desbrousses, Celine3, Liu, Jing3,
Ottaviani, Daniela3, Reichman, Sacha1, Goureau, Olivier1
1Institut de la Vision, Sorbonne Universite, INSERM, CNRS,
Institut de la Vision, Paris, France, 2Institute for Stem Cell
Therapy and Exploration of Monogenic Diseases, INSERM
U861, UEVE, CECS, AFM, Corbeil-Essonnes, France, 3UMR144
CNRS, Institut Curie, Paris, France
Photoreceptor degenerative diseases are a major cause of
blindness, for which there are currently no effective
treatments. For stem cell-based therapy using human
induced pluripotent stem cells (hiPSCs) is crucial to obtain a
homogenous photoreceptor cell population. We previously
showed that the cell surface antigen CD73 is specically
expressed in photoreceptors in hiPSC-derived retinal
organoids. Flow cytometry analysis in dissociated cells from
retinal organoids indicated that the percentage of CD73+
cells increased with organoid maturation, with CD73+ cells
representing more than 60% of cells at day 180 of
differentiation. Targeting of CD73 by Magnetic-Activated Cell
Sorting (MACS), led to enrichment to 90% of CD73+ cells
in the positive sorted fraction. Nanostring analysis on
CD73-sorted cells conrmed expression of photorecep-
tor-specic genes, while showing downregulation of genes
expressed in other retinal lineages compared to dissociated
retinal cells before MACS. We conrmed that CD73 targeting
by MACS is an effective strategy to separate a homogenous
population of photoreceptors by using a uorescent Cone
rod homeobox (Crx) reporter hiPSC line. Freeze-thawing of
whole retinal organoids resulted in a source of viable cells
and did not affect MACS effectiveness. Finally, transplanta-
tion studies in a rat model of photoreceptor degeneration
demonstrated the capacity of CD73-sorted cells to survive
and mature in close proximity to host inner retina during
several weeks. Human cells were identied by a combina-
tion of human-specic cytoplasmic and nuclear markers,
morphological features and specie-specic uorescence in
situ hybridization (FISH) probes, excluding the occurrence of
cytoplasmic material exchange between donor human cells
and recipient rat cells. Functional analysis by full-eld
electroretinogram (ERG) recording failed to detect an
improvement of the visual function in transplanted eyes
compared to controlateral uninjected eyes. In conclusion,
although the ability of donor cells to establish functional syn-
aptic connections and mediate a signicant rescue of the
visual function remains to be assessed, these data demon-
strate that CD73+ photoreceptor precursors hold great
promise for the development of a future clinical translation.
Keywords: photoreceptors; organoid; transplantation
68
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Poster Abstracts
093
DEVELOPMENT OF A HUMAN 3D VASCULARIZED
SKIN ORGANOID MODEL
Peking, Patricia1, Hochreiter, Anna1, Scharler, Cornelia1,
Wimmer, Monika2, Krisch, Linda3, Wolf, Martin1, Hochmann,
Sarah1, Schallmoser, Katharina3, Strunk, Dirk1
1Experimental and Clinical Cell Therapy Institute, Spinal
Cord Injury and Tissue Regeneration, Paracelsus Medical
University, Salzburg, Austria, 2EB House Austria, Research
Program for Molecular Therapy of Genodermatoses,
Department of Dermatology, University Hospital of the
Paracelsus Medical University, Salzburg, Austria,
3Department of Blood Group Serology and Transfusion
Medicine, Paracelsus Medical University, Salzburg, Austria
Stem cell-derived organoids provide self-organized 3D tissue
models, representing the ex vivo organ physiology but
frequently lacking stromal vascular support. Here, we
aimed at developing suitable metabolic conditions for
iPSC-derived as well as adult skin cell re-organization that
support functionality of the different cell types in a 3D skin
organoid model. Adult interfollicular epidermal skin
keratinocytes (KC), skin broblasts (FB) for stromal support,
and endothelial cells (EC) for prevascularisation, were
isolated and propagated in 2D under animal serum-free
conditions. Umbilical cord blood-derived induced pluripotent
cells (iPSC) were differentiated into iPS-KC, -FB and –EC in
addition. Cell viability, identity and purity was conrmed by
ow cytometry and clonal assays indicated their
stem/progenitor potential. Triple cell type organoid formation
required optimized media selection and human
platelet-derived growth factors to support the regenerative
and angiogenic metabolic condition. 3D confocal microscopy
revealed viable skin organoids with a size of 150 μm after 4
days in culture. Using uorescent probes and life cell
tracking, the cell organization process was monitored. Live
cell imaging revealed sequential organoid assembly starting
from stromal-vascular aggregation and followed by the
supercial anchorage of KC, indicating an organized structure
already 50h after cell seeding. These data indicate that
optimized metabolic conditions required for skin regeneration,
potentially promoting skin re-organization and angiogenesis
in vivo, can be determined in a 3D organoid model that may
be suitable for drug development as well as establishing
novel skin transplantation strategies.
Keywords: skin organoid; stem cell self-organization; skin
regeneration
094
MAYO CLINIC’S HIGH-RISK MAMMARY GLAND-
FALLOPIAN TUBE ‘LIVING’ ORGANOID BIOBANK
FOR WOMEN’S CANCER RESEARCH
Kannan, Nagarajan1, Couch, Fergus1, Boughey, Jodi2,
Sherman, Mark3, Jamie, Bakkum-Gamez4
1Department of Laboratory Medicine and Pathology,
Mayo Clinic, Rochester, MN, USA, 2Surgery, Mayo Clinic,
Rochester, MN, USA, 3Health Sciences Research, Mayo Clinic,
Jacksonville, FL, USA, 4Obstetrics and Gynecology, Mayo
Clinic, Rochester, MN, USA
A woman’s life-time risk for breast cancer and ovarian
cancer are more than 10% and 1.5% respectively. This risk is
highly elevated in patients with strong family history of such
cancers and/or deleterious mutations in genes such as
BRCA1/2. A signicant proportion of these high-risk patients
succumb to their disease due to lack of effective therapies.
The rst malignant cells of highly aggressive triple negative
breast cancer and high-grade serous ovarian cancer are
believed to be from epithelial cells lining the mammary gland
and fallopian tubes. The prevention strategy currently available
to these patients involve highly invasive and least desirable
surgical debulking of breast and/or ovaries/fallopian tubes.
Patient tissue-organoids and isolated epithelial stem cell
derived-orga noids generated from high-risk patients are vital
resource to study tissue regeneration and track disease
origin. With a vision to boost women’s cancer prevention
research at Mayo, we have established the rst and largest
clinically and genetically-annotated patient-derived organoid
biobank for mammary gland and fallopian tube tissues from
average and high-risk patients undergoing surgeries at
different sites of Mayo Clinic. We have viably frozen >1000
vials of mammary tissue-organoids from 100 patients
including carriers of BRCA1/2 and other breast
cancer-associated mutations who underwent prophylactic
mastectomy, reduction mammoplasty or autopsy, and
>16,000 individual fallopian tube stem cell derived organoids
from 14 patients who underwent salpingectomy. This
continuing effort to create largest and well-annotated ‘living’
organoid biobank for women’s cancer prevention program,
attendant challenges in its establishment, preliminary
characterization studies of epithelial stem cells and
resource sharing plan will be discussed in detail.
Funding Source
This work is partly supported by grants to N.K. from Mayo
Clinic’s Breast Cancer SPORE and Ovarian Cancer SPORE.
Keywords: Organoid Biobank; Mammary gland; Fallopian
tubes
69
AMSTERDAM NETHERLANDS
Poster Abstracts
095
BMP ANTAGONISTS SECRETED BY MSC INDUCE
COLONIC ORGANOID PROLIFERATION: APPLICA-
TION TO SIDE EFFECTS OF RADIOTHERAPY
TREATMENT
Mathieu, Noelle1, Moussa, Lara2, Squiban, Claire2,
Lapierre, Alexia2, Demarquay, Christelle2, Milliat, Fabien2
1IRSN, Fontenay Aux Roses, France, 2IRSN, Paris, France
Radiation therapy is used in at least 50% of cancer patients
and plays a critical role in 25% of cancer cures. Many cancer
patients have undergone radiation therapy of tumors in the
abdomen and the bowel is an organ at risk due to the
presence of some parts of it in the irradiation eld. Delayed
bowel radiation toxicity is a highly important issue for long
term cancer survivors. It is a progressive condition with
substantial long-term morbidity and mortality. Today, there is
no unied approach for the assessment and treatment of
this disease. The aim of this study is to use cultured adult
stem cell from colon (CSC) to reduce colonic injuries induced
after colorectal irradiation. We also try to improve the
therapeutic outcome by co-injection with mesenchymal
stromal cells (MSC) involved in the function of niche and
widely used in clinical trials. In mice model reproducing
radiation-induced histological damage observed in patients
suffering from severe side effects after radiotherapy, epithelial
cells from colonic organoids of C57/Bl6 mice expressing
GFP were locally injected. We determined that CSC in vitro
amplied, implant and proliferate in irradiated colonic
mucosa. CSC were readily detectable in the colon until 21
days after injection. We also highlighted that CSC injections
improve colonic regeneration as determined by a reduction
of the ulcer size on histologic slides. Moreover, we also
determined the benet of using CSC in association with
MSC. First of all, in vitro analysis demonstrated that co-cul-
ture of MSC with CSC increase the number, proliferation and
size of colonic organoids in normal and irradiated conditions.
Gene expression analysis on MSC revealed huge expression
of two BMP-antagonists, and inhibition using siRNA demon-
strated the involvement of these molecules in organoids
formation. Then, we co-injected CSC with MSC in irradiated
colon. This study provides evidence of the potential of CSC to
limit radiation effects on the colon and open perspectives on
combined strategies to improve their therapeutic benet.
Keywords: Colonic organoid, mesenchymal stromal cells;
Treatment, Cell therapy; Irradiation, pelvic radiotherapy side
effect
096
HUMAN THYROID-DERIVED ORGANOIDS GENERATE
HORMONE-PRODUCING GLANDULAR TISSUE
Ogundipe, Vivian1, Groen, Andries2, Hosper, Nynke1,
Nagle, Peter2, Hess, Julia3, Faber, Hette1, Jellema, Anne1,
Baanstra, Mirjam1, Links, Thera4, Unger, Kristian3, Plukker,
John5, Coppes, Rob6
1Biomedical Sciences of Cells and Systems, University
Medical Center Groningen, Netherlands, 2Surgical Oncology
and Biomedical Sciences of Cells & Systems, University
Medical Center Groningen, Netherlands, 3Radiation
Cytogenetics, Helmholtz Zentrum München, Germany,
4Endocrinology, University Medical Center Groningen, Nether-
lands, 5Surgical Oncology, University Medical Center Groningen,
Netherlands, 6Radiation Oncology and Biomedical
Sciences of
Cells & Systems, University Medical Center
Groningen, Netherlands
Treatment of hypothyroidism with thyroid hormone replace-
ment often results in unbalanced thyroid hormone levels which
results in fatigue, constipation, weight gain and potential
cardiovascular diseases or osteoporosis severely compromis-
ing the quality of life of patients. Organoid transplantations
could be used to regenerate and restore tissue functionality.
We isolated and cultured primary murine and human thyroid
gland tissue as single cells to form thyroid organoids. Tran-
scriptomic analysis of these human thyroid gland organoids
demonstrated the expression of several stem cell markers,
such as Sca-1, CD133 and EpCAM, and the thyroid-specic
markers Nkx2-1, thyroglobulin and T4. After prolonged passag-
ing, upregulation of stem cell markers, FUT4 and SOX2, and
proliferation markers, PCNA and Ki67 was observed. Induced
differentiation of murine and human organoids in vitro induced
the development of tissue-resembling mini-glands that abun-
dantly expressed thyroid gland markers and produced T4
hormone. (Xeno-) transplantation of dissociated organoids
underneath the kidney capsule of athyroid mice resulted in the
generation of hormone-producing murine and human thy-
roid-resembling follicles, and increased survival. These studies
provide the rst proof of principle that primary thyroid
gland-derived organoids can be cultured, and are capable of
developing into a functional mini-gland in vivo, thereby sug-
gesting a promising applicability for thyroid gland regeneration.
Funding Source
This research was supported by the Dutch Cancer Society,
grant number 10650.
Keywords: Hypothyroidism; Hormone-producing thyroid gland
organoids; T4 hormone
097
IDENTIFYING HUMAN SALIVARY GLAND STEM
CELLS SIGNATURE THROUGH A MOLECULAR
NETWORK BASED APPROACH
Rocchi, Cecilia1, Barazzuoi, Lara3, Boekhoudt, Jeunard1,
Jellema, Anne3, Baanstra, Mirjam3, Os, Ronald2, Guryev,
Victor2, Coppes, Rob3
1Department of Biomedical Sciences of Cells and Systems,
University Medical Center Groningen, Netherlands, 2European
Research Institute for the Biology of Aging, University Medical
Center Groningen, Netherlands, 3Radiation Oncology and
Biomedical Sciences of Cell and Systems, University Medical
Center Groningen, Netherlands
Radiotherapy treatment for head and neck cancer increases
patient survival but comes with the frequent cost of xerosto-
mia. This is caused by radiation-induced hypo-salivation,
resulting from sterilization of the salivary gland stem/progeni-
tor cell compartment. Therefore, salivary gland stem cell
therapy could hold great potential to restore tissue functional-
ity. Although we previously showed that salivary glands contain
a pool of cells exhibiting stem/progenitor cell properties, the
slow turnover of the salivary gland epithelium, the scarcity of
tissue specic stem cells and their niche complexity have made
their characterization until now particularly challenging. We
70
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Poster Abstracts
performed bulk RNA sequencing and weighted gene co-expres-
sion network analysis to dene the molecular signature of
human salivary gland stem cells and the molecular cues that
control their self-renewal and differentiation. Using salivary
gland organoid, we generated samples corresponding to a
heterogeneous human salivary gland stem/progenitor popula-
tion, a more pure stem cell population (by adding CHIR99021
and Valproic acid (CV)), and a more differentiated population
showing budding and branching features. Stem cell potency
was assessed by organoid forming efciency (OFE). RNA
proling data were used to construct a molecular network
where modules of co-expressed genes were directly correlated
with OFE,CV treatment and differentiation potential. The
analysis allowed us to identify 3 modules strongly correlated
with high OFE and CV treatment and weakly correlated with
differentiation ability, indicating that these modules could be
associated with a more primitive stem cell prole, showing the
involvement of the Hippo and Wnt pathways and several
unique genes expressed in the salivary gland stem cell
compartment. In particular, YAP overexpression in salivary
gland organoid cultures showed increased OFE revealing a key
role of YAP in maintenance of salivary gland stem cells.
Functional high-throughput genetic screens will be used for the
validation of driver genes in these modules, and could poten-
tially lead to stem cell surface markers. These markers could
be used to isolate the most potent stem cell population and
therefore could be used in clinical trials for patients suffering
from radiation-induced hypo-salivation.
Funding Source
Dutch Cancer Society Grant RUG2013-5792.
Keywords: Salivary Gland Stem Cells; Organoid; Weighted
Gene Co-Expression Network Analysis
098
BILE AS A NON-INVASIVE SOURCE OF BILE
CHOLANGIOCYTE ORGANOIDS FOR DEVELOPING
PATIENT-SPECIFIC DISEASE MODELS
Roos, Floris1, Verstegen, Monique M.A.1, Poley, Jan-Werner2,
Bruno, Marco M.J.2, Tetteroo, Geert W.M3, Ijzermans, Jan N.
M.1, van der Laan, Luc J.W.1
1Department of Surgery, Erasmus Medical Center, Rotterdam,
Netherlands, 2Gastroenterology and Hepatology, Erasmus
Medical Center, Rotterdam, Netherlands, 3Surgery, Ijsselland
Hospital, Capelle aan den IJssel, Netherlands
Bile duct related diseases are the leading cause for
pediatric liver transplantation and adult re-transplantation of a
liver graft. Studying biliary diseases has long-term been
hampered by the inability to culture bile duct lining
cholangiocytes long-term. Recently was shown that
Extra-hepatic Cholangiocyte Organoids (ECOs) that are
derived from extra-hepatic bile duct (EHBD) tissue can be
long-term expanded in culture. However, with the current
limitation for disease modeling or personalized regenerative
medicine applications highly invasive bile duct biopsies are
required to obtain these ECOs from individual patients.
Therefore the aim of the current study is to investigate
whether ECOs can be cultured from less invasively obtained
bile uid. Bile-derived ECOs were cultured, according to the
previous published protocol, either from gallbladder bile
obtained from donor livers for transplantation, or from bile
obtained by endoscopic retrograde cholangiopancreatography
(ERCP) or percutaneous transhepatic cholangiography drain
(PTCD). Next, ECO’s were initiated from three different patients
and compared to bile-derived cholanchiocyte organoids
(BCO’s) on the genetic level (qrt-PCR), protein level (either
immunohistochemistry), immunouorescence or western
blotting) and functional level by testing the cholangiocyte
specic transporter channels (Ussing chamber). Cultures
were initiated from 1 ml of bile obtained from all different
sources. ECOs could beeffectively (8/9 attempts) expanded
from all sources of bile from patients with a variety of
diseases. Bile-derived ECOs expressed similar cholangiocyte
markers on gene and protein level as
tissue-derived ECOs and both lacked either stem cell- or
hepatocyte markers. Furthermore, these cells expressed and
responded similarly to stimulation and inhibition of different
cholangiocyte ion-channels. Interestingly, bile-derived ECOs
from a patient with cystic brosis (CF) clearly lacked CFTR
channel activity, showing that ECOs can be used as a disease
model to study biliary diseases. Our study showed that bile
provides a novel and less-invasive source of patient-specic
ECOs. This creates new opportunities to study autologous
bile duct regeneration and develop patient-specic disease
models.
Keywords: Bile duct; Disease model; Bile cholangiocytes
099
HIGH-THROUGHPUT MICROFLUIDIC PLATFORM
FOR STUDYING VASCULARIZATION OF IPSC-
DERIVED KIDNEY ORGANOIDS
Previdi, Sara1, Koning, Marije2, Wiersma, Loes2, Kurek, Dorota1,
van den Berg, Cathelijne W.3, Vulto, Paul1, Rabelink, Ton J.2
1MIMETAS B.V., Leiden, Netherlands, 2Department of Internal
Medicine-Nephrology, Leiden University Medical Centre, Leiden,
Netherlands, 3Leiden University Medical Centre, Leiden,
Netherlands
Kidney organoids derived from human induced pluripotent
stem cells (iPSCs) represent a powerful in vitro model for
studying kidney development, disease mechanisms and
drug testing. Despite the great level of structural complexity
reached in vitro, these kidney organoids are immature possibly
due to the lack of a functional vascular system.
Transplantation of kidney organoids under the kidney
capsule of a mouse can signicantly improve their
maturation. However, alternative approaches are valuable for
studying these processes in vitro. Microuidic techniques
show great potential in bridging the gap between 2D in vitro
cultures and animal models. Here, we present the use of a
high-throughput in vitro ‘grafting’ platform which allows
co-culture of vessels with kidney organoids. One unit of the
Mimetas Organoplate® Graft is made of two microuidic
channels in which endothelial cells can be patterned against
ECM. Presence of a tissue chamber allows endothelial cell
co-culture with 3D tissues. When kidney organoids are used,
extensive vascular remodeling occurred with formation of a
complex 3D network of angiogenic sprouts growing towards
the tissue. Moreover, vessel stabilization can be monitored
overtime by real time imaging and perfusion with 150 kDa
Dextran. The established kidney organoid-on-a-chip system
provides a promising platform for drug testing and disease
modeling.
Keywords: Kidney Organoids; Microuidics; Vascularization
71
AMSTERDAM NETHERLANDS
Poster Abstracts
100
HUMAN BILE DUCT-DERIVED ORGANOID
CULTURES FOR STEM CELL CHARACTERIZATION
AND DISEASE MODELING
Verstegen, Monique M.A.1, Burka, Ksenia1, de Wolf, Maaike1,
Bijvelds, Marcel J.1, de Jonge, Hugo R.1, Fuchs, Sabine A.2,
Gehart, Helmuth3, Roos, Floris1, Roest, Henk J.1,
de Jager, Myrthe4, Ijzermans, Jan N.M1,
Cuppen, Edwin P.J.G.5, van der Laan, Luc J.W.1
1Department of Surgery, Erasmus Medical Center, Rotterdam,
Netherlands, 2Metabolic disorders, University Medical Centre
Utrecht, Netherlands, 3Hubrecht Institute, Utrecht, Netherlands,
4Center for Molecular Medicine and Oncode Institute, University
Medical Center Utrecht, Netherlands, 5Center for Molecular
Medicine, University Medical Center Utrecht, Netherlands
Integrity of the biliary tree is imperative for liver function.
Though evidence suggests that peribiliary glands harbor
stem cells that contribute to bile duct homeostasis and
repair during disease or injury, these are not well characterized.
Therefore, the aim of our study is to expand and characterize
human biliary stem cells using 3-dimensional organoid. For
this, human extrahepatic bile ducts and paired liver biopsies
(n=40) were collected from donor liver grafts at time of liver
transplantation. Bile duct organoid cultures were initiated
and propagated by weekly passaging using similar conditions
as described for liver biopsies. The bile duct-derived
organoids were characterized and compared to paired
liver-derived organoids by phenotypic (EM, light sheet
microscopy), genomic (q-PCR, RNAseq) and proteomic
(MassSpec, immunohistochemistry) analysis. Growth
characteristics and genetic stability was determined and
functional transporter channel function was measured using
Ussing chamber technology and Forskolin Induced Swelling
(FIS) assays. In addition, the ability of hepatocyte and
cholangiocyte differentiation was studied. Organoids were
efciently grown from the bile ducts and expanded for >9
months. These organoids stained positive for biliary cell
markers CK19, EpCAM and MUC1. RNAseq analysis
demonstrated expression of stem cell markers LGR5, PDX1
and Sox9. Functional transporter channels activity was
detected for CFTR and AE2. Although bile duct-derived
organoids were less prone to differentiate towards hepatocyte
lineage as compared to their liver-derived counterparts, they
had good differentiation capacity towards cholangiocyte
lineage. To demonstrate that these bile duct organoids could
be used for disease modeling, we observed lack of CFTR
channel activity in bile duct organoids from a cystic brosis
patient. In conclusion: This study showed the presence of
LGR5-positive stem/progenitor cells in human extra hepatic
bile ducts which can be expanded long-term as bile duct
organoids. These organoids express hepato-biliary genes
and proteins, and show functional transporter channel
activity and can be used for disease modeling and tissue
engineering applications.
Keywords: Bile duct-derived organoids; Disease modeling;
Extrahepatic bile duct
101
THE RENIN-ANGIOTENSIN SYSTEM IS PRESENT
AND FUNCTIONAL IN IPSC-DERIVED KIDNEY
ORGANOIDS
Shankar, Anusha S.1, Korevaar, Sander S.1, van den Berg –
Garrelds, Ingrid2, Gribnau, Joost3, Baan, Carla C.1, Danser,
Jan A.H2, Hoorn, Ewout J.1, Hoogduijn, Martin J.1
1Department of Internal Medicine, Division of Nephrology and
Transplantation, Erasmus Medical Center, Rotterdam,
Netherlands, 2Department of Internal Medicine, Division of
Pharmacology and Vascular Medicine, University Medical
Center Rotterdam, Erasmus MC, Rotterdam, Netherlands,
3Department of Developmental Biology, University Medical
Center Rotterdam, Erasmus MC, Rotterdam, Netherlands
The intrarenal renin-angiotensin system (RAS) arises early
during kidney development and is important for proper
nephrogenesis. The lack of a suitable human model to study
the intrarenal RAS hampers investigation into the relevance
of this system. Recently protocols for the in vitro generation of
induced pluripotent stem cell (iPSC)-derived kidney organ-
oids were developed. In this study we investigated the
presence and functionality of the RAS in iPSC-derived kidney
organoids. 4 human iPSC lines were grown on Geltrex and
treated with CHIR99201 and broblast growth factor 9, after
which the cells were transferred to a transwell membrane.
The resulting organoids showed a 10- to 40-fold increase in
mRNA for kidney-specic markers after 25 days of culture,
including a marker for renin-producing stromal cells, FOXD1.
Renal structures were observed using immunohistochemistry.
Moreover, there was a 10-fold increase in the expression of
the organic anion transporters OAT1 and OAT3, suggesting
tubular function. Interestingly, the mRNA level of
angiotensinogen (AGT) increased more than 100-fold as
early as day 7 of the culture in comparison to iPSC and
remained stable until day 25. Also, angiotensin receptor
type 1 and type 2 mRNA expression increased and remained
highly expressed throughout the culture, while high levels
of ACE were maintained in kidney organoids. Finally, a
10- to 100-fold increase in the mRNA expression of renin
was observed at day 25. The use of an indirect
enzyme-kinetic assay revealed the functionality of renin in
the kidney organoids at day 25, as measured by the
conversion of exogenously administered AGT to angiotensin
I. Moreover, analysis of the medium harvested from kidney
organoid cultures at day 25 exhibited varying amounts of
renin activity, ranging from 12 to 200 ng angiotensin I/ml per
hour. The addition of the cyclic AMP-elevating agents forsko-
lin and dibutyryl cyclic AMP to the culture for 24 hours
increased the mRNA expression of renin drastically (up to
1000-fold), indicating that the production of renin in the kidney
organoids may be a regulated and inducible process. In
summary, we demonstrate the presence and functionality of
components of the RAS in human iPSC-derived kidney
organoids. This provides the opportunity to study the
intrarenal RAS and its regulation in an in vitro human model.
Keywords: Renin-angiotensin system; Induced pluripotent
stem cells; Kidney organoid
72
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Poster Abstracts
102
CHARACTERIZATION OF THE IMMUNOGENICITY
OF IPSC-DERIVED KIDNEY ORGANOIDS
Shankar, Anusha S.1, Korevaar, Sander, S.1, Van Den Bosch,
Thierry P. P2, Clahsen-van Groningen, Marian2, Gribnau,
Joost3, Baan, Carla C.1, Hoorn, Ewout J.1, Hoogduijn, Martin J.1
1Department of Internal Medicine, Division of Nephrology and
Transplantation, Erasmus Medical Center, Rotterdam,
Netherlands, 2Department of Pathology, Erasmus Medical
Center, Rotterdam, Netherlands, 3Department of
Developmental Biology, Erasmus Medical Center,
Rotterdam, Netherlands
There is an increasing interest in iPSC-based therapies for
kidney regeneration. Recently protocols for the in vitro
generation of kidney organoids have been developed. For
successful implementation into clinical practice,
immunological acceptance of the iPSC-derived cells is
crucial. Therefore, our aim was to study the immunogenicity
of iPSC-derived kidney organoids. Three human iPSC lines
were grown on Geltrex and treated with CHIR99201 and
fibroblast growth factor 9. The resulting organoids showed a
10- to 40-fold increase in mRNA for kidney-specific markers
after 25 days of differentiation. Immunostaining confirmed
that organoids contained essential renal structures. The
kidney organoids were cultured together with peripheral
blood mononuclear cells from two healthy donors for 7
days. Subsequently, a >100-fold increase in the mRNA
expression of the leukocyte marker CD45 was observed in
the organoids. Immunostaining confirmed the presence of
infiltrating CD45+ cells in the organoids. The response
appeared to be T-cell mediated as there was a 10-fold mRNA
increase in CD4 and CD8, while macrophage marker CD68
was also highly expressed. At the protein level, T-cells
predominantly clustered around glomerular structures while
macrophages were diffusely distributed throughout the
organoid. A mixed pattern of macrophages could be
observed, as both pro-inflammatory (M1) and anti-
inflammatory (M2) macrophage markers were substantially
increased at the mRNA level. Even though a 10-fold mRNA
increase of inflammatory factors such as TNFα and
Granzyme B was suggestive of a pro-inflammatory response,
immunofluorescence showed that the infiltrating cells did
not proliferate as observed by the absence of Ki-67+CD45+
cells. The mRNA expression of kidney differentiation
markers remained stable throughout the co-culture. Yet,
immunohistochemistry revealed that the expression of WT1,
a podocyte marker, was decreased 4-fold in comparison to
control organoids, indicating that specifically podocytes may
be a target of the immune response. Although further
characterization of the immune response to kidney
organoids and its influence on differentiation is required,
these preliminary results offer novel insights into the in vitro
interaction of immune cells with iPSC-derived kidney
organoids.
Keywords: Immunogenicity; Induced pluripotent stem cell;
Kidney organoid
103
THE ARYL HYDROCARBON RECEPTOR PATHWAY
DEFINES THE TIME FRAME FOR RESTORATIVE
NEUROGENESIS
Durovic, Tamara1, DiGiaimo, Rossella2, Barquin, Pablo2,
Kociaj, Anita2, Lepko, Tjasa2, Irmler, Martin3, Schotta, Gunnar4,
Beckers, Johannes3, Wurst, Wolfgang5, Stricker, Stefan6,
Ninkovic, Jovica7
1Cell Biology Department, Biomedical Center, Planegg,
Munich, Institute of Stem Cell Research, Helmholtz Zentrum
München, Munich, Germany, 2Institute of Stem Cell Research,
Helmholtz Center Munich, Munich, Germany, 3Institute of
Experimental Genetics, Helmholtz Zentrum München,
Munich, Germany, 4Division of Molecular Biology, Biomedical
Center, Faculty of Medicine, LMU Munich, Germany, 5Institute
of Developmental Genetics, Helmholtz Zentrum München,
Munich, Germany, 6MCN Junior Research Group, Munich
Center for Neurosciences, Munich, Germany, 7Department for
Cell Biology and Anatomy, Biomedical Center of LMU, Munich,
Germany
Zebrash have a high capacity to replace lost neurons after
brain injury. New neurons involved in repair are generated by
a specic set of glial cells, known as ependymoglial cells
that represent stem cells in the zebrash telencephalon. We
analysed changes in the transcriptome of ependymoglial
cells and their progeny after injury to infer the molecular
pathways governing restorative neurogenesis. We
identied the aryl hydrocarbon receptor (AhR) as a
regulator of ependymoglia differentiation towards
post-mitotic neurons. In vivo imaging showed that high AhR
signalling promotes the direct conversion of a specic
subset of ependymoglia into post-mitotic neurons, while low
AhR signalling promotes ependymoglial proliferation.
Interestingly, we observed the inactivation of AhR signalling
shortly after injury followed by a return to the basal levels 7
days post injury. Interference with timely AhR regulation
after injury leads to aberrant restorative neurogenesis. Taken
together, we identied AhR signalling as a crucial regulator
of the neurogenic fate of ependymoglia and of the timing of
restorative neurogenesis in the zebrash brain.
Keywords: Regeneration, Stem Cells; Neurogenesis, AhR
signalling; Differentiation, proliferation
73
AMSTERDAM NETHERLANDS
Poster Abstracts
104
3D CARDIAC ORGANOIDS FOR STUDYING
MULTINUCLEATION OF HUMAN CARDIOMYO-
CYTES DERIVED FROM INDUCED PLURIPOTENT
STEM CELLS LACKING HEME OXYGENASE-1
Jez, Mateusz1, Stępniewski, Jacek1, Andrysiak, Kalina1,
Kania, Alan2, Chrobok, Łukasz2, Palus-Chramiec, Katarzyna2,
Lewandowski, Marian H.2, Józkowicz, Alicja1, Dulak, Józef1
1Department of Medical Biotechnology, Jagiellonian University,
Krakow, Poland, 2Department of Neurophysiology and
Chronobiology, Jagiellonian University, Krakow, Poland
During myocardial infarction up to 25% left ventricle
cardiomyocytes (CMs) are lost. Non-functional scar tissue is
formed in damaged area which weakens the heart muscle
and might lead to heart failure. Therefore it is reasonable to
understand the mechanisms of CMs proliferation. To date,
mechanisms of CM proliferation were investigated mainly in
murine model. However, there are several limitations of this
model. One of them is different proportion of multinucleated
CMs. In mice percentage of terminally differentiated,
non-dividing multinucleated CM is up to 90%, whereas in
human it’s 25-60%. This might be linked to higher energy
demand of mice hearts, as their heart beating rate is approx.
10x faster than in human. High energy production is linked
with elevated ROS production, which in turn can damage
DNA and result in cell cycle arrest and formation of
multinucleated cells. Due to mentioned differences,
molecular pathways responsible for proliferation in murine
model might be different in human. CMs derived from human
induced pluripotent stem cells serve as a platform for
investigating CMs proliferation mechanisms in human.
By modulating Wnt/β-catenin pathway using small molecular
inhibitors, virtually pure (95%) CM population is obtained.
Expression of cardiac markers i.e Cardio Troponin T and I
and action potential measured by patch clamp conrmed
their cardiac properties. What is important, we also detected
multinucleated CMs. CRISPR/Cas9 mediated knock down of
cytoprotective enzyme heme oxygenase-1 (HO-1), crucial for
embryonal heart development, resulted in less mature
phenotype of CMs, basing on shortened action potential and
lower expression of ion channels. To partially mimic in vivo
process of heart development, we have modied 2D culture
protocol. At different days of differentiations, cells were
formed into organoids and cultured in 3D up to 45 days.
Interestingly, CMs from organoids, compared to 2D cultured
cells were characterised by more mature phenotype, basing
on expression pattern of Titin N2BA and N2B and increased
expression of potassium channel KCNH2.
Spatially-patterned CMs in organoids might facilitate
formation of multinucleated cells, thus are more suitable
platform for investigating multinucleation and proliferation
of CMs, compared to 2D culture.
Funding Source
This study was supported by Harmonia grant from the
National Science Centre (2014/14/M/NZ1/00010) and grant
for Young Researchers funded by the Faculty of Biochemis-
try, Biophysics and Biotechnology of the Jagiellonian
University.
Keywords: Cardiomyocytes; Organoids; Multinucleation
106
HUMAN PLURIPOTENT STEM CELL DERIVED LIVER
ORGANOIDS
Sullivan, Gareth J.1, Siller, Richard1, Harrison, Sean1, Sharma,
Kulbhushan2, Zweigardt, Robert3, Gadegaard, Nikolaj4, Park,
Inhyun5, Takebe, Takanori6, Morkry, Jaroslav7, Wolf, Roland8
1Molecular Medicine, Institute of Basic Medical Sciences,
University of Oslo, Norway, 2Department of Cardiac, Thoracic,
Transplantation and Vascular Surgery, Hannover Medical
School, Hannover, Germany, 3Engineering, University of
Glasgow, UK, 4Genetics, Yale University, New Haven, CT, USA,
5Gastroenterology, Hepatology and Nutrition, Cincinnati
Children’s Hospital, Cincinnati, OH, USA, 6Department of
Histology and Embryology , Charles University in Prague,
Hradec Králové, Czech Republic, 7Medicine, University of
Dundee, UK
Human pluripotent stem cell derived hepatocytes have
immense potential to revolutionise the eld of hepatotoxicity
and represent a paradigm shift for the treatment of liver
disease and regenerative medicine. Unfortunately, hPSC
derived hepatocytes do not fully recapitulate the full cellular
and metabolic repertoire of a fully functional, adult liver. With
the advent of hPSC derived organoids marking a critical
turning point in this regard. Current liver organoid models,
while useful, generally provide the opportunity to study one
or two cell types of the liver (traditionally hepatocytes and/or
cholangiocytes). While these model systems can be used to
interrogate drug metabolism, liver tissue regeneration, and
generate large number of hepatocytes, the lack of functional
and cellular equivalency when compared to the liver sinusoid
makes them insufcient to accurately model liver toxicity.
In order to address these shortcomings we have developed
a 3D liver organoid model system from hPSCs that accurately
present with all the expected cell types of the liver (hepato-
cytes, cholangiocytes, stellate, LSECs, endothelial cells etc).
The hPSC derived liver organoids demonstrate signicantly
enhanced drug metabolism activity and inducibility, and
critically this enhanced function can be maintained during
long-term culture. This simple and robust method of
generating large number of liver organoids is of potentially
great interest to the eld as it is readily scalable at low cost
due to the exclusive use of small molecules as the drivers of
differentiation.
Funding Source
This work was supported by the Research Council of
Norway project 247624 and partially supported by the
Research Council of Norway through its Centres of Excel-
lence scheme, project number 262613.
Keywords: Liver Organoids; Human pluripotent stem cells;
Small molecule driven procedure.
74
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Poster Abstracts
107
THREE-DIMENSIONAL BIOPRINTING IPSCS TO
FORM ORGANOIDS TO RECAPITULATE HUMAN
LIMB BUD DEVELOPMENT – AN OSTEOARTHRITIS
DISEASE MODEL
Simonsson, Stina
Clinical Chemistry & Transfusion Medicine/Biomedicine,
Gothenburg University, Goteborg, Sweden
Cartilage lesions that can develop into degenerated
osteoarthritis (OA) cartilage are a worldwide burden. As a
prospective treatment for such lesions, we have previously
shown that human-derived induced pluripotent stem cells
(iPSCs) can be 3D bioprinted into cartilage-mimics. The
advantages by using an established iPSC line developed
from chondrocytes are unlimited, immortal characterized
cell source with a differentiation bias towards cartilage.
Designing protocols that generates hyaline cartilage from
pluripotent cells in vitro is still a challenge, due to that joint
formation are late in development and far from the pluripotent
state. There are recent protocols for hyaline-like cartilage
generation from iPSCs. Herein our 3D differentiation of
bio-printed iPSC line or organoids experience that resemble
limb bud formation will be discussed. Also, results from
screening by use of a molecular library on our genetically
modied iPSC-line expressing green uorescence protein
(GFP) controlled by an OA-marker will be presented.
Funding Source
Financial support was received from Swedish research
council VR 2015-02560, granted Stina Simonsson.
Keywords: iPSC; Organoid; Osteoarthritis
108
EXPLORING THE HOMING POTENTIAL OF HEMATO-
POIETIC STEM CELLS THROUGH BONE MARROW
ORGANOIDS
Giger, Sonja1, Hoehnel, Sylke1, Kleiner, Esther2, Ehrbar, Martin2,
Lutolf, Matthias1
1IBI, Ecole Polytechnique Federale de Lausanne, Switzerland,
2Forschung Geburtshilfe, University Hospital Zurich,
Switzerland
A crucial initial step for the successful transplantation of
hematopoietic stem cell (HSCs) is their ‘homing’, the
trafcking of the stem cells and the subsequent engraftment
in their bone marrow niches. Several approaches are available
to improve the homing ability of HSCs, including the pre-treat-
ment with drugs that increase the expression of CXCR4, a
surface marker mediating homing. Rodent models are
routinely used to functionally test the homing capacity of
human HSCs. However, these models are very costly,
time-consuming and only poorly recapitulate the human
bone marrow niches. Here we explored the possibility of
building a 3D bone marrow-like culture system that could be
used to quantify the homing capacity and maintenance of
cord blood (CB)derived-HSCs in a more tractable in vitro
setting. To this end, we established a bioengineered
microtissue platform in which human mesenchymal
stem/progenitor cells (hMSPCs) and human endothelial
cells (hECs) can be aggregated and co-cultured with high
reproducibility and throughput. The two cell types were
found to form compact spheroids that expanded over time
in culture and maintaining key phenotypic markers of the
two cell types. Intriguingly, hECs self-organized into
interconnected, vessel-like networks within the hMSPCs
environment. A small fraction of CB-HSCs showed the
capability of migrating into compact spheroids and colonized
mostly in regions close to the vasculature network.
Interestingly, CB-HSCs of different donors showed clear
varieties of their homing capacity. These data suggest that
the bone marrow organoids hold potential for assaying stem
cell homing in vitro. Ongoing experiments aim at validating
these initial ndings by manipulating the molecular machinery
that controls homing in vivo, by capturing stem cell dynamics
through time-lapse microscopy, and by characterizing
interactions between CB-HSCs and their putative
in vitro niches.
Keywords: Homing; Hematopoietic stem cells; Bone marrow
niche
109
THE ROLE OF GRAVITY MECHANOTRANSDUCTION
IN REGULATING STEM CELL TISSUE REGENERATIVE
POTENTIAL AT THE SINGLE CELL EXPRESSOME
LEVEL
Almeida, Eduardo1, Juran, Cassandra M.2, Zvirblyte, Justina1,3
1Space Biosciences Division, NASA Ames Research Center,
Moffett Field, CA, USA, 2Cell and Molecular Biology, USRA,
Moffett Field, CA, USA, 3Life Sciences Center, Vilnius
University, Vilnius, Lithuania
Forces generated by gravity in load-bearing tissues such as
bone marrow promote stem cell-based tissue-regenerative
processes by increasing proliferation and differentiation of
tissue progenitors. Conversely, in microgravity, mouse bone
marrow mesenchymal and hematopoietic precursors
down-regulate differentiation markers and up-regulate
stemness maintenance genes. In-vitro, mechanical loading
of mesenchymal stem cell-derived osteoprogenitors on a
collagen matrix also promotes proliferation and differentiation
leading to increased mineralized tissue via a
p21/CDKN1a-regulated mechanism. In this work we sought
to test the hypothesis that gravity mechanotransduction
regulates stem cell tissue regenerative processes by
modulating stem cell proliferation and differentiation fates
at specic cell cycle stages. To do this we subjected
clonally-derived mouse embryonic stem cell cultures
on a collagen matrix to either a 60 min pulse of gravity
mechanotransduction, or no stimulation. Six hours
post-stimulation, we used a 10X Genomics Chromium/Single
Cell controller to generate bar-coded single cell Illumina
libraries and sequenced expressomes for 5,000 1g control
cells, and 5,000 cells pulse-stimulated with 50g, simulating
running/jumping bone marrow hydrostatic pressures. Initial
analyses of unstimulated cells show seven major graph-based
clusters of cells, corresponding to different cell cycle stages,
with cluster seven showing a unique highly over-expressed
(3-5 log2 fold) pattern of cytoskeletal and lipid membrane
trafc genes including actin, and actin regulating proteins,
cytokeratins and annexins (Acta2, Tagln, Ker8, Ker9, Ker19,
Anxa2, Anxa3, Anxa5). Mechanostimulation of cells in
75
AMSTERDAM NETHERLANDS
Poster Abstracts
cluster seven also resulted in maintenance of the
cytokeratin/annexin pattern with additional highly elevated
expression levels of differentiation markers for neuroblasts
and muscle as well as pH/chloride regulation (Ahmak,
Cald1, Lgals3, Sct, Clic1), with other clusters showing much
smaller or no alterations. The results suggest that gravity
mechanostimulation results in increased cell proliferation
and differentiation, and that these effects at the expressome
level are most notable on a specic cell-cycle cluster
encompassing 10% of the total mouse embryonic stem cell
population.
Funding Source
NASA Space Biology and NASA grant NH14ZTT001N-0063
to E. Almeida.
Keywords: gravity; mechanotransduction; mouse embryonic
stem cells
110
METABOLIC REGULATORY PATHWAYS IN KIDNEY
ORGANOID DIFFERENTIATION
Hekman, Katherine1, Sarami, Iman2, Ivancic, David1,
Wertheim, Jason1
1Department of Surgery, Northwestern University, Chicago, IL,
USA, 2Department of Pathology, Geisinger Health System,
Danville, PA, USA
The role of metabolites in regulating differentiation of
pluripotent stem cells to kidney organoids is a eld of
growing interest. Mapping these intrinsic regulatory pathways
can aid in understanding embryonic development, as well as
provide insight to improve the directed differentiation
process for tissue and organ engineering. We performed a
screening assay to assess which amino acids play a
regulatory role in the development of kidney organoids.
Advanced RPMI media was generated that lacked each of
the 22 distinct amino acid components, and directed
differentiation to nephron progenitor cells was performed
using these media on pluripotent stem cells. Deprivation
conditions for arginine, cystine, histidine, isoleucine, leucine,
lysine, methionine, phenylalanine, serine, threonine, tryptophan,
tyrosine or valine were incompatible with cell survival from
pluripotency to day 3 of the differentiation process. Depriva-
tion of alanine, asparagine, aspartate, glycine, glutamine,
glutamate, hydroxyproline, or proline allowed for cell survival
until completion of the protocol at the end of day 9. All
conditions displayed expected morphologic development
demonstrated six2 expression. Most notable were the
deprivation conditions for glutaminergic pathway. Depriva-
tion of members of the glutaminergic pathway advanced in
vitro differentiation more rapidly than complete media.
These data further illustrate the differing effects of discrete
metabolic pathways in regulating differentiation of pluripo-
tent stem cells to nephron progenitor cells.
Funding Source
NIH 1F32HL137292; Northwestern Medicine Transplant
Endowment Grant; McCormick Foundation.
Keywords: kidney organoid; metabolism; directed differentia-
tion
111
HUMAN IPSC-DERIVED CARDIAC ORGANOIDS
MIMIC EARLY EMBRYONIC HEART DEVELOPMENT
De Andrade E Silva, Ana Catarina, Joy, David A., Matthys,
Oriane B., Kauss, Mara A., Natarajan, Vaishaali, Turaga,
Diwakar, McDevitt, Todd C.
Gladstone Institute of Cardiovascular Disease, Gladstone
Institutes, San Francisco, CA, USA
The development of cardiac organoids is crucial to
model human cardiac development and disease. Heart
morphogenesis initiates from a simple structure and
composition, which evolves into a more complex organ
through the temporal emergence of distinct cells populations
that contribute to heart substructures. Human induced
pluripotent stem cells (hiPSCs) are a valuable cell source,
due their ability to model ex vivo aspects of human
cardiogenesis. Previous efforts to study cardiac development
with hiPSCs lack the co-emergence of multiple cardiac cell
populations, whose temporal differentiation, growth,
morphogenesis and maturation processes are inherent to
heart development. In order to generate heterogeneous,
self-organized, functional cardiac organoids from hiPSCs,-
cardiac mesoderm progenitor cells were specied by
modulation of Wnt signaling prior to aggregation and 3D
suspension culture thereafter in hydrodynamic conditions.
By day 10 of differentiation, organoids consisted of a
cardiomyocyte (CM) core surrounded by stromal cells
embedded within a proteoglycan-rich extracellular matrix and
an outer layer of Tbx18+ epicardial-like cells. The organoids
grew signicantly with culture duration, from ~250µm to
>1mm in diameter by day 100 and survive for more
than 1 year. Gene expression and patch clamp analysis
revealed that the organoids were comprised of >70-80%
atrial/nodal-like CMs and dissociated CMs exhibited greater
sarcomere alignment and more elongated morphology than
typical hiPSC-CMs. After 40 days of differentiation, CD31+
endothelial cells were interspersed among CM and in-
creased in number by day 100. As early as day 10 of
differentiation, organoids exhibited spontaneous contractili-
ty and calcium ux transients which became more respon-
sive to external eld stimulation over time. After 100 days,
organoids exhibited little spontaneous beating but were
capable of being paced up to 4-6 Hz, revealing an increased
functional maturation state. Overall, these results describe
the rst cardiac organoid from human iPSCs that recapitulates
the co-emergence and self-organization of multiple cardiac
cell populations as well as physiological heart function,
thereby establishing an in vitro model to examine mechanisms
regulating human embryonic morphogenesis and cardiac
maturation.
Funding Source
CIRM.
Keywords: Organoids; cardiogenesis; stem cell-derived
cardiomyocytes
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AMSTERDAM NETHERLANDS
Poster Abstracts
114
REGENERATION OF FULL THICKNESS SKIN AND
HAIR FOLLICLES IN WOUNDS TREATED WITH AN
AUTOLOGOUS HOMOLOGOUS SKIN CONSTRUCT
IN PRECLINICAL PORCINE MODEL
Baetz, Nicholas1, Miller, Devin2, Yalanis, Georgia2,
Labroo, Pratima1, Ifediba, Marytheresa1, Irvin, Jennifer1,
Kirk, Natalie2, Sieverts, Michael2, Stauffer, Kendall2,
Miess, James1, Robinson, Ian1, Roth, Stephanie1, Tracy, Lon1,
Lambert, Lyssa2, Krishnan, Naveen3, Abdo, Joe1,
Sopko, Nikolai1
1Research & Development, PolarityTE, Inc, Salt Lake City, UT,
USA, 2Translational Medicine and Product Development,
PolarityTE, Salt Lake City, UT, USA, 3Clinical Operations,
PolarityTE, Salt Lake City, UT, USA
Standard of care for cutaneous full-thickness wounds is
limited by incomplete healing, graft failure, scarring and
contraction, donor site morbidity and lack of appendage
formation. A novel approach using an autologous homologous
skin construct (AHSC) (SkinTETM, PolarityTE, Salt Lake City,
UT) was evaluated for full-thickness skin regeneration in a
pre-clinical porcine model. AHSC, which is comprised of
minimally polarized functional units created from a piece of
full thickness skin involving a process of micro-wounding
that activates the endogenous stem cells that can generate
all components of the skin and are returned immediately to
the wound bed to expand within the wound itself and not ex
vivo. 36 full-thickness wounds on the dorsum of female
Yorkshire swine were treated with AHSC or split-thickness
skin graft (STSG). For 6 months, healing was documented with
digital single-lens reex (DSLR) and stereoscopic imaging.
Excised defect areas were evaluated by brighteld, confocal,
and scanning electron microscopy (SEM). Molecular
composition of wounds was assessed with Raman
spectroscopy. Gene expression was analyzed using stem cell,
apoptosis, and extracellular pathway PCR arrays. Wounds
treated with the AHSC demonstrated improved healing,
decreased contracture, and development of hair follicles and
dermal appendages on macroscopic and uorescent imag-
ing. Collagen organization characterization via SEM was
consistent with native skin. Raman intensity proles demon-
strate similar bond energy peaks corresponding to collagens in
native and AHSC-treated wounds. RNA extracted from
AHSC-treated wounds demonstrated that stem cell, apoptotic,
extracellular matrix and adhesion gene expression is similar
to native tissue. Stereoscopic, uorescent, and SEM imaging
of hair follicles from AHSC-treated areas have morphology,
organization, and nuclear content consistent with hair follicles
from native skin. Raman intensity proles have correlative
peaks among native and AHSC regenerated hair. Preclinical
application demonstrated complete healing with appendage
and pigmentation development along with improved function-
al outcome compared to STSGs suggesting AHSC is capable
of regenerating fully functional skin in pre-clinical models.
Keywords: autologous homologous skin construct; wound
healing; functional full thickness skin regeneration
115
CARTILAGE MICRO-TISSUES MIMIC BONE DEVEL-
OPMENT AND REGENERATE LONG-BONE DEFECTS
Nilsson Hall, Gabriella1, Mendes, Luís F.1, Gklava, Charikleia1,
Geris, Liesbet2, Papantoniou, Ioannis1, Luyten, Frank1
1Skeletal Biology and Engineering Research Center, KU
Leuven, Belgium, 2Biomechanics Research Unit, Université
de Liège, Belgium
A major limitation in Tissue Engineering is the ability to control
complexity within 3D engineered constructs. Diffusion
limitations lead to the development of uncontrolled or even
adverse environments leading to inappropriate stem cell fate
decisions and cell death. Recently, bottom-up strategies have
been introduced advocating the use of smaller tissue modules
as building blocks for the formation of larger implants. This
strategy in combination with recapitulation of the develop-
mental process of bone formation; endochondral ossication,
where a cartilage intermediate is transformed into bone may
possess great potential for bone regeneration. Human perioste-
al derived progenitor cells were seeded on non-adherent
agarose surfaces containing microwells. This allowed initial
condensation and the formation of µ-aggregates with con-
trolled-size. Chondrogenic differentiation resulted in 3D
cartilage intermediate µTissues, positive for safranin-o,
indicating deposition of mature cartilaginous extracellular
matrix while RNA sequencing analysis revealed the onset of
hypertrophy following signaling patterns analogous to
endochondral ossication. When single µTissues were
implanted subcutaneously, they developed into µBone organs
exhibiting a central compartment with blood vessels. Interest-
ingly, when these µTissues were fused via self-assembly into
larger implants (2mm Ø) in vitro they formed one larger bone
organ containing a cortex and a prominent bone marrow
compartment after subcutaneous implantation. As control we
used implants formed by cells cultured in macro-pellet format
in the same media formulation and containing the same
amount of cells as the bottom-up assembled implant. In
contrary to the fused µTissues, the macro-pellet demonstrat-
ed a large brotic tissue domain at the center of the implant.
Finally, using fused µTissues, we evidenced reproducible
successful healing of a murine critical-sized bone defect with
presence of implanted human cells after 8 weeks. Our
ndings provide a rst step in establishing a biomanufactur-
ing pipeline for robust production of fracture callus-like
cartilage tissue intermediates for bone regeneration. Although
demonstrated for a skeletal application, the strategy present-
ed could be applied for the manufacturing of a variety of
tissues.
Funding Source
Research funded by Research Foundation Flanders (FWO)
1S05116N and 12O7916N, the European Research Council
under the European Union’s 7th Framework Program
(FP/2007-2013)/ERC n.279100 and 249191 and the KU
Leuven fund (GOA/13/016).
Keywords: Developmental Tissue Engineering; Endochondral
ossication; Fracture healing
77
AMSTERDAM NETHERLANDS
Poster Abstracts
116
DEVELOPMENT OF SUBSTRATE PLATFORMS FOR
GENERATING HUMAN PLURIPOTENT STEM
CELL-DERIVED OLIGODENDROCYTES
Cho, Ann-Na1, Cho, Younghak2, Im, Sung Gap2, Cho, Seung-Woo1
1Biotechnology, Yonsei University, Seoul, Korea, 2Department
of Chemical and Biomolecular Engineering, Korea Advanced
Institute of Science and Technology (KAIST), Daejeon, Korea
Oligodendrocyte poses a crucial role to facilitate transmission
of electrical signals by forming myelin on neurons in central
nerve system (CNS). Thus, defects of oligodendrocytes cause
critical neurodegenerative disease, cognitive and behavioral
disorders. Despite the important roles of oligodendrocyte in
CNS, there have been few studies to develop effective culture
platforms for scalable production of functional oligodendro-
cytes from stem cells. In this study, we developed a novel
substrate platform to efciently generate oligodendrocytes
from human induced pluripotent stem cells (hiPSCs). Through
a suspension culture of hiPSC-derived oligodendrocyte
progenitors on the engineered substrate platform, we could
shorten time required for oligodendrocyte differentiation and
maturation, and produce a large quantity of functional
oligodendrocytes. Oligodendrocytes generated from our
substrate platforms showed increased expression of several
oligodendrocyte markers including myelination binding
protein (MBP), one of the most important indicators of
oligodendrocyte maturation and myelination functionality.
Here we may conclude that our novel culture platform enables
efcient and scalable production of oligodendrocytes from
stem cells, which would be applied for demyelination disease
model and regenerative medicine for the treatment of
demyelination diseases.
Funding Source
This research was supported by a grant (2015M3A9B4071076)
from the Bio & Medical Technology Development Program of
the National Research Foundation of Korea funded by the
Korean government, MSIT and Brain Korea 21 plus program.
Keywords: Human induced pluripotent stem cell; Oligoden-
drocyte; Culture substrate
117
ESTABLISHMENT OF THREE-DIMENSIONAL IN
VITRO DIGESTIVE SYSTEM WITH A TRI-CULTURE
OF ORGANOIDS
Kim, Suran, Jin, Yoonhee, Kim, Jin, Min, Sungjin, Lee, Jung
Seung, Cho, Senug-Woo
Biotechnology, Yonsei University, Seoul, Korea
Recently, there is an increasing interest on the researches
of the digestive systems which play important roles in
the human pathophysiology. The development of
three-dimensional (3D) organoid culture technology enables
to establish in vitro models that possess cellular complexity
and resemble native organs, leading to more precise in vitro
studies of the digestive systems. Despite promising aspects
of organoid technology, a single organoid culture is insufcient
to reconstitute the dynamic communications and interactions
between various digestive organs in the body. Therefore, here
we developed a multi-organ cultivation platform of digestive
systems with stem cell-derived gastric organoids, intestinal
organoids, and hepatic organoids. We conrmed that
phenotypes of each organoid were maintained during the
tri-culture. We also observed cross-talk between
organoids in this digestive system such as bile acid‐mediated
regulation of the hepatic enzyme for bile acid synthesis in
co-culture of intestinal organoids and hepatic organoids. Our
in vitro digestive system with multiple organoid culture can
recapitulate in vivo-like complex phenomena and interactions
more precisely than a single organoid model.
Funding Source
This research was supported by Brain Korea 21 plus (BK-
21PLUS) program, and a grant (2018M3A9H1021382) from
National Research Foundation of Korea (NRF) funded by the
Ministry of Science and ICT (MSIT), Republic of Korea.
Keywords: organoid; digestive system; tri-culture
118
CULTIVATION OF HUMAN BRAIN-LIKE TISSUE
MODELS IN A THREE-DIMENSIONAL PRINTABLE
MINIBIOREACTOR
Erharter, Anita1,2, Hagenbuchner, Judith3,
Außerlechner, Michael J.4, Edenhofer, Frank1,2
1Center for Molecular Biosciences, University of Innsbruck,
Austria, 2Genomics, Stem Cell Biology and Regenerative
Medicine, University of Innsbruck, Austria, 3Department of
Pediatrics II, Medical University Innsbruck, Austria,
4Department of Pediatrics I, 3D Bioprinting Lab,
Medical University Innsbruck, Austria
Standard three-dimensional (3D) tissue-like in vitro
cultivation involves agitation-mediating devices such as
orbital shakers or stirred-tank bioreactors, which facilitate
oxygen and nutrient uptake and help forming compact
spheroids. However, these devices do not support
parallelization while allowing minimal condition testing only.
Here we show a customized, 3D-printed mini-bioreactor that
can be utilized for brain-like tissue cultivation in up to 48
(12-well format) or 96 (24-well format) conditions in parallel
due to its stackable 4-levels setup. We nd that our
mini-bioreactor in combination with a modied cultivation
protocol for cerebral organoids is benecial for standardized
growth, shape and nutrient supply. These optimized conditions
result in the formation of brain-like tissues with an overall
more physiological, non-spheroid morphology as compared
to standard agitation cultivation. Moreover, we show that
human neuroblastoma-derived spheroids proted from
cultivation in the mini-bioreactor judged by an average 2-fold
increase in diameter. We anticipate our optimized procedure
to be of high value for cost-efcient and robust realization of
in vitro 3D models of brain and tumor development needed to
parallelize drug screening at small scale.
Keywords: Organoid; 3D-printing; Neuroscience
78
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Poster Abstracts
119
ENTEROENDOCRINE CELL-DERIVED HORMONE A
IS INVOLVED IN THE INTESTINAL HOMEOSTASIS
BY DIRECTING DIFFERENTIATION OF THE MOUSE
INTESTINAL ORGANOID
Seo, Yoojin1, Yeo, Dain1, Ahn, Ji-Su2, Oh, Su-jeong1,
Yang, Ji Won1, Shin, Ye Young1, Kim, Hyung-Sik1
1Department of Life Science in Dentistry, School of Dentistry,
Pusan National University, Yangsan, Korea, 2Biomedical
Research Institute, Pusan National University School of
Medicine, Yangsan, Korea
Organoids can be utilized as a modeling system for the
investigation of stem cell biology, organ development and
disease progression, as well as for drug discovery.
Here, we isolated mouse small intestinal crypts and cultured
intestinal epithelial organoids, one of the most well-established
organoid system. It develops a highly organized structure
with both adult stem cell niches and fully differentiated
populations, which enable us to study the nature of stem cell
homeostasis. Interestingly, as the largest endocrine system
in the body, enteroendocrine cells (EECs) produce the highest
level of hormones and bioactive molecules despite that they
comprise only 1% of the intestinal epithelium. In this study,
we focused on EEC-derived endogenous signals to evaluate
their impact on the intestinal homeostasis. It was noted that
one of EEC-secreted hormone A was impeded the normal
generation of intestinal organoids; upon treatment of A,
organoid growth was retarded and the typical budding
pattern was almost disappeared, resulting in the round to
oval shaped-organoids. The epithelial lining was intact and
budding ability was restored after A withdrawal, suggesting
that A did not induce epithelial cell death. We found that the
morphology of A-treated organoid was similar to that of
IWP-2(Wnt inhibitor)-, DAPT (Notch inhibitor)-and
U0126(MEK inhibitor)-treated organoid. Also, re-budding
process after A removal was accelerated with Chir99021
and Epiregulin, which activates Wnt and EGF pathway,
respectively. Since those signaling are important to maintain
ISC population, we performed qPCR to screen the ISC and
differentiated intestinal cell markers. Importantly, both active
ISC and proliferation markers are down-regulated, while
secretory lineage markers such as Neurogenin3, defensin-a
and Gob5 were increased upon A treatment. Immunohisto-
chemistry analysis also revealed that lineage-specic
differentiation was induced after A treatment. These data
suggest the novel endogenous impact of EEC-derived
hormones on ISC maintenance, differentiation and intestinal
homeostasis.
Funding Source
This work was supported by the National Research Founda-
tion of Korea (NRF) grant funded by the Korea government
(MSIT) (No. NRF-2018R1A5A2023879) and and partially
supported by a grant from Pusan National University.
Keywords: intestinal organoid; enteroendocrine cell; wnt
signaling
120
IN VITRO MODELING OF UROLOGICAL DISEASES
BY THREE-DIMENSIONAL PRINTED MINIATURE
BLADDER
Kim, Eunjee, Kim, Seungeun, Kim, Yubin, Kwon, Jeongwoo,
Shin, Kunyoo
Life Sciences, Pohang University of Science and Technology
(POSTECH), Pohang, Korea
Despite tremendous efforts for studying in vivo physiology
and disease modeling with organoid system, organoids do
not faithfully recapitulate in vivo tissues because of the lack
of niche or microenvironment. Here, we rst established a
novel in vitro co-culture platform that structurally and
functionally similar with in vivo tissues, normal bladder or
bladder tumor. We demonstrated that urological diseases,
such as urinary tract infection (UTI) or bladder cancer, can
be modeled in our co-culture system, recapitulating in vivo
phenomena. Our 3D model provides an advanced tool
representing in vivo tissues with implication of potential
use for personalized medicine and disease modeling.
Keywords: Organoid; Co-culture; Disease modeling
121
DEVELOPMENT AND CHARACTERISATION OF
CHOROID PLEXUS ORGANOIDS
Pellegrini, Laura1, Chadwick, Jessica1, Begum, Farida2,
Skehel, Mark2, Lancaster, Madeline1
1Cell Biology, Laboratory of Molecular Biology - MRC,
Cambridge, UK, 2Mass Spectrometry, Laboratory of Molecular
Biology - MRC, Cambridge, UK
The choroid plexus is a highly conserved and surprisingly
understudied secretory tissue in the brain. This tissue is
enlarged in humans proportionally to our brain size and
displays a number of important functions in the brain such
as forming a protective epithelial barrier and secreting the
cerebrospinal uid (CSF). The CSF is important for the
maintenance of physiological levels of nutrients in the brain,
for the transport of signalling molecules and growth factors
and for its protective role in the regulation of intracranial
pressure. This uid regulates several aspects of develop-
ment in two fundamental ways: by exerting ventricular
pressure that may be necessary for driving brain expansion
and by regulating neural stem cell proliferation and differen-
tiation in the brain. To explore the role of the choroid
plexus-CSF system in early stages of human brain develop-
ment, we recently established a protocol to generate choroid
plexus organoids using a combination of signalling molecules
that are physiologically present during the stages of devel-
opment of this tissue. More interestingly, not only do these
organoids develop the choroid plexus but they also recapitu-
late fundamental functions of this tissue, namely secretion
and formation of a tight epithelial barrier. We detected the
presence of choroid plexus specic water channels and
79
AMSTERDAM NETHERLANDS
Poster Abstracts
transporters localised on the apical brush border of the
choroid plexus epithelium by histological and EM analysis.
These tissues displayed tight junctions forming the epithelial
barrier, and we noticed the formation of large uid-lled
cysts protruding from the organoids, the contents of which,
analysed by mass spectrometry, highly resembles human
embryonic CSF. In conclusion, we believe this system
represents an excellent tool to study the role of the choroid
plexus-CSF system in human brain development.
Keywords: Organoids; Choroid plexus; Cerebrospinal Fluid
122
TBI MODELING THROUGH SELF-ORGANIZED
HUMAN FORE-BRAIN ORGANOIDS FROM SINGLE
CELL SOURCE
Kim, Min-Soo, Choi, Soon Won, Kang, Kyung-Sun
College of Veterinary Medicine, Seoul National University,
Seoul, Korea
Three-dimensional structure brain organoid represents
human brain in vitro models that can be usefully utilized to
study early neuronal development, neurodegenerative
disease, and human based drug screening. However,
long-term culture and heterogeneous generations of brain
organoid are challenges to overcome. Moreover, in vitro
traumatic brain injury model has not been previously
reported in human brain organoids. Here, we generate
self-organized human forebrain organoid from adult dermal
broblast-derived neural stem cells, which can recapitulate
traumatic brain injury model using a 3D cell culture system.
For homogenous brain organoid generation, similar-sized
organoids were produced using same number of cells
embedded in matrigel and maintained in stationary culture.
These organoids were characterized by apical-basal polarity
and self-formation of neuroepithelium, which display
premature neuronal development. For maturation of the
organoids, self-organized neuroepithelium was transferred
to spinner ask and maintained in agitated culture.
To analyze these fore-brain organoids, we performed
immunohistochemistry and quantify cell composition,
division rate, and relative gene expression level. Based on
our data, cortical plates in mature fore-brain organoids
were self-organized, displaying two distinct layers; inner
layer (ventricular zone) and outer layer (early and late
cortical-plate zone). In addition, we applied our fore-brain
organoid to in vitro traumatic brain injury model system. In
this model, we conrmed cell apoptosis and microglia
activation in the traumatic lesions of the organoids.
We also investigated neuroregenerative patterns in
fore-brain organoids during recovery. Overall, this study
suggests that human fore-brain organoids generated from
neural stem cells can provide new opportunities to develop
drug screening platforms and personalized disease
modeling.
Keywords: Fore-brain; NSC; TBI
123
FUNCTIONAL DIFFERENCES AND SIMILARITIES
BETWEEN HIPSC- AND PERIPHERAL BLOOD-
DERIVED MONOCYTES AND MACROPHAGES
Cao, Xu, Yakala, Gopala K., van den Hil, Francijna, Cochrane,
Amy, Mummery, Christine L., Orlova, Valeria V.
Anatomy and Embryology, Leiden University Medical Centre,
Leiden, Netherlands
The ability to obtain high quality, functionally characterized
monocytes and macrophages from human induced
pluripotent stem cells (hiPSCs) would be of great benet for
modelling diseases in which inammation plays an important
role. Here we developed a fully dened, efcient differentiation
protocol to derive monocytes and macrophage subtypes
from multiple hiPSC lines. We compared hiPSC-macrophages
functionally and for gene expression and cytokine
production with primary peripheral blood-derived
monocytes/macrophages (PBDMs) and found similarities
but also important differences. hiPSC-macrophages showed
higher endocytotic activity for AcLDL and efferocytosis than
PBDMs but similar bacterial phagocytosis. Interestingly,
hiPSC-macrophages also showed high tumor cell
phagocytosis activity indicating potential value of these cells
for drug screening in cancer immunotherapy. In summary,
hiPSC-macrophages and PBDMs showed signicant
similarities but some functional differences, indicating that
they could be complementary to PBDMs in many functional
assays in vitro but with the additional advantage that they
could be derived from patients with inammatory diseases
of interest to understand the nature of inammatory defects.
Keywords: macrophages; hiPSCs; phagocytosis
124
INDUCED PLURIPOTENT STEM CELLS (IPSC)
GENERATED FROM ERYTHROID CELLS OF
POLYCYSTIC KIDNEY DISEASE 1 (PKD1) PATIENT
DIFFERENTIATED INTO THREE GERM LAYERS:
POTENTIAL SOURCE FOR RENAL ORGANOIDS
Villela, Roberta F.1, Boim, Mirian2, Lojudice, Fernando3,
Sogayar, Mari Cleide3
1UNIFESP, Renal Division, Department of Medicine Federal
University of São Paulo, Brazil, 2Nefrologia, UNIFESP, Sao
Paulo, Brazil, 3Biotecnologia, NUCEL - USP, Sao Paulo, Brazil
Human iPSC (hiPSC) can be obtained from any somatic cell.
Here we showed that it is possible to generate hiPSC from
circulating erythroid progenitors cells of patients with PKD1.
The choice of erythroid cells for reprogramming is due
minimally invasive procedure, genomic integrity and epigenetic
memory. From these hiPSC it will be possible to generate
renal organoids. Blood samples was donated by a patient
with PKD1 and by a control health person, according to
approved institutional procedures. Erythroid progenitor cells
were separated from whole blood and expanded in vitro.
Erythroid cell colonies were identied by expression of the
transferrin receptor (CD71) and Glycophorin A (235A). The
80
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Poster Abstracts
efciency of the method was evaluated as 28.6% according to
GFP expression, being 50% CD71 positive) and 67% positive
for 235A. Dedifferentiation was induced by transfection
(electroporation) with episomal vectors containing 5
reprogramming factors (Oct-4, Sox2, Lin28, L-Myc and klf4).
After transfection, typical images of iPSCs-like colony began
to appear as large size, tight cell packing and well dened
borders. After 10 passages, endogenous pluripotency was
assessed by Imunouorescence using specic markers
as Nestin, ATX2, GATA4 and SOX2. hiPSC were able to
differentiated into the three layers, ectoderm, mesoderm and
endoderm. Each layer was characterized by the expression
of specic markers for endoderm (GATA4 and SOX147),
mesoderm (CXCR4) and ectoderm (PAX6) detected by
RT-PCR. Additionally, through immunostaining technique,
differentiated cells of each layer were labeled by specic
markers, including FOXA2 and OTX2 for endoderm, smooth
muscle actin (SMA) and Brachyury (T) for mesoderm and
Nestin for ectoderm. These results indicate that the easily
obtained circulating erytroid progenitor cells from either, a
PKD1 patient and a health person, could be reprogrammed
to reach a pluripotency phase. As a perspective, from these
hiPSC it will be possible to generate organoids, which similar
architecture to the organ of interest as for example the kidney.
The success in generate organoids from PKD1 patients will
rise the possibility to better understand the PKD1
pathophysiology and the mechanisms of renal cysts formation
Funding Source
Research supported by FAPESP and cNPQ.
Keywords: iPS cells; erythroid cells; organoids
125
SIMPLIFIED DIFFERENTIATION OF TRANSPLANT-
ABLE NEURONAL CELLS FROM HES-CELLS
Fosby, Anne Helene
Department of Physiology, University of Oslo, Norway
Human embryonic stem cells are an attractive renewable
source of various cell-types for transplantation and
mechanistic studies. The existing protocols for making
neuronal cells from stem cells are however mostly
inefcient, time-consuming, intricate, pricey and/or giving
low yields - resulting in low availability of relevant cells.
Here we show how human embryonic stem cells can be
differentiated into aggregates of neuronal cells of spinal
cord identity by simple means – resulting in efcient, rapid
differentiation of sufcient functional transplantable cells for
downstream applications. As this gives around 3 million
cells for under 30$ and 3h of hands-on time,
purpose-specic optimisation should ensure its
usefulness regarding both research and clinical applications
relevant to pathological conditions affecting the spinal cord.
Keywords: human embryonic stem cells; organoids; spinal
cord
126
MODELLING REACTIVE GLIOSIS IN HUMAN
RETINA ORGANOIDS
Voelkner, Manuela1,2, Wagner, Felix1, Ebner, Lynn1, Del Toro
Runzer, Claudia1, Hermann, Andreas3, Khattak, Shahryar4,
Karl, Mike O.1,2
1AG Karl, German Center for Neurodegenerative Diseases
Dresden, Germany, 2Technische Universität Dresden, Center for
Molecular and Cellular Bioengineering, Center for Regenerative
Therapies Dresden, Germany, 3Neurology, German Center for
Neurodegenerative Diseases Dresden and Technische Universi-
tät Dresden, Faculty of Medicine Carl Gustav Carus, Department
of Neurology, Dresden, Germany, 4Stem Cell Platform, Tech-
nische Universität Dresden, Center for Molecular and Cellular
Bioengineering, Center for Regenerative Therapies Dresden,
Germany
Retinal organoids derived from human induced pluripotent
stem cell (hiPSC) are a potential experimental model system
for studies on neuronal degeneration and regeneration. Here,
we sought to develop a human retinal organoid model to study
reactive gliosis, a glial response commonly associated with
neurodegenerative diseases of the mammalian central
nervous system and potentially related with regenerative
capacity, at least in some types of vertebrates. We established
that the major types of retinal neurons, rod and cone photore-
ceptors, and glia, the radial Müller glia, develop and become
postmitotic by culture day 150. Of note, human photoreceptors
and Müller glia in retina organoids under baseline culture
conditions did not present hallmarks of cell death or reactive
gliosis, e.g. glial brillary acidic protein (GFAP), respectively. In
contrast, reactive gliosis is well-known to be strongly induced
upon organotypic culture of primary animal and human retina
and brain tissues. Thus, we hypothesized that the retinal
organoid system might provide a powerful tool for disease
modeling. To test this hypothesis, we challenged retinal
organoids with signaling factors previously associated with
retinal damage, reactive gliosis and glial derived neuronal
regeneration, at least in animal models. Thereby, we estab-
lished a human retinal organoids model replicating several
histopathologies that develop at the same time dynamically
within 10 days (N>4 experiments, > 20 oragnoids per N in >3
different hiPSC lines): Of note, we observed acute and severe
loss of photoreceptor neurons, hallmarks of reactive gliosis –
including GFAP expression and cell proliferation of Müller glia;
as well as impaired retinal stratication. In sum, our data
suggest that acute, severe and complex histopathologies can
be modeled in human retinal organoids. We conclude, that the
organoid system offers a powerful model system and opportu-
nity to study potential mechanisms of retinal degeneration,
reactive gliosis and regeneration.
Keywords: organoid; disease model; retina
81
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Poster Abstracts
127
CHARACTERIZATION OF HUMAN PANCREAS
ORGANOIDS FOR CELL-BASED THERAPY OF
TYPE 1 DIABETES
Piras, Roberta1, Dossena, Marta1, Cherubini, Sander1,
Barilani, Mario1, Salanitro, Francesca1, Rusconi, Francesco1,
Pera-Munoz, Marta1, Buono, Giuseppe1, Colombo, Beatrice1,
Santana-Gomes, Luana1, Dugnani, Erica2, Piemonti, Lorenzo 3,
Lazzari, Lorenza1
1Unit of Regenerative Medicine - Cell Factory, Fondazione
IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milano, Italy,
2Beta Cell Biology Unit, Diabetes Research Institute IRCCS San
Raffaele Scientic Institute, Milano, Italy, 3Beta Cell Biology Unit,
Vita-Salute San Raffaele University, Milano, Italy
Organoids are three-dimensional in-vitro-grown cell clusters
that recapitulate many key features of the native organ. In
the groundbreaking European project LSFM4LIFE
(www.lsfm4life.eu) directed towards the therapy of type 1
diabetes (T1D) with human pancreas organoids (hPO). The
aim is the isolation and the therapeutic-scale manufacture
of hPO for cellular therapy of T1D. In this context, starting from
discarded pancreatic tissues we developed a large-scale
process in order to obtain large quantities of undifferentiated
organoids and we set up a standardized protocol for passag-
ing and culturing the hPO skipping enzymatic digestions and
operator-dependent hPO progenitor picking. To give a new
identity to our hPO we dened an experimental strategy
aimed to deeply characterize our product. We rst have
designed a quality control gene card containing expression
proles for each developmental and differentiation pancreat-
ic stages. We analyzed pancreatic markers for the bipotent
progenitors (PDX1, SOX9, HNF1B), endocrine progenitors
(NGN3, NEUROD1), adult epithelial stem cells (LGR5), acinar
cells (AMY), ductal cells (MUC1, KRT19, EPCAM) and mature
endocrine cells (INS, GCG, IAPP, SST). Concerning the
immunophenotype we have determined the identity of the
starting material and the hPO purity during passages by ow
cytometry. We tested relevant markers of acinar (UEA-1),
ductal (MUC1, CXCR4, EPCAM), endothelial (PECAM,
MCAM) and mesenchymal (CD90, CD73) compartments. To
address the healthy/quiescent status of hPO during passag-
es, we performed a RT2 Proler PCR-array till passage 11. In
conclusion, we have been able to dene a new hPO prole at
the cellular and molecular level that will pave the way to
dening GMP quality controls.
Keywords: Pancreas Organoids; Diabetes; GMP quality
control
128
P21WAF1/CIP1, MUSASHI-1, AND KRÜPPEL-LIKE
FACTOR 4 REGULATE ACTIVATION OF MOUSE
RESERVE INTESTINAL STEM CELLS AFTER GAMMA
RADIATION-INDUCED INJURY IN VIVO
Orzechowska, Emilia J., Bialkowska, Agnieszka B., Yang,
Vincent W.
Department of Medicine, Stony Brook University, Stony Brook,
NY, USA
The intestinal epithelium is a rapidly self-renewing tissue,
maintained by active intestinal stem cells. After injury, BMI1
expressing reserve intestinal stem cells (rISCs) serve as a
source of regeneration, but the molecular mechanisms
regulating rISC remains unclear. Previously, we showed that
Krüppel-like factor 4 (KLF4) plays a radioprotective role in the
intestinal epithelium by modulating BMI1+ cells’ response
to
radiation injury. Here, we investigated the mechanisms by
which p21Waf1/Cip1, Musashi-1 (MSI1) and KLF4 coordinate to
regenerate the intestinal epithelium. Bmi1CreER;Rosa26eYFP and
Bmi1Cre
ER;Rosa26eYFP;Klf4/ mice were exposed to 12 Gy
total-body γ-irradiation (TBI). Tamoxifen was administered 48
h before or 24 h after TBI to induce recombination in BMI1+
cells for lineage tracing (YFP+ cells). Small intestine was
collected 0, 6, 48, 72 or 96 h after TBI and examined by
immunouorescent stain. We observed that during apoptotic
phase (0-48 h post-TBI), surviving rISC-lineage YFP+ cells
were located around +4 position and in transit-amplifying
zone. Shortly after injury YFP+ cells started expressing p21
with a peak at 48 h post-TBI at nearly 60% of YFP+ cells.
During regenerative phase (48-96 h post-TBI) p21 expression
started to diminish while 70% of YFP+ cells co-expressed
MSI1. RT-qPCR analysis of FACS-isolated YFP+ cells con-
rmed the inverse relationship between Cdkn1a and Msi1
expression. Luciferase assay showed that mouse MSI1
inhibits p21 translation, which was further conrmed by
western blot analysis of irradiated HCT116 cells with or
without human MSI1 overexpression. Furthermore, we
observed that 96 h post-TBI KLF4 expression was increased,
whereas MSI1 started to diminish following regenerative
phase. Using luciferase assay, we showed that mouse KLF4
negatively regulates Msi1 transcription, suggesting KLF4
plays an important role in the initiation of the normalization
phase. Collectively, these data suggest that p21, MSI1, and
KLF4 are key factors that regulate survival, proliferation, and
normalization of rISC-lineages during regeneration of the
intestinal epithelium.
Funding Source
This study is supported by CA084197 and DK052230 grants
awarded to V. W. Yang.
Keywords: mouse reserve intestinal stem cells (rISCs);
gamma radiation-induced injury; KLF4, MSI1, p21
82
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Poster Abstracts
129
EFFECTS OF L-TYPE CALCIUM CHANNEL
REGULATORS ON CHONDROGENIC POTENTIAL OF
3D CULTURED HUMAN MESENCHYMAL STEM
CELLS UNDER MECHANICAL LOAD
Bernotiene, Eiva1, Uzieliene, Ilona2, Gudiskyte, Giedre2,
Denkovskij, Jaroslav2, Porvaneckas, Narunas3, Aleksiuk,
Viktorija2, Bagdonas, Edvardas2, Bironaite, Daiva2, Kvederas,
Giedrius3, Mobasheri, Ali2
1Department of Regenerative Medicine, Centre for Innovative
Medicine, Vilnius, Lithuania, 2Department of Regenerative
Medicine, State Research Institute Centre for Innovative
Medicine, Vilnius, Lithuania, 3Clinic of Rheumatology,
Orthopaedics-Traumatology and Reconstructive Surgery,
Faculty of Medicine, Vilnius University, Lithuania
Osteoarthritis (OA) is characterized by the progressive
deterioration and loss of articular cartilage and is actually
one of the most common, costly, and disabling forms of
articular diseases, while there is no effective treatment for it.
Under physiologic conditions, moderate physical activity
reduces the risk of cartilage deterioration, while in an altered
biochemical and/or biomechanical setting, mechanical
factors can lead to the OA development. One of the earliest
chondrocyte responses to extracellular mechanical input is
a transient increase in intracellular Ca2+ levels. Voltage-
dependent Ca2+ channels that are regulated by different
physical signals play critical role in controlling the intracellular
Ca2+ responses of in situ chondrocyte in the loaded
cartilage. Arterial hypertension is a comorbidity in more than
75% of patients with OA. Long-term consumption of antihy-
pertensive drugs L-type Ca2+ channel blockers may lead to
the altered performance of chondrocyte chanellome and
modulate Ca2+ oscillations, resulting in altered synthesis of
extracellular matrix (ECM). We analyzed the effects of
abundantly used anti-hypertensive agents – Ca2+ channel
blockers on human mesenchymal stem cell (hMSC)-derived
organoids in hydrogel scaffolds under chondrogenic
conditions, and compared them to the effects in human OA
cartilage explants. Mechanical compression was applied
using Flexcell Fx500 for 1 hour daily. Increased production
of ECM has been determined by immunohistochemistry in
the 3D cultured hMSCs in the presence or absence of
Nifedipine or Bay K8644. MSC organoids and OA cartilage
explants differentially responded to the mechanical com-
pression and Ca2+ regulators. Mechanical compression in
the presence of Nifedipine resulted in modulated expression
of Sox9, MMPs and other genes related to chondrogenesis
and catabolism. These data are important for consideration
of chondroprotective approach in patients having comorbidi-
ties of cardiovascular diseases and osteoarthritis.
Funding Source
This research is funded by the European Social Fund
according to the activity ‘Improvement of researchers’
qualication by implementing world-class R&D projects’
through Measure No. 09.3.3-LMT-K-712; 2017-2021.
Keywords: Chondrogenesis of MSCs; L-type Ca2+ channel
regulators; Osteoarthritis
130
HUMAN ATRIAL TISSUES FOR TESTING ATRIAL-
SPECIFIC PHARMACOLOGICAL COMPOUNDS OR
MODELING ATRIAL FIBRILLATION
Schwach, Verena, Ribeiro, Marcelo C., Arbelaez, Jose,
ten Den, Simone, Passier, Robert
Department of Applied Stem Cell Technologies, University
of Twente, Enschede, Netherlands
Atrial brillation (AF) is the most common condition with
abnormal atrial rhythm affecting more than 33 million
patients worldwide. Unfortunately, most antiarrhythmic
drugs are non-selective for the atria and risk fatal ventricular
pro-arrhythmic events. Thus, it is very important to improve our
understanding of underlying causes of AF and to develop
more advanced models to predict atrial selectivity. By
modulating retinoic acid signaling during differentiation
of human embryonic stem cells (hESCs) towards
cardiomyocytes (CMs), we generated atrial-like and
ventricular-like CMs. Expression of atrial-specic genes,
including the ion channel genes KCNA5 (encoding Kv1.5
channels) and KCNJ3 (encoding Kir 3.1) was strongly
enriched in hESC-atrial CMs when compared to their
ventricular counterparts. These ion channel genes were
regulated by atrial-enriched chick ovalbumin upstream
promoter (COUP)-TF transcription factors I and II.
Importantly, these hESC-derived atrial CMs could predict
atrial-selectivity of pharmacological compounds.
To generate pure populations of atrial- and ventricular-like
CMs, we then targeted mCherry to the COUP-TFII genomic
locus in hESCs expressing GFP from the NKX2.5 locus.
This dual atrial NKX2.5eGFP/+-COUP-TFIImCherry/+ reporter
allowed identication and selection of GFP+/mCherry+ atrial
CMs from heterogeneous cultures following cardiac
differentiation. These GFP+/mCherry+ cells exhibited
transcriptional and functional properties of atrial CMs,
whereas GFP+/mCherry- CMs displayed ventricular
characteristics. To develop a more advanced model for drug
testing or modeling AF, in a next step, we now generated
3-dimensional atrial and ventricular cardiac tissues which
allow measurement of absolute contraction force. These
atrial and ventricular tissues not only offer the possibility to
compare functional aspects of pure cardiac tissues with
cardiac tissue composed of CMs and non-CMs like
endothelial cells and broblasts, but also allow functional
testing of atrial-specic drugs, such as carbachol or
DPO-1. Preliminary functional data on atrial versus
ventricular tissues will be presented. In the future, it will be
of interest to generate tissues using CMs differentiated from
mutated hESC or hiPSC lines to model genetic facets of AF.
Keywords: Atrial cardiac tissue; Drug testing; Atrial disease
modeling
83
AMSTERDAM NETHERLANDS
Poster Abstracts
131
RETINAL ORGANOIDS: AN ACCELERATED METHOD
OF GENERATING GANGLION CELLS FOR DISEASE
MODELS
Wagstaff, Philip1, ten Asbroek, Anneloor LMA1, Boon,
Camiel JF2, Karnebeek, van, Clara D.3, Wijburg, Frederik3,
Wanders, Ronaldus JA4, Meijers-Heijboer, Elizabeth J.1,
Jansonius, Nomdo M.5, Bergen, Arthur AB1
1Department of Clinical Genetics, Academic Medical Center,
Amsterdam, Netherlands, 2Department of Ophthalmology,
Academic Medical Center, Amsterdam, Netherlands,
3Department of Pediatric Metabolic Diseases, Academic
Medical Center, Amsterdam, Netherlands, 4Department of
Genetic Metabolic Diseases, Academic Medical Center,
Amsterdam, Netherlands, 5Department of Ophthalmology,
University Medical Center Groningen, Netherlands
Retinal diseases, like glaucoma and age-related macular
degeneration, affect more than 253 million people worldwide.
Since there is no effective cure for most of these disorders,
they are becoming an increasingly large problem in global
health. A subset of patients rely on treatments that only slow
down the progression of the disease, but do not stop the
impending visual impairment. Only recently, new experimental
treatments such as gene-, stem cell-, and small molecule
therapies are being developed. A complicating factor in the
development of such treatments is that the various retinal
diseases are associated with different cell types. Lately, a lot
of research is focused on the development of a new type of
in vitro model: retinal organoids. These retinas-in-a-dish
contain all retinal cell types and are useful for investigating
multiple disorders. Although the basic protocol has been
established, methods are still continuously being improved.
During our glaucoma research, we have developed a more
rapid and efcient protocol for retinal organoid differentiation,
focused on ganglion cell generation. Most published retinal
organoid protocols initially use a oating embryoid body
culture, and develop ganglion cells within 34-50 days of
differentiation, promoted by certain factors that modulate
developmental pathways. For research purposes, a faster and
more robust protocol is desirable. We replaced the oating
method with an encasement in a 3D matrigel matrix, which
gives an initial boost to the differentiation of stem cells
towards a retinal fate. With this method, we have managed to
generate ganglion cells within 28 days of differentiation,
without the addition of other external factors to direct cells
towards a retinal fate. We have also been able to generate all
other retinal cell types, conrmed by sq-PCR and immunohis-
tochemistry using both developmental and cell specic
markers. These cells include retinal pigmented epithelium
and photoreceptors. Utilizing this protocol will not only allow
us to study the development and morphology of the emerg-
ing eye with different degenerative disease models, but also
gives us the opportunity to generate multiple different cell
types that can be used for transplantations in a variety of
animal disease models and ultimately, human transplanta-
tions in the future.
Funding Source
I’d like to thank the European Comission for funding this
project under the Marie Skłodowska-Curie Horizon 2020
Innovative Training Networks program, Project ID 675033.
Keywords: Retina; 3D-matrix; Organoids
132
HYDROGEL ENCAPSULATION OF MESENCHYMAL
STEM CELL USING TWO-PHASE SYSTEM FOR
CHONDROGENIC DIFFERENTIATION
Park, Hyun Sook1, Lee, Sunray1, Lee, Dong-Mok2
1Research Center, CEFO Co, Ltd., Seoul, Korea, 2Technology
Convergence R&BD Group, Korea Institute of Industrial
Technology, Dae-Gu, Korea
In this study we optimized the encapsulation of mesenchymal
stem cells (MSC) with hydrogel to enhance chondrogenic
differentiation. We encapsulated MSC with alginate and
gelatin using two phase system. Micro-beads were passed
through a mineral oil to form uniform size aggregates using
syringe pump and micro size needle uniform size. Cell
encapsulated micro-beads were precipitated by gravity force
and crosslinked with crosslinking agent (ex. Ca2+, Mg2+).
We further conrmed the survival and production of
extracellular matrix of micro-bead cultured cells were
enhanced. These results implicated that hydrogel
encapsulation provides useful platform technology for
chondrogenic differentiation of MSC and enhanced cell
survival after transplantation and consequently can be
applied for cartilage defect repair.
Funding Source
We would like to acknowledge the nancial support from the
Korea Healthcare technology R&D Project (HI16O1002 &
HI18C2186), Ministry for Health, Welfare & Family Affairs,
Republic of Korea.
Keywords: Hydrogel; encapsulation; micro-beads
133
GENERATION OF HAIRY-SKIN ORGANOIDS FROM
PLURIPOTENT STEM CELLS
Lee, Jiyoon1, Rabbani, Cyrus1, Pum, Zachary1, Steinhart,
Matthew2, Kim, Alexander Y.1, Shipchandler, Taha1, Koehler,
Karl1
1Otolaryngology - Head and Neck Surgery, Indiana University
School of Medicine, Indianapolis, IN, USA, 2Stark
Neurosciences Research Institute, Indiana University
School of Medicine, Indianapolis, IN, USA
Over one hundred million people worldwide suffer from
injury or loss of skin due to burns, diseases, or genetic
defects. Skin is essential for protecting body by regulating
uid retention and temperature, guarding against external
stresses, and mediating touch and pain sensation. Human
skin develops from coordinated interactions between
multiple cell lineages and is vulnerable and difcult to be
reconstructed once damaged. Despite repeated attempts for
decades, however, a method of reproducing the full cellular
diversity of skin in tissue cultures or in bioengineered skin
equivalents has been elusive. Here we report a skin organoid
culture system that generates complex skin from human
pluripotent stem cells and recapitulate key features of skin
development. We found that skin organoids are composed
of stratied epidermis, fat-rich dermis, dermal condensate,
pigmented hair follicles equipped with sebaceous glands
and bulge stem cells, and sensory neuronal cells forming
synapses with Merkel cells in organoid hair follicles, mimicking
84
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Poster Abstracts
human touch circuitry. Furthermore, the skin organoids are
comparable to human fetal facial skin and capable of
reconstituting hairy skin in a xenograft mouse model.
Together, our results demonstrate that the skin organoids
produced in our culture system are the most functional and
fully equipped human skin tissue that can be generated in
vitro, to date. We anticipate our study provides a foundation for
using skin organoids in studying skin development, modeling
skin disease processes, or supplying cell source for skin
regeneration and transplantation.
Funding Source
Ralph W. and Grace M. Showalter Trust (to Karl R. Koehler).
Keywords: Skin; Organoids; Hair Follicles
134
IDENTIFICATION AND CHARACTERIZATION OF
MATURE DOPAMINERGIC NEURON SUBTYPES AT
SINGLE-CELL RESOLUTION IN HUMAN VENTRAL
MIDBRAIN-PATTERNED ORGANOIDS
Fiorenzano, Alessandro, Zhang, Yu, Birtele, Marcella,
Sharma, Yogita, Jarl, Ulla, Mattsson, Bengt, Parmar, Malin
Developmental and Regenerative Neurobiology Wallenberg
Neuroscience Center, Lund University, Sweden
Parkinson’s disease (PD), the most common neurodegenerative
disorder, is characterized by progressive loss of dopamine
(DA) neurons in midbrain. Although the relatively focal
degeneration in PD makes it a good candidate for cell-based
therapies, the inaccessibility of functional human brain
tissue and the inability of two-dimensional in vitro cultures to
recapitulate the complexity and function of dopaminergic
circuitries have made the study of human midbrain functions
and dysfunctions challenging. Despite intensive research
efforts in recent years, the molecular mechanisms controlling
the developmental program and differentiation of DA neuron
subtypes remain largely unknown. In this study, we designed
a method for differentiating human pluripotent stem cells
into three-dimensional (3D) dopaminergic organoids, which
mimic features of human ventral midbrain (VM) develop-
ment by recreating authentic and functional DA neurons.
Immunolabelling-enabled 3D imaging of solvent-cleared
organs (iDISCO) of whole organoids provides an anatomical
perspective useful for reconstructing regional identities,
spatial organization and connectivity maps. By combing
CRISPR-Cas9 gene editing – used for generation of trans-
genic tyrosine hydroxylase (TH)-Cre reporter cell line – with
unbiased transcriptional proling at single-cell resolution, we
showed that TH+ neurons exhibit molecular and electro-
physiological properties of mature DA neurons expressing
functional receptors of A9 and A10 neurons, which are
severely affected in PD. Importantly, we also conducted a
direct comparison with fetal VM organoids, which may serve
as a valuable reference for creating the optimal conditions to
differentiate pluripotent stem cells into human midbrain
organoids, underscoring developmental similarities and
differences.
Keywords: Stem cell differentiation; Dopaminergic Organ-
oids; Parkinson’s disease (PD)
135
USING OLIGOCORTICAL SPHEROIDS TO
STUDY DEVELOPMENT AND DISEASE OF
OLIGODENDROCYTES IN THE BRAIN
Madhavan, Mayur C., Nevin, Zachary, Clayton, Benjamin LL,
Tesar, Paul J.
Department of Genetics and Genome Sciences,
Case Western Reserve University, Cleveland, OH, USA
Cerebral organoids provide an accessible system to exam-
ine developmental aspects of cell specication, cell interac-
tions and organization. Most research has focused on gener-
ating neurons and astrocytes in cerebral organoids, but we
have focused on generating oligodendrocytes, the myelinat-
ing glia of the central nervous system. We have reproducibly
generated oligodendrocytes and myelin in human pluripotent
stem cell-derived “oligocortical spheroids”. We have used
this newly developed system in conjunction with single cell
RNA sequencing and molecular characterization to study the
development of oligodendrocytes within these organoids.
Additionally we have used this system to study new aspects
of leukodystrophies such as Pelizaeus-Merzbacher disease.
Keywords:
Cerebral organoids; Oligodendrocyte; Disease
modeling
136
THREE-DIMENSIONAL SPHEROID CULTURE
WORKFLOW USING PURE NEURONAL SUB-TYPES
DERIVED FROM HUMAN IPSCS
Laha, Kurt, Du, Zhong-wei, Xu, Kaiping, Dungar, Ben
BrainXell, Inc., Madison, WI, USA
Human induced pluripotent stem cells (iPSC) derived
neurons are now considered a more relevant in vitro model
system for psychiatric and neurological diseases. They can
be used for the development of physiological cell models,
human disease models, and drug library screening. Three
dimensional (3D) cultures are also being pursued as a more
physiologically relevant system because they provide a
microenvironment, cell-to-cell interactions, and biological
processes that better represent in vivo conditions. The
implementation of 3D spheroid culture plays an important
role as an alternative approach for drug development and
therapeutic applications in central neural system (CNS)
disorders. To develop a neuronal 3D spheroid culture
system, we tested iPSC-derived human motor neurons and
cortical glutamatergic neurons using the S-BIO PrimeSurface
Ultra Low Attachment Micoplates. After 2 hours, plated
neurons started to settle down at the bottom of the well and
form large clusters. On day 3, 3D spheroids could be seen
clearly under phase contrast. On day 7, the spheroids were
more condensed. The size of the 3D spheroids was proportional
to the number of neurons seeded per well. In addition to the
morphological assessments, a cytotoxicity assay and MEA
assay were performed to demonstrate the suitable of 3D
spheres as a platform for various applications. In both
assays, the spheroids yielded expected results. The results
presented here demonstrate the feasibility of generating
uniform and reproducible spheroids using human neurons
and the potential application for neurotoxicity studies.
Keywords: Spheroids; iPSC-Derived Neurons; 3D
85
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Poster Abstracts
137
COMBINING NGN2 PROGRAMMING AND
DEVELOPMENTAL PATTERNING TO RAPIDLY
GENERATE CORTICAL ORGANOIDS
Smith, Kevin S.1, Nehme, Ralda2, Eggan, Kevin1
1Harvard Department of Stem Cell and Regenerative Biology,
Harvard University, Cambridge, MA, USA, 2Stanley Center for
Psychiatric Research, Broad Institute, Cambridge, MA, USA
The current models for investigating psychiatric or
neurodegenerative diseases rely on animal and/or neuronal
cell culture based models. While these formats have
been integral for elucidating many underlying disease
mechanisms, this often leads to a failure to translate into
effective therapeutics. Many of these failures can be
attributed to the biological differences between human
disease and animal models, or the lack of proper neuronal
development/maturation in vitro (e.g. dendritic spine
formation). Our lab has pioneered a rapid and robust
protocol for differentiating human stem cells into functional
excitatory cortical neurons, providing a species-specic
model for investigating human disease. The recently
published protocol (Nehme et al. 2018) combines twopotent
neuralizing components: expression of the transcription
factor NGN2, and small molecule patterning (DUAL SMAD
and Wnt inhibition). Here we apply the neuronal differentia-
tion protocol to 3D cultures (e.g organoids) and characterize
their composition. The patterned NGN2-organoids show
high expression of multiple upper layer cortical neuronal
markers (e.g. Cux1, Brn2), few deep layer neuronal marker
(Ctip2), as well as GFAP expressing cells. Interestingly, the
spheres also do not require embedding in Matrigel droplets,
as they naturally keep their structural integrity. These results
show that the coupling of the 3D organoid protocol with
NGN2/patterning allows for easy, rapid formation of neural
organoids that could be used as a cortical model. However,
further analysis (e.g. single cell RNA-seq) is necessary for
determining the absolute identity of the cells within the
spheres, as well as understanding the maturation stage of
the neurons.
Keywords: NGN2; Organoid; Cortical
139
INDUCED 2C EXPRESSION AND IMPLANTATION-
COMPETENT BLASTOCYST-LIKE CYSTS FROM
PRIMED PLURIPOTENT STEM CELLS
Kime, Cody
Lab of Retinal Regeneration, RIKEN, BDR, Kobe, Japan
Soon after fertilization, the few totipotent cells of mammalian
embryos diverge to form a structure called the blastocyst
(BC). Although numerous cell types, including germ cells and
extended pluripotency stem cells, have been developed from
pluripotent stem cells (PSCs) in-vitro, generating functional
BCs only from PSCs remains elusive. Here we describe
induced self-organizing 3D BC-like cysts (iBLCs) generated
from mouse PSC culture. Resembling natural BCs, iBLCs
have a blastocoel-like cavity and were formed with outer
cells expressing trophectoderm (TE) lineage markers and
with inner cells expressing pluripotency markers. iBLCs
transplanted to pseudopregnant mice uteruses implanted,
induced decidualization, and exhibited growth and
development before resorption, demonstrating that iBLCs
are implantation-competent. iBLC precursor intermediates
required the transcription factor Prdm14 and concomitantly
activated the totipotency-related cleavage stage MERVL
reporter and 2C genes. Thus, our system may contribute to
understanding molecular mechanisms underpinning
totipotency, embryogenesis, and implantation.
Keywords: primed pluripotent stem cell germ; 2C totipotency
MERVL early embryo genesis; reprogramming cell identity
140
CELL TYPES OF THE HUMAN RETINA AND ITS
ORGANOIDS AT SINGLE CELL RESOLUTION:
DEVELOPMENTAL CONVERGENCE, TRANSCRIP-
TIONAL
IDENTITY, AND DISEASE MAP
Renner, Magdalena1, Cowan, Cameron2, Roska, Botond2
1Novartis Institutes of Biomedical Research (NIBR) and
Institute of Molecular and Clinical Ophthalmology Basel (IOB),
Basel, Switzerland, 2IOB, Friedrich Miescher Institute for
Biomedical Research, Basel, Switzerland
Organoids are stem cell-derived articial organs that mimic
aspects of organ development, function and disease. How
closely cell type diversity and cell type maturation in human
organoids recapitulate that of their target organ is not well
understood. We performed histochemistry and sequenced
the RNA of 163,971 single cells from improved human retinal
organoids at different developmental stages and from
donated healthy adult human retinas and choroid. Cell types
in mature organoids had morphologies and transcriptomes
that resembled their adult equivalents. Remarkably, organoids
developed at a similar rate to the fetal retina and the
transcriptome trajectory of cell types contained a progression
of key developmental markers. Mapping disease-associated
genes to cell types revealed cellular targets for studying
disease mechanism in organoids and performing targeted
repair in adult retinas.
Keywords: Retinal Organoid; Single-cell RNA sequencing;
Cell type developmental convergence
86
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AMSTERDAM NETHERLANDS
Poster Abstracts
141
UNCOVERING REGULATORS OF HUMAN
EMBRYONIC STEM CELL DIFFERENTIATION BIAS
TOWARDS DEFINITIVE ENDODERM
Dziedzicka, Dominika1, Tewary, Mukul2, Tilleman, Laurentijn3,
Keller, Alex1, Östblom, Joel2, Couvreu De Deckersberg,
Edouard1, Markouli, Christina1, Spits, Claudia1,
Van Nieuwerburgh, Filip C.3, Zandstra, Peter2, Sermon, Karen1,
Geens, Mieke1
1Research Group Reproduction and Genetics, Vrije Universiteit
Brussels, Belgium, 2Institute of Biomaterials and Biomedical
Engineering, University of Toronto, ON, Canada, 3Laboratory
of Pharmaceutical Biotechnology, Ghent University, Ghent,
Belgium
Recently launched clinical trials aim to use human pluripotent
stem cell (hPSC) mesendodermal (ME) derivatives to treat
patients with heart failure and type 1 diabetes. As individual
hPSC lines can signicantly vary in their ME differentiation
efciencies, acquiring a deeper understanding about this
phenomenon and the development of tools to rapidly screen
hPSC differentiation bias is of great value to the eld
of regenerative medicine. In this study, we used ve
karyotypically normal human embryonic stem cell (hESC)
lines to investigate molecular mechanisms of hESC
differentiation bias towards denitive endoderm (DE). We
quantied DE differentiation efciency using both a classic
adherent differentiation and an in vitro model of early
gastrulation-associated fate patterning in geometrically-
conned micropatterned colonies, and identied VUB04 as a
hESC line with signicantly lower DE differentiation efcien-
cy when compared to the other lines. As our DE differentia-
tion protocol employs a strong activator of WNT signalling,
we hypothesize that differentially expressed genes in VUB04
prevent its proper activation for endodermal specication.
Bulk mRNA-sequencing at the undifferentiated stage
showed that the main pluripotency genes were expressed a
t comparable levels between hESC lines, however, pathway
enrichment analysis of differentially expressed genes in
VUB04 pointed at MAPK/ERK signalling. GO-term analysis of
mRNA-sequencing samples 6 and 24 hours after the onset
of DE differentiation showed that VUB04 fails to upregulate
genes responsible for gastrulation, endoderm formation and
BMP signalling. We are currently modifying expression levels
of candidate genes selected from a list of 120 genes which
were differentially expressed in VUB04 at all three mRNA-
sequencing timepoints, and investigating if they have
regulatory effects on early lineage specication to DE. Our
work provides preliminary insight into the molecular mecha-
nisms of how hESC may manifest DE differentiation bias and
provides experimental validation of an in vitro platform that
can be employed for high-throughput screens of hPSC
differentiation propensities.
Keywords:
Differentiation bias; Denitive endoderm; Micropat-
terned
culture
142
MODELING IN VIVO KIDNEY CELL FATE SPECIFI-
CATION PROCESSES BY REACAPITULATING
KIDNEY ORGANOGENESIS IN VITRO
Taguchi, Atsuhiro1, Yoshimura, Yasuhiro2, Nishinakamura,
Ryuichi2
1Department of Genome Regulation, University Medical
Center Groningen, Berlin, Germany, 2Department of Kidney
Development, Institute of Molecular Embryology and
Genetics, Kumamoto University, Japan
Recent progress in developmental biology identied
metanephros, the embryonic kidney, develops by the
interaction of nephron progenitors (NPs), ureteric bud (UB),
and stromal progenitors, each of which nally gives rise to
distinct cell types. However, the cell fate specication
processes before and after the anlage formation remains
largely unknown, in part due to the limited time resolution
of genetic studies and difculty to address the in vivo signal
redundancy. We recently took advantage of in vitro directed
differentiation approach and identied distinct signal require-
ment for the differentiation of NPs and UB from pluripotent
stem cells (PSCs). Assembly of the PSCs-derived NPs, UB
together with mouse embryonic stromal progenitors
recapitulated “higher-order kidney organogenesis” in a dish,
conrming the reliability of NPs and UB induction signals.
We further addressed the cell fate specication process
during the post-metanephros formation stage. NPs
differentiate into 5 types of nephron segments, including
parietal epithelial cells, podocytes, proximal tubules,
loop of Henle and distal tubules, which form along the
proximo-distal axis in this order. Here we focused on the
podocytes differentiation process, which are one of the
major targets in the kidney disease research eld. We rst
cultured embryonic nephron progenitors in the presence of
growth factors/inhibitors and identied the differentiation
stage dependent signal requirement including, mesenchy-
mal-to-epithelial transition, proximalization and podocyte
specication. Further application of these cues to our
human PSC-derived NPs enabled the highly-selective (more
than 90% purity) induction of podocytes, which is much
higher than the unbiased nephron differentiation method, in
which podocytes consisted 5-20% of the kidney organoid.
The induced podocytes exhibited comparable global
signature gene expressions to those in adult human podo-
cytes and had podocyte morphological features including
foot process-like and slit diaphragm-like structures, which
suggested identied signals were sufcient to induce
podocyte fate. Taken together, in vitro directed differentia-
tion approaches not only produce organotypic kidney
organoid but also give novel insights into cell-type specica-
tion processes in vivo.
Keywords: Kidney organoid; Cell fate specication; Pluripo-
tent stem cell
87
AMSTERDAM NETHERLANDS
Poster Abstracts
143
MOUSE FETAL ORGANOIDS: NEW MODEL TO
STUDY INTESTINAL EPITHELIAL MATURATION
FROM SUCKLING TO WEANING
Martins Garcia, Tânia1, Navis, Marit1, Martins Garcia, Tânia1,
Renes, Ingrid B.2, van den Brink, Gijs3, Vermeulen, Jacqueline1,
Meisner, Sander1, Wildenberg, Manon1, van Elburg, Ruurd4,
Muncan, Vanesa1
1Tytgat Institute for Liver and Intestinal Research, Amsterdam
UMC, University of Amsterdam, Netherlands, 2Danone
Nutricia Research, Utrecht, Netherlands, 3GlaxoSmithKline,
Medicines Research Center, London, UK, 4Department of
Pediatrics, Amsterdam UMC, University of Amsterdam,
Netherlands
During the suckling-to-weaning transition the intestinal
epithelium adapts to the change in diet from milk to solid
food by altering its gene expression prole. Previous studies
have suggested that at least part of this transition is
intrinsically programmed. In this context, we used mouse
fetal intestinal organoids as a model and aimed to determine
whether fetal intestinal organoids in vitro mimic the in vivo
gut epithelial maturation process that takes place from birth
to weaning. Organoids were cultured from fetal intestinal
epithelial cells (E19) for one month, with adult intestinal
organoids as control. Global gene expression proles of
fetal and adult organoids at day 3 and day 28 of culture were
identied by micro-array and compared to gene expression
proles of fetal and adult intestinal tissue. Expression of
specic maturation markers was evaluated weekly in fetal
organoids by qPCR, enzyme activity assay and immunohis-
tochemistry. To investigate whether gut maturation in fetal
intestinal organoids can be modulated, organoids were
treated with dexamethasone (DEX), a factor known to
stimulate gut maturation. Global gene expression proles
showed an overall shift from fetal towards adult epithelium
in fetal organoids cultured for 28 days, compared to day 3 of
culture. Markers of neonatal intestinal epithelium could be
detected in fetal organoids at day 3 but were progressively
lost in time and were completely absent at day 28 of culture
as well as in adult control organoids. In contrast, markers
characteristic for the adult intestinal epithelium were absent
in the fetal organoids during the rst two weeks of culture
and gradually increased to adult levels after 4 weeks of
culture. Results were conrmed at enzyme activity level
indicating that the organoids develop a functional adult
brush border over time. Finally, DEX accelerated certain
aspects of in vitro maturation in fetal intestinal organoids.
Our data show that mouse fetal intestinal organoids mature
into adult epithelium in vitro in a process that recapitulates
the hallmarks of in vivo intestinal epithelial maturation. Fetal
intestinal organoids can therefore be used to elucidate the
molecular mechanisms of postnatal epithelial development
and identify novel factors that inuence the timing of epithelial
maturation.
Keywords: Intestinal epithelium; Maturation; Fetal organoids
144
3D IN VITRO MODEL FOR HUMAN BRAIN
DEVELOPMENT IN AN ENVIRONMENT OF
BRAIN VASCULATURE AND SYSTEMIC MICRO-
ORGANOIDS NEURONAL DEVELOPMENT
Wang, Tongguang1, Bagnell, Anna1, Johnson, Kory2,
Nath, Avindra1
1Translational Neuroscience Center, NIH/NINDS, Bethesda,
MD, USA, 2NINDS, NIH, Bethesda, MD, USA
Compared to 2D neuronal cultures, brain organoids provide
a more relevant model of in vivo neural development and
neurological disorders. However, 3D brain organoids
developed using current protocols lack critical cell types of
brain such as mature astroglia and endothelial cells, or
include a combination of the separately differentiated cell
types, and thus is not a perfect representation of natural
neural development. Further, interactions among different
organs also play an important role in the condition of the
neural system, thus asking for better models involving
multiple micro organs. To address these critical issues,
we determined if blood vessel building endothelial cells
may play a critical role in organ development. We rst made
embryoid bodies from human iPSCs derived from CD34
cells, then initiated endothelial cell differentiation using a
combination of small molecules and growth factors Activin
A, BMP-4 and VEGF in a 3D environment consisting of
Matrigel. The resulting spheroids were further cultured in
media to induce neural development. The resulting organoids
were collected at 2 weeks and 8 weeks and characterized by
immunouorescent staining for endothelial and neuronal
markers. Gene expression was studied by Real-time PCR
and RNA-Seq analysis. The organoids thus developed
showed a spectacular morphology consisting of a vesicular
sac and dense mass within. Immunostaining showed that the
organoids were positive for the endothelial cell marker CD31,
mostly in the outside layer and neuronal marker βIII-tubulin
mostly in the mass. RT-PCR and RNA-Seq conrmed the
existence of both arterial and venous endothelial cell
markers and mature neural cells, as well as glial cell markers
such as GFAP and MBP. Furthermore, transcripts specic for
other organs such as kidney, liver and lung were also
detected, indicating tracks of multiple organ development in
the sac, which makes it a model for multiple organ interac-
tions. This model holds great promise for a more in vivo
relevant 3D model, suitable for applications for studying
organ development, disease modeling, drug screening and
development.
Funding Source
Sponsored by NIH intramural fund.
Keywords: 3D organoid; Neuronal development; Multiple
organ interactions
88
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Poster Abstracts
145
CO-DEVELOPING HUMAN HEART MUSCLE AND
NEURONAL STRUCTURES IN BIOENGINEERED
HEART MUSCLE
Raad, Farah1, Zafeiriou, Maria-Patapia1, Zeidler, Sebastian1,
Roa, Angelica1, Hudson, James E.2, Uchida, Shizuka3,
Wingender, Edgar4, Zimmermann, Wolfram H.1
1Institute of Pharmacology and Toxicology, University Medical
Center Goettingen, Germany, 2Cell and Molecular Biology,
QIMR Berghofer Medical Research Institute, Brisbane,
Australia, 3University Louisville School of Medicine,
Louisville, KY, USA, 4University Medical Center Goettingen,
Department of Bioinformatics, Goettingen, Germany
Recent work in our lab showed the ability of human pluripotent
stem cells (hPSC) to self-assemble into a force generating
bioengineered heart muscle (BHM) with spontaneous
co-development of sympathetic neuron-like structures in
long-term cultures (day 60). We hypothesized that the neuronal
co-development in BHM can be guided by directed differenti-
ation of neurogenesis, resulting in a complete heart-neuron
tissue with enhanced contractile performance. BHM was
generated from hPSCs (5x105 cells/BHM) under stage-spe-
cic directed cardiac differentiation. Mechanical loading
was introduced on day 13 followed by dened neuronal
induction from culture day 29 onwards. Isometric force
measurement was performed on day 60 to assess BHM
contractility under dened conditions. Enzymatic dispersion
of BHMs was performed to determine total cell count and
composition via ow cytometry. Neuronal gene expression
was conrmed using qPCR and immunohistochemistry.
Previous results revealed that BHMs closely mimic key steps
of in utero heart development with the induction of meso-
derm, followed by specication towards cardiac lineages. At
culture day 60, BHMs subjected to neuronal induction (8
days), neuronal expansion (7 days) followed by neuronal
differentiation (14 days), showed improved contractility
(maximal force of contraction in mN: 0.38±0.05 vs 0.25±0.03
for Control; n=75; p<0.05). Flow cytometry results revealed
signicantly higher survival of cardiomyocytes in the
neuro-enhanced BHM group (% α-actinin+ cells: 19±3 vs
10±2 for Control; n=27/30; p<0.05). On the molecular level,
neuronal markers such as PAX6 and MAP2 were highly
upregulated in neuro-enhanced BHM. Detailed morphologi-
cal studies conrmed MAP2 and NF positive staining,
presence of neuronal rosettes and ne neuronal outgrowths,
suggesting innervation in day 60 as well as day 90 neuro-en-
hanced BHMs. Taken together, our study provides rst
evidence for pro-survival effects and enhanced contractility
in human BHM with an engineered neuro-cardiac interface.
The engineered co-development of cardiac mesoderm and
ectoderm appears to recapitulate the complex process of
autonomic innervation of heart muscle during fetal heart
development.
Keywords: Pluripotent Stem Cells; Cardiac Differentiation;
Neuronal Differentiation
146
MTOR AND LIF SIGNALING IN HUMAN CORTICAL
DEVELOPMENT
Andrews, Madeline G., Subramanian, Lakshmi, Bhaduri,
Aparna, Kriegstein, Arnold
Regeneration Medicine, University of California,
San Francisco, CA, USA
The cerebral cortex, the folded exterior of the brain, is
expanded in humans and is important for our cognitive
abilities. Abnormal cortical development is a leading cause
of epilepsy and developmental delay and can lead to
malformations where the brain does not fold appropriately or
grow as in Lissencephaly, Microcephaly, and Megalenceph-
aly. The cortex expands by directing appropriate proliferation
of its resident neural stem cells, radial glia cells. There are
multiple distinct types of radial glia, and the outer radial glia
(oRG) population is dramatically expanded in humans,
suggesting its contribution to human cortical expansion.
Currently we have a limited understanding of the developmen-
tal processes that regulate oRG generation and maintenance.
We used single cell RNA sequencing and discovered an
association between oRG cells and mTOR signaling. Interest-
ingly, the Lif/Stat3 pathway, which regulates pluripotency in a
variety of stem cell populations, is also transcriptionally asso-
ciated with oRG cells. We assessed the functional role of
mTOR signaling in oRG formation and the relationship
between mTOR and Lif signaling utilizing two human-specic
models of cortical development: ex vivo culture of developing
human cortical tissue and in vitro culture of human induced
pluripotent stem cells (iPSCs) directed toward cortical
organoids. We found that Lif activity is sufcient to increase
the number of oRGs and also activates mTOR signaling in
these models of human cortical development. Manipulation
of mTOR signaling using small molecules resulted in a
decrease in oRG basal ber length and a reduction in the
number of oRGs. These studies provide insight into develop-
mental mechanisms guiding the generation of the oRG
population and have implications for the treatment of cortical
disease.
Keywords: Cortex; Development; Signaling
89
AMSTERDAM NETHERLANDS
Poster Abstracts
147
MEX3A DELETION IMPAIRS LGR5+ STEM CELL
MAINTENANCE IN THE MOUSE GUT
Pereira, Bruno1, Amaral, Ana L.1, Dias, Alexandre1, Mendes,
Nuno1, Muncan, Vanesa2, Silva, Ana R.1, Thibert, Chantal3,
Radu, Anca G.3, David, Leonor1,4, Máximo, Valdemar1,4,
van den Brink, Gijs R.5, Billaud, Marc7, Almeida, Raquel1,3
1Institute of Molecular Pathology and Immunology of the
University of Porto, i3S - Institute for Research and Innovation in
Health, Porto, Portugal, 2Department of Gastroenterology and
Hepatology, Tytgat Institute, Amsterdam UMC, Amsterdam,
Netherlands, 3Institute for Advanced Biosciences, INSERM
U1209, CNRS UMR5309, University Grenoble Alpes, Grenoble,
France 4Faculty of Medicine of the University of Porto,
Portugal, 5Medicines Research Center, GSK, Stevenage, UK,
6Clinical and Experimental Lymphomagenesis, INSERM
U1052,CNRS U5286, Universite Claude Bernard Lyon, France
Intestinal stem cells (ISCs) fuel the lifelong self-renewal of
the intestinal tract and are paramount for epithelial repair.
Genetic studies have dened the Wnt signalling as a crucial
regulator of ISC identity. One of the most reliable ISC
markers is the Wnt/β-catenin pathway member LGR5. Still,
the effort to reveal ISCs identity and regulatory networks
remains a challenge. It is becoming clear that RNA-binding
proteins (RBPs) and the post-transcriptional mechanisms
they rule underpin stem cell fate decisions in response to
different stimuli. We have generated the rst Mex3a knockout
mouse model and report that the function of this RBP is
crucial for the in vivo maintenance of the Lgr5+ ISC pool
and for efcient epithelial turnover during intestinal postna-
tal development. Mex3a null mice exhibit growth retardation
and postnatal mortality due to impaired epithelial turnover,
underlined by a dramatic decrease in Lgr5+ ISCs and KI67+
cells. Transcriptomic proling revealed that Mex3a ablation
induces activation of the peroxisome proliferator-activated
receptor (PPAR) signalling at the crypt level, with a concomi-
tant loss of the Lgr5+ stem cell signature. Furthermore, we
provide evidence that PPARgamma is a target ofMEX3A-
mediated repression, thus uncovering a new layer of
post-transcriptional regulation operating in ISCs that
critically contributes to intestinal homeostasis.
Funding Source
This work has been supported by the INFRAFRONTIER-I3
project under the EU Grant Agreement No. 312325 of the EC
FP7 Capacities Specic Programme and by Portuguese
funds through FCT (SFRH/BPD/109794/2015; POCI-01-
0145-FEDER-031538).
Keywords: Lgr5+ intestinal stem cells; RNA-binding protein
MEX3A; Intestinal homeostasis
148
HUMAN IPSC DERIVED 3D-NEUROSPHERE ASSAY
TO STUDY DEVELOPMENTAL NEUROTOXICITY
Dinnyes, Andras1, Kobolák, Julianna1, Téglási, Annamaria1,
Bellák, Tamas1, Molnár, Kinga2, Bock, István1, Janstova,
Zoa1, László, Lajos2, Dinnyes, Andras1
1BioTalentum Ltd, Godollo, Hungary, 2Department of Anatomy,
Cell and Developmental Biology, Eötvös Loránd University,
Budapest, Hungary
Neuronal stem cells (NSCs) are considered as a multipotent
and self-renewing pool of cells of the mammalian central
nervous system (CNS) and have the capacity to differentiate
into all neuronal cell types, as well as glial cells. They are
able to form spherical, free-oating 3D cell clusters, the
neurospheres, and differentiate into neurons, glial cells and
oligodendrocytes, forming a complex tissue, mimicking the
events of early neurodevelopment. NSCs can be differentiated
from pluripotent stem cells, provide an attractive in vitro tool
for studying CNS disorders or for drug development purpos-
es. Here, we present a 3D human induced pluripotent stem
cell (hiPSC)-based in vitro toxicology assay that can be used
to test developmental neurotoxicity. Human iPSCs derived
neurospheres grown in 3D culture were characterised timew-
ise to monitor their complexity and homogeneity over a
7-weeks-long period using immunocytochemistry and
electron microscopy. 3D neurospheres were exposed to 10
different toxicants (e.g. Paraquat, VPA, acrylamide, mercury
chloride) activating different toxicity pathways. Samples
were examined at different developmental time points (21,
28 and 42 days after plating), representing different develop-
mental stages and maturity, with an ATP-based cell viability
assay optimised for 3D-tissues in 96-well plate format.
Concentration-responses were investigated after acute (72
hours) exposure and the effect of toxicants were determined
by histology as well. In addition, Transcriptional activity of
major developmental, structural and cell type specic
markers was investigated at weekly intervals. The results
demonstrated that the acute exposure to different classes of
toxicants resulted in distinct cell susceptibility proles in
different developmental stages, indicating that hiPSC-based
in vitro neurodevelopmental models might be used effective-
ly to evaluate developmental neurotoxicity. This will open
new avenues for 3Rs replacement of animal models with in
vitro assays in various academic and
pharma-, chemical- and cosmetics industry applications.
Funding Source
This project has received funding from the European Union’s
Horizon 2020 research and innovation programme under
grant agreement No 681002.
Keywords: Induced pluripotent stem cells; 3D neurospheres;
Developmental neurotoxicity
90
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Poster Abstracts
149
DEVELOPMENTAL NEUROTOXICITY (DNT) OF
GAMMA-RADIATION IN A 3D EARLY BRAIN
DEVELOPMENTAL MODEL GENERATED FROM
HUMAN INDUCED PLURIPOTENT STEM CELLS
(HIPSC)
Klatt, Annemarie K K, Schneider, Lisanne, Kadereit, Suzanne
Life Science, Albstadt-Sigmaringen University, Sigmaringen,
Germany
Due to increasing medical imaging, radiation therapy and
long-distance ights humans are increasingly exposed to
ionizing radiation (IR). The sensitivity of cells to IR differs a
lot between different cell types. Neural progenitor cells are
known to have low radiation resistance. Low dose IR may
induce permanent damage with potential consequences for
the developing brain. However, data on impact of IR on very
early human brain development is scarce. We had previously
published a three dimensional (3D) model from hESC
recapitulating the very early stages of predominantly
fore-brain development. Altered neural marker gene
expression after exposure to known DNT chemicals validated
the 3-D DNT model. Here we established the same model
from human iPSCs and investigated the effects of IR
(1Gy X-ray). To evaluate the capacity of self-renewal of the
surviving, irradiated neural stem/progenitor population
neurosphere (NS)-forming assays were performed. We also
evaluated long-term effects by measuring the diameter of
NS over time. In addition, the expression levels of 19 different
marker genes crucial for normal neural development such as
patterning, structure, neural/neuronal development and
stress response genes were determined 2 and 9 days post
IR (p.IR) by qRT-PCR. NS formation was reduced after
irradiation and NS were found to have signicantly smaller
diameters starting 16 days p.IR. Signicant changes were
also present at the molecular level. Most analyzed marker
genes were reduced 2 days p.IR but interestingly went back
to control levels by day 9 p.IR. 7 genes were however not
expressed at control levels 9 days p.IR, including 3 markers
for stress. Among the 6 reduced markers were also genes
known to be involved in neurodevelopmental syndromes
(microcephaly, Mowat-Wilson syndrome, growth abnormali-
ties). We were thus able to identify a signicant impact of
radiation in the surviving cells. The detected reduction in
marker expression points towards potential detrimental
effects of IR on early brain development and contradict the
long held hypothesis that in development irradiated cells die,
or survive with no later appearing major damage. Our data
suggest that IR, even at low doses could have a negative
neurodevelopmental impact on the human brain.
Funding Source
Federal Ministry for Education and Research Germany
(BMBF); Ministry for Science, Research and Arts of
Baden-Württemberg.
Keywords: Irradiation; Neurogenesis; Neurospheres
150
TOWARDS IN VITRO MODELING OF EARLY MOUSE
DEVELOPMENT
Girgin, Mehmet U.1, Beccari, Leonardo2, Moris, Naomi3,
Duboule, Denis4, Martinez-Arias, Alfonso3, Lutolf, Matthias1
1Institute of Bioengineering, Ecole Polytechnique Federale
de Lausanne, Switzerland, 2Department of Genetics and
Evolution, University of Geneva, Switzerland, 3Department
of Genetics, University of Cambridge, UK, 4Swiss Cancer
Research Institute, Ecole Polytechnique Federale de
Lausanne, Switzerland
In-vitro differentiation approaches of mouse embryonic stem
cells (ESC) have attempted to generate cell types that occur
during embryonic development. However, early mouse
development is a complex system comprising interaction
of various cell types in a spatially and temporally controlled
manner. Therefore, such differentiation approaches failed
to faithfully recapitulate early mouse development in vitro.
Recently, it became possible to generate self-organizing
multicellular tissue constructs termed organoids. While
important aspects of the complex in-vivo organization
have been recreated in these organoid systems, most
of these studies have focused on the recapitulation of
organogenesis, not of the early embryonic development.
In this study, we aim to study certain features of the
developing mouse embryo by using an embryonic organoid
model called ‘gastruloids’. Gastruloids, small aggregates of
mouse embryonic stem cells, demonstrate symmetry
breaking, axial elongation and germ layer specication in a
reproducible manner. Moreover, gastruloids are capable of
establishing anterior-posterior (A-P), dorso-ventral (D-V)
and medio-lateral (M-L) axes in vitro. Surprisingly, full
implementation of spatial and temporal colinearity of Hox
genes is recapitulated in these self-organizing aggregates of
mESCs. We hope that this model would be useful to provide
an alternative way to study development and to generate
rare cells types that arise during embryogenesis in vitro.
Keywords: Gastruloids; Self organisation; Embryonic
development
91
AMSTERDAM NETHERLANDS
Poster Abstracts
151
EFFECTS OF MEDIUM SUPPLEMENTS ON
EXPANSION AND CHONDROGENIC POTENTIAL
WITH CAPACITY TO UNDERGO ENDOCHONDRAL
OSSIFICATION OF MESENCHYMAL STEM CELLS
Ikeda, Masa-Aki1, Ikeda, Yayoi2, Tagami, Ayako2,
Kasugai, Shohei3
1Department of Molecular Craniofacial Embryology, Graduate
School of Medical and Dental Sciences, Tokyo Medical and
Dental University, Tokyo, Japan, 2Department of Anatomy,
Aichi-Gakuin University School of Dentistry, Nagoya, Japan,
3Department of Oral Implantol. & Regenerative Dentistry,
Tokyo Medical and Dental University, Bunkyo-ku, Japan
Mesenchymal stem cells (MSCs) offer a promising source
of cells for bone regeneration by mimicking the process of
endochondral ossication. However, their chondrogenic
differential potential and stem cell properties decreases
during in vitro expansion. In this study, we examined effects
of various medium supplements on MSC self-renewal and
chondrogenic differentiation potential with a capacity to
undergo endochondral ossication. Human bone
marrow-derived MSCs (passage 3, P3) were cultured until
conuent and split continuously until reaching senescence.
Medium supplementation including broblast growth factor
(FGF)-2, transforming growth factor (TGF)-β1, and
dexamethazone (Dex) increased cell numbers before
reaching senescence (from P3 to P8), which was associated
with loss of chondrogenic differentiation potential in pellet
culture for 3 weeks. However, for maintaining chondrogenic
differentiation potential up to P8, two other supplements
were required in addition to these supplements. The MSCs
(P6) expanded with 5 supplements were subjected to
chondrogenic differentiation for 3 weeks and hypertrophic
maturation for 2 weeks. The resultant MSCs formed bone
ossicles, including blood vessels, when subcutaneously
implanted into nude mice for 8 weeks, indicating that the
long-term cultured MSCs retained the capacity to undergo
endochondral ossication. We are currently performing
further experiments to conrm these observations.
Funding Source
This study was supported in part by JSPS KAKENHI
Grant Number 26293419 and 15K15722.
Keywords: Mesenchymal stem cells; chondrogenic differen-
tiation; endochondral ossication
152
WNT1/B-CATENIN SIGNALING REGULATES
SUBSET-SPECIFIC MIDBRAIN DOPAMINERGIC
NEURON DIFFERENTIATION IN A DOSE DEPENDENT
MANNER IN THE MOUSE
Nouri, Parivash1, Götz, Sebastian2, Rauser, Benedict2,
Irmler, Martin3, Trümbach, Dietrich2, Sharma, Yojet1,
Dlugos, Andrea 1, Grosschedl, Rudolf4, Beckers, Johannes5,
Prakash, Nilima1
1Hamm-Lippstadt University of Applied Sciences, Hamm,
Germany, 2Institute of Developmental Genetics, Helmholtz Center
Munich, German Research Center for Environmental Health,
Neuherberg, Germany, 3Institute of Experimental Genetics,
Helmholtz Center Munich, German Research Center for
Environmental Health, Neuherberg, Germany, 4Department of
Cellular and Molecular Immunology, Max Planck Institute of
Immunobiology and Epigenetics, Freiburg, Germany, 5Chair of
Experimental Genetics, Technical University of Munich,
Freising-Weihenstephan, Germany
Mesodiencephalic dopaminergic (mdDA) neurons, which
comprise the substantia nigra pars compacta (SNc) and the
ventral tegmental area (VTA), play signicant roles in the
neural circuitry responsible for movement, cognition, and
emotion. Considering their critical functions in neuropsychiatric
and neurodegenerative disorders, such as Parkinson disease
(PD), considerable attention has been paid to the development
of mdDA neurons. A growing body of literature has revealed
the importance of the WNT1/b-catenin signaling pathway in
the differentiation and survival of these neurons and showed
that a disrupted activation or inhibition of the WNT1/b-catenin
signaling pathway leads to an incorrect specication or
differentiation of the mdDA neurons. This study aims to
unravel the identity of the cells responding to this pathway in
the murine ventral midbrain (VM) and the precise mechanism
of WNT/b-catenin action in these cells. We found that the
WNT/b-catenin-responsive cells constitute only a fraction of
all mdDA progenitors, precursors, and neurons in the murine
VM. Located mostly in the Wnt1+, Rspo2+ and Lef1+ lateral
oor plate of the medial and caudal midbrain, these
WNT/b-catenin-responsive cells develop preferentially into
caudomedial (VTA) mdDA neurons. We also show that the
differentiation of WNT/b-catenin-responsive mdDA progenitors
into mature mdDA neurons is inhibited by a strong
activation of the WNT/b-catenin signaling pathway via
RSPO2, a WNT/b-catenin agonist, and LEF1, a nuclear
effector of this pathway. Furthermore, we show that this
inhibition is due to the repression of the murine Pitx3 gene
promoter via conserved LEF1/TCF binding sites in this
promoter. Our results indicate that the correct differentiation
of mdDA progenitors into specic mdDA neuron subsets,
particularly into SNc DA neurons, relies on the attenuation of
WNT/b-catenin signaling in these cells and provide new
means for stem cell-based regenerative therapies of PD and
in vitro models of neuropsychiatric diseases.
Keywords: WNT1/b-catenin signalling pathway; Mesodien-
cephalic dopaminergic neurons; Pitx3
92
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Poster Abstracts
153
IDENTIFICATION OF KEY REGULATORS DURING
THE ESTABLISHMENT OF MOUSE INTESTINAL
ORGANOID FROM EMBRYONIC STEM CELLS
Kim, Min-Seon, Yoo, Mijoung, Kim, Hyo-Min, Ko, Hyunjoo,
Roh, Tae-Young
Division of Integrative Bioscience and Biotechnology,
POSTECH, Pohang, Korea
Direct lineage specication of pluripotent stem cell is one of
the valuable systems for studying cell fate decision as well
as developmental process. We successfully established a
robust methodology for intestinal organoids formation from
mouse embryonic stem cells by treatment of specic growth
factors and inhibitors. During this cell lineage process, we
sought key regulators responsible for the cell fate determina-
tion in embryonic stem cells, denitive endoderm, mid-gut
and intestinal organoid by single cell transcriptomic analysis.
Also we compared the temporal-spatial single-cell
transcriptome landscapes of our mouse intestinal
organoid with that of the public human fetal digestive tract
to understand the cellular mechanism of stemness,
differentiation, and cell-to-cell communication. From this
study, we can provide a gene regulation network focused on
the master regulators at a single cell resolution during the
differentiation.
Keywords:
Embryonic stem cell; Intestinal organoid; transcrip-
tomics
154
CARDIAC ORGANOIDS FROM HUMAN PLURIPO-
TENT STEM CELLS RESEMBLE KEY FEATURES OF
HUMAN HEART DEVELOPMENT
Drakhlis, Lika1, Sklarek, Jana1, Franke, Annika1,
Hegermann, Jan2, Zweigerdt, Robert1
1Leibniz Research Laboratories for Biotechnology and
Articial Organs, Hannover Medical School, Germany, 2Central
Laboratory of Electron Microscopy, Hannover Medical School,
Germany
Organoids are three-dimensional (3D) cellular aggregates
that better resemble features of native organs regarding
functionality and morphology compared to conventional cell
culture. They can be used as in vitro models for organ
development and diseases, drug development, and potentially
for future regenerative therapies. Although comprehensive
organoids have already been published for a wide range of
tissues including small intestine, kidney and brain, advances in
the cardiovascular eld are limited. Ideally, cardiac organoids
should resemble heart morphology at early developmental
stages. This should include proper formation of the three
heart layers (epi-, myo-, and endocardium) and an
organ-typical tissue composition, in particular cardiomyocytes,
myobroblasts and endothelial cells. However, up to
date such cardiac organoids do not exist and recent
approaches failed to mimic the human embryonic heart
properly. Therefore, the aim of this project is to overcome
these prior limitations. We have established a protocol,
which leads to the highly reproducible generation of cardiac
organoids from human pluripotent stem cells, which contain
at least two heart layers in an organized 3D pattern. We
show that these structures are composed of all cell types
expected for the heart, including the formation of endothelial
networks. Moreover, we show that these organoids could be
used as an informative model for drug screening e.g. in a
teratogenicity assay and for genetic disease modeling as
well. Together, we provide evidence that these novel organ-
oids represent a superior in vitro model for human embryon-
ic heart development, open new perspectives in pharmaco-
logical research and are potentially of interest for
organ-specic lab-on-chip approaches.
Keywords: Heart model; Cardiac organoid; Heart develop-
ment
155
DEFINING THE SINGLE-CELL DEVELOPMENTAL
LANDSCAPE OF HUMAN INNER EAR ORGANOIDS
Steinhart, Matthew R., Lee, Jiyoon, Koehler, Karl
Department of Otolaryngology, Indiana University School of
Medicine, Indianapolis, IN, USA
Inner ear development requires the complex interaction of
numerous cell types arising from multiple lineages. Most of
what is known about inner ear development has been
derived from animal studies; thus, the cellular and molecular
idiosyncrasies of human inner ear development are unknown. Our
group recently showed how to recapitulate human inner ear
organogenesis in vitro using 3D self-organizing stem cell
cultures. Our method promotes development of an entire
sensorineural circuit, including hair cells, inner ear-like
neurons, and Schwann cells. Although we have characterized
many of these cell types, we have not fully dened the process
by which individual cells commit to lineages represented in
inner ear organoids. Here, our goal was to reconstruct a
time-based map of the origin of cell lineages during inner ear
organoid development. We analyzed inner ear organoids
using single-cell RNA sequencing at various time points
during the rst two weeks of our protocol. We identied
expression patterns of inner ear organoids as they progress
from undifferentiated stem cells to surface ectoderm then to
otic placode following treatment with FGF, BMP, and WNT
signaling modulators. The rst major cell group identied
was surface ectoderm, indicated by TFAP2A and ECAD. At a
later time point, we observed lineage commitment to
posterior cranial placode (PAX8, SOX2, TFAP, ECAD and
NCAD). Further along differentiation, we observed cells
expressing markers specic to otic placode (PAX2, PAX8,
SOX2, SOX10, JAG1). Interestingly, we also mapped a
population of cells with expression patterns similar to
cranial neural crest (TFAP2A, SLUG, SOX10), which
bifurcated into mesenchymal and neuroglial cells. By
elucidating organoid developmental lineages, we have
created a platform upon which to perturb developmental
signaling mechanisms. We plan to use this platform to
model genetic disease using gene editing and test new drug
targets for hearing loss and balance disorders.
Funding Source
National Institute of Health grants R03 DC015624 and R01
DC017461-01.
Keywords: inner ear; organoid; lineage trajectory
93
AMSTERDAM NETHERLANDS
Poster Abstracts
156
THE RNA METHYLTRANSFERASE TRDMT1
COORDINATES EARLY EMBRYONIC STAGE
CELL-FATE SPECIFICATION
Garcia-Outeiral, Vera1, Fuentes-Iglesias, Alejandro1, Guallar,
Diana2, Fidalgo, Miguel1
1Physiology, Center for Research in Molecular Medicine and
Chronic Diseases (CiMUS), Universidade de Santiago de
Compostela, Spain, 2Biochemistry, Universidade de Santiago
de Compostela, Spain
Proper cytosine methylation of nucleic acids to give rise to
5-methylcytosine is vital for the regulation of developmental
processes, but the molecular underpinnings of this epigenetic
modication in RNA species remain poorly understood.
tRNA aspartic acid methyltransferase 1 (Trdmt1), which
acts by catalyzing tRNA methylation, is the most conserved
and enigmatic member of the DNA methyltransferase family.
In this study we attempt to understand Trdmt1 functions
during early embryo development using mouse embryonic
stem cells (ESCs) stimulated to undergo gastrulation-like
events. For this purpose, we employed combinations of
RNAi and CRISPR/Cas9 approaches for Trdmt1 loss-of-func-
tion in pluripotent stem cells. Remarkably, we found that
while Trdmt1 is dispensable for the maintenance of the
pluripotent cell state, the absence of this protein impairs
the establishment of the three germ layers (ectoderm,
mesoderm and endoderm) and therefore hampers proper
gastrulation process in vitro. At the molecular level, we
show that absence of Trdmt1 in in vitro organoids affects
transcriptional landscapes required to modulate early cell
fate decisions during embryo developmental processes.
Taken together, our results reveal an unanticipated role of
an epitranscriptomic writer, Trdmt1, in coordinating gene
expression programs that are essential for exiting
pluripotency.
Funding Source
This research was funded by the the Agencia Estatal de
Investigación (BFU2016-80899-P) (AEI/FEDER, UE) and the
Consellería de Cultura, Educación e Ordenación Universitaria
(ED431F 2016/016) to M.F.
Keywords: RNA methylation; Organoids; Differentiation
158
HISTONE AMINOTRANSFERASE 1 IS A NOVEL
PLAYER GOVERNING EARLY ECTODERM LINEAGE
COMMITMENT
Escudero, Adriana, Souto, Yara, Fuentes-Iglesias, Alejandro,
Pardavila, Jose Angel, Guallar, Diana, Fidalgo, Miguel
Physiology, Center for Research in Molecular Medicine and
Chronic Diseases (CiMUS), Universidade de Santiago de
Compostela, Spain
Proper epigenetic regulation of gene expression programs is
critical for orchestrating the cell-fate transitions occurring in
every organism during embryonic development. Although
histone acetylation is well known to play a central role in many
nuclear processes such as transcriptional competence, the
specic mechanisms by which histone acetyltransferases
regulate developmental processes remain to be addressed.
Here, using 3D in vitro cultures we identied the Histone
aminotransferase 1 (Hat1) as a new key regulator of bona de
pluripotency commitment. We show that both short and
stable Hat1 loss-of-function signicantly alters the histone
acetylation-dependent control of gene expression in mouse
embryonic stem cells. Importantly, we found that Hat1
ensures accurate expression of ectoderm lineage regulators
during early stages of in vitro gastrulation process. Thus, our
data suggest that Hat1 plays an essential role through the
establishment of histone acetylated landscapes that are
required for exiting pluripotency. Overall, our ndings link Hat1
as a histone acetyltransferase enzyme that exerts a funda-
mental role in ectodermal cell specication. Furthermore, our
results may open new avenues to understand developmental
diseases linked to faulty development of the ectoderm during
embryogenesis.
Funding Source
This research was funded by the the Agencia Estatal de
Investigación (BFU2016-80899-P) (AEI/FEDER, UE) and the
Consellería de Cultura, Educación e Ordenación Universitaria
(ED431F 2016/016) to M.F.
Keywords: Histone Acetyltransferase 1; development;
gastrulation
94
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Poster Abstracts
159
GENERATION AND CONFIRMATION OF HIGH
THROUGHPUT MODELED CEREBRAL BRAIN
ORGANOIDS AS A FUTURE SCREENING MODEL
Jung, Yong Hun, Jun, Yesl, Shin, Seungchul, Choi, Donghee,
Yang, Jihun, Chung, Seok
Mechanical Engineering, Korea University, Seoul, Korea
Generating brain organoid have allowed researchers to
understand actual human developments. Upon the onset of
brain organoids, many new possibilities have been opened,
such as ZICA virus, Alzheimer, Parkinson’s, and many more
models. Furthermore, brain organoids have given us a new
option to replace PDX mouse models. As PDX mouse
models are expensive, time consuming and irrelevant to
actual humans, brain organoids offer far more understand-
ings and choices. Even though all seems great, brain
organoids are still yet to be generated as a high throughput
model. Here, a new method of generating brain organoids as
a high throughput model will be introduced. By culturing
cerebral organoids without Matrigel on an ultra-non-adhesive
well plates, size controlled, quantiable, and identical
cerebral brain organoids were grown. Conrming with the
immunouorescence data, not only do MAP2 and TUJ1 well
show the recapitulation of cortical plate in the cerebral
cortex area, but PAX6 also shows the ventricular zone in the
brain. Furthermore, when the electrical cues were applied to
the brain organoids made in high throughput manners,
various signals such as action potential, LFP waves, burst
waves and random waves that of in vivo brain’s were all
detected; assuring that the brain organoids cultured in a high
throughput method well represent actual in vivo cerebral
cortex. In the future, it is believed that high throughput
cerebral brain organoids will be utilized in many areas such
as disease modeling, screening, transplanting and many
more.
Funding Source
This work was supported by the Technology Innovation
Program (10067407, Development of high throughput
organoid clearing system and 3D imaging system for drug)
funded By the Ministry of Trade, industry & Energy.
Keywords: high throughput; cerebral organoids; Matrigel
160
IMPACT OF SOX11 ON HUMAN EARLY
NEURODEVELOPMENT
Kavyanifar, Atria1, Turan, Soeren1, Boerstler, Tom2, Winner,
Beate2, Lie, Dieter Chichung1
1Department of Biochemistry, Friedrich–Alexander University
Erlangen–Nürnberg, Erlangen, Germany, 2Department of Stem
Cell Biology, Friedrich–Alexander University Erlangen–
Nürnberg, Erlangen, Germany
The SoxC family of transcription factors controls a number
of key processes in murine CNS development including
neuronal fate determination, migration, neurite growth,
neuronal survival and synapse development. While the
function of SoxC in mice has been well characterized, the
role of SoxC members in human neurodevelopment remains
largely unknown. Heterozygous missense mutations or
deletions in SOX11 have recently been identied as the
underlying genetic cause in children with a Cofn-Siris
Syndrome (CSS) like syndrome. Hallmarks of CSS include
developmental disability, skeletal abnormalities, and charac-
teristic facial features. The involvement of multiple organ
systems in CSS, indicate that SOX11 fullls critical functions
in the development of different germ layers. Here, we
investigate the function of SOX11 in human development
using human embryonic stem cells (hESCs) as a model
system. Homozygous SOX11-decient hESCs lines with a
SOX11 frameshift mutation were generated using CRISPR/
Cas9 genome editing. In recent years different approaches
have been optimized to study the development of human
brain: 2D hPCS (human pluripotent stem cells) neural
differentiation models, which mimics the environment that
produces the neuroectoderm though embryoid body
formation, and hPCS- derived 3D brain organoids. In both
models we found that Sox11 deciency was associated
with decreased expression of Nestin, Pax6 and Sox1 upon
induction of neural differentiation. Moreover, we observed
that Sox 11 deciency resulted in strong impairment in the
generation of neurons. Collectively, our data point at an
essential role for Sox11 during early stages of human neural
development.
Funding Source
This work was supported by the German Research Founda-
tion (DFG) Research training group; GRK2162 “Neurodevel-
opment and Vulnerability of the CNS.”
Keywords: SOX11; human neurodevelopment; organoid
95
AMSTERDAM NETHERLANDS
Poster Abstracts
161
TRANSCRIPTIONAL REGULATION OF CELL
STATES IN THE DEVELOPING MOUSE INTESTINAL
EPITHELIUM
Hansen, Stine Lind, Larsen, Hjalte List, Pikkupeura, Laura
Maarit, Guiu, Jord1, Müller, Iris, Sahadevan, Sudeep, Helin,
Kristian, Sandelin, Albin, Jensen, Kim
1Biotech Research and Innovation Centre, BRIC, Universitiy of
Copenhagen, Denmark
The processes that govern the development of functional
organs, such as the gastrointestinal tract, are complex and
require accurate regulation of cell fate decisions. Once
formed, a series of events ensure that fetal tissues transition
into mature organs that are subsequently maintained
throughout life. One of the signicant differences that
discriminate tissues during homeostasis versus develop-
ment is the balance between gain and loss of cells, whereas
the developmental state is characterized by unbalanced cell
fate decisions with limited natural loss of cells in order to
fuel constant tissue growth. Stem cells are well character-
ized in the adult intestinal epithelium, but the mechanisms
that regulate the transition of the cells from a fetal to an
adult state remain largely unknown. By utilizing the organoid
culture system, we aim to identify gene regulatory networks
that control the transition between fetal and adult stem cells
states in the intestinal epithelium. We have previously
demonstrated that intestinal epithelial cells derived from the
fetal epithelium are distinct from their adult counterpart
when cultured in vitro. When cultured in a 3D matrix fetal
epithelial cells form enterosheres that resemble the fetal
state in vivo with respect to differentiation states. Moreover,
fetal enterospheres retain the capacity to mature into adult
epithelium upon transplantation. Transcriptional analysis
reveal that more than 3000 genes are differently expressed
between the fetal and adult organoids, suggesting that
distinct gene regulatory networks control the different
cellular states. In order to identify key gene regulatory
networks, we are currently performing a CRISPR/Cas9 KO
screen for transcription factors and epigenetic regulators
differentially expressed between fetal enterospheres and
adult organoids. Identied factors will subsequently be
characterized for their role in maturation of the intestinal
stem cells in vitro and in vivo in order to provide molecular
insight into the mechanisms that govern tissue maturation
of the intestinal epithelium.
Keywords: Intestinal development; CRISPR/Cas9; Organoids
162
UNCOVERING THE ROLE OF PLANAR CELL POLARI-
TY
DURING MOUSE INTESTINAL MORPHOGENESIS
Rao-Bhatia, Abilasha1, Coquenlorge-Gallon, Sabrina2,
Zhu, Min2, Yin, Wen-Chi2, Zhang, Xiaoyun 2, Hui, Chi-Chung2,
Hopyan, Sevan2, McNeill, Helen3, Kim, Tae-Hee2
1Department of Molecular Genetics, University of Toronto,
The
Hospital for Sick Children, Toronto, ON, Canada, 2Developmen-
tal and Stem Cell Biology, PGCRL, The Hospital For Sick
Children, Toronto, ON, Canada, 3Developmental Biology,
Washington University School of Medicine, St. Louis, MO, USA
The mammalian intestine is lined with millions of nger-like
projections, termed villi. These villi are critical for maximiz-
ing nutrient absorption, digesting food and serving as a
barrier from the harsh luminal environment. As such
compromised villi can lead to serious diseases including
malabsorption, short bowel syndrome, celiac and others.
Therefore, understanding how intestinal morphogenesis
occurs is essential for regenerative therapies for these
diseases. Although villi are precisely patterned by a network
of signalling pathways during embryogenesis, most notably
Hedgehog (Hh), it remains unclear as to how these signals
translate into distinct morphogenetic transformations. Using
the mouse model, our RNA-seq analyses coupled with GLI2
(Hh-transcriptional activator) ChIP-seq reveal that planar cell
polarity (PCP) genes such as Fat4, Dchs1 and Vangl2 are
direct targets of Hh in the gut mesenchyme. Notably, mice
deleted and/or mutated for these genes exhibit severe villus
fusions and fail to form mesenchymal clusters, demonstrat-
ing for the rst time the importance of PCP in villication.
Furthermore, genetic interaction studies reveal that the
core-PCP axis (Vangl2) acts in parallel to the atypical
cadherin axis (Fat4, Dchs1) in maintaining PCP. By utilizing
live light-sheet uorescence microscopy, we have visualized
and tracked stromal cell behavior during villication ex vivo
and have identied perturbed cell migration and orientation
upon Fat4 knockout. Additionally, we have been able to
model this stromal behavior in vitro using 3D-culture based
approaches. Together, we introduce Hh-activated stromal
PCP as novel mechanisms required for morphogenetic cell
behaviour and subsequent epithelial rearrangement, critical
for villication.
Keywords: Intestinal Morphogenesis; Planar Cell Polarity;
Live imaging and 3D-culture systems
96
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Poster Abstracts
163
TARGETING DECISION NODES DURING DIRECT
LINEAGE REPROGRAMMING
Lohrer, Benjamin1, Falk, Sven1, Schichor, Christian2,
Berninger, Benedikt3, Karow, Marisa1
1Physiological Genomics, Ludwig Maximilian University of
Munich, Planegg-Martinsried, Germany, 2Department of
Neurosurgery, Klinikum Grosshadern, Ludwig Maximilians
University Munich, Germany, 3Institute of Psychiatry,
Psychology & Neuroscience, Centre for Developmental
Neurobiology, MRC Centre for Neurodevelopmental
Disorders, King´s College London, UK
Ectopic expression of dened transcription factors can force
direct cell fate conversion from one cell lineage to another in
the absence of cell division. Several transcription factor
cocktails have enabled successful reprogramming of
various somatic cell types into induced neurons (iNs) of
distinct neurotransmitter phenotypes. However, the interme-
diate stages bridging starter cell and iN population have
been largely unstudied. We addressed this aspect by
studying the transcriptional landscape during the course of
pericyte-to-neuron conversion by using single cell RNA-se-
quencing (scRNA-seq). We could thereby recently show that
successful reprogramming of adult human brain pericytes
into functional iNs by Ascl1 and Sox2 (AS) encompasses
activation of a neural stem cell-like gene expression program
which is characterized by the expression of a particular set
of genes referred to as switch genes. Building up on this
work, we show here using scRNA-seq, that modulating the
neural stem cell-like intermediate stage by manipulating
signaling pathways specically active in this switch-state
greatly impacts on the reprogramming outcome. Our
ndings suggest a novel unanticipated entry point to
navigate the reprogramming trajectory towards specic
target cell populations. These results for the rst time
provide evidence for the possibility to specically target an
intermediate neural stem cell-like state during AS-mediated
reprogramming allowing the modulation of the reprogram-
ming outcome. The investigation of cellular intermediates
during iN reprogramming will provide handles to improve
lineage conversion towards therapeutically relevant cell
types.
Keywords: direct lineage reprogramming; neural stem cells;
scRNA-sequencing
164
LEPTIN RECEPTOR IDENTIFIED MESENCHYMAL
STEM CELL IN ENDOCHONDRAL BONE
FORMATION AND FRACTURE REPAIR
Yen, Yu-Ting1, Hung, Shih-Chieh2, Chien, May1
1Integrative Stem Cell Center, China Medical University,
Taichung City, Taiwan, 2Institute of Biomedical Sciences,
Academia Sinica, Taipei, Taiwan
Endochondral ossication is an important process for both
skeletal development and bone repair. In the long bones,
cartilage anlage is rst formed by mesenchymal condensa-
tion, followed by chondrocyte differentiation and maturation
and subsequently bone mineralization. A common molecular
marker for mesenchyme progenitors has not been fully
identied. Here, by lineage-tracing experiments in mice, we
discover that leptin receptor (Lepr+) cells distribution during
endochondral bone formation from fetus to adult. Pp2a is
cell-autonomously required for the maintenance of MSCs. To
assess the consequences of ppp2r1a deletion from MSCs, we
generated Lepr-cre; ppp2r1a / mice. As expected, YAP
phosphorylation increased in LepR+ stromal cells isolated
from Lepr-cre; ppp2r1a / mice. Pp2a is thus cell-autono-
mously required to positively regulate YAP activation and to
maintain normal numbers of quiescent Lepr+ MSCs in adult
bone marrow. To investigate the contribution of Lepr+ cells to
the repair of perforated cartilage, we created a hole at
diaphysis of the femur to observe fracture healing in Lepr-cre;
tdTomato mice. Six days following perforation, the injured
region of the diaphysis was covered Lepr+ cells inltration.
Thus, Lepr marks mesenchymal progenitors responsible for
both normal bone formation and fracture repair.
Keywords: leptin receptor; mesenchymal stem cell; endo-
chondral ossication
97
AMSTERDAM NETHERLANDS
Poster Abstracts
165
THE EFFECT OF DIFFERENT OXYGEN CONDITIONS
ON MITOCHONDRIAL DYNAMICS IN THE EARLY
STAGE OF DEVELOPMENT OF HUMAN CEREBRAL
ORGANOID
Buzanska, Leonora1, Liput, Michal1, Stachowiak, Ewa2,
Stachowiak, Michal K.2, Zayat, Valery1, Zychowicz, Marzena1
1
Department of Stem Cell Bioengineering, Mossakowski
Medical Research Centre, Warsaw, Poland,
2
Department of
Pathology and Anatomical Sciences, State University of New
York at Buffalo, NY, USA
Biomimetic in vitro conditions of 3D culture together with
lowering oxygen level to 5% (physiological normoxia) were
used to study mitochondrial dynamics and neuronal differ-
entiation during early development using human cerebral
organoids model. In this report we will elucidate whether the
constant 5% O2 conditions or the short-term changes
(pulses) in the oxygen level affect cortical development and
mitochondrial dynamics in cerebral organoids. For that
purpose cerebral organoids were generated from induced
pluripotent stem cells (hiPSC) in feeder-free conditions and
cultured either in 21% (control) or 5% O2. For short-term
pulse of 5% O2 2-week cerebral organoids were transferred
from 21 to 5%O2 and cultured for one week, followed by
transition back to the 21% O2 for an additional week. Next,
cerebral organoids were xed and processed for immunohis-
tochemistry (IHC) with mitochondrial and neural-specic
antibodies and transmission electron microscopy (TEM). To
quantify mitochondrial dynamics and neuronal networks in
selected Regions of Interest (ROIs) within ventricular zone
(VZ) and cortical zone (CZ) of cerebral organoids imaging
software and Mitochondrial Network Analysis toolset were
applied. Data analysis revealed signicant changes of
mitochondrial number and branches in mitochondrial
networks in different regions of cerebral organoids in all test-
ed groups, suggesting that lower level of O2 may affect
mitochondrial dynamics. The number of individual mitochon-
dria in cortical zone was lowered in the cerebral organoids
cultured in 21%O2. Staining against non-glycosylated protein
of mitochondria surface membrane showed differences in
their uorescence intensity between ventricular and cortical
zones in different oxygen conditions. IHC detection of
neuronal processes with Pan Neuronal Marker showed
reduced uorescence intensity in cortical zone in 5% O2 as
compared to control. Analysis of TEM images suggests that
short-term pulses of low O2 may induce neuronal differentia-
tion within cerebral organoids. Further studies are ongoing
to compare mitochondrial dynamics and structure between
VZ and CZ in organoids cultured in constant physiological
normoxia conditions. The cerebral organoid model offers a
unique opportunity to study early human neural develop-
ment.
Funding Source
National Science Foundation (CBET-1706050), Patrick P. Lee
Foundation and USA Fulbright Foundation to MKS and
Statutory Funds from MMRC PAS to LB.
Keywords: Cerebral organoids; neurogenesis; mitochondrial
dynamics
166
NONVIRAL REPROGRAMMING OF HUMAN
ASTROCYTES TO DOPAMINERGIC NEURONS
Grigsby, Christopher L.1, Rivetti di Val Cervo, Pia1, Arenas,
Ernest1, Stevens, Molly M.2
1
Department of Medical Biochemistry and Biophysics,
Karolinska Institutet, Stockholm, Sweden,
2
Department of
Materials; Department of Bioengineering; and Institute of
Biomedical Engineering, Imperial College London, UK
Parkinson’s disease (PD) is currently treated with dopami-
nergic drugs or deep brain electrical stimulation. However,
these treatments are symptomatic, do not slow the progres-
sion of disease, and their clinical benets inevitably decline
as the loss of dopaminergic neurons progresses. Disease
modifying strategies such as transplantation of stem
cell-derived midbrain dopaminergic cells or direct repro-
gramming of resident glia to dopaminergic neurons are
currently being developed. We recently reported that
astrocytes can be converted into neurons, both in vitro and
in vivo, using viral delivery of transcription factor genes that
recapitulate developmental expression patterns. Now, we
have developed a virus-free, nanoparticle-based reprogram-
ming protocol that eliminates the risk of deleterious genetic
integration associated with viral methods. We delivered
cocktails of transcription factor gene and microRNA
constructs packaged in polymer nanoparticles derived from
the synthetic bioreducible poly(amidoamine) p(CBA-ABOL).
Programmed degradation of the polymer backbone releases
the payload efciently, while diminishing toxicity of the
carrier so as to enable repeated application without com-
pounding cytotoxicity. Using a serial dosing protocol, we
could efciently convert human astrocytes to Tuj1+/TH+
cells. Conversion efciencies were assayed using high
content imaging and automated analysis, which also
allowed us to quantify neuronal morphometrics including
neurite number, length, and branching. These nonvirally-in-
duced dopaminergic neurons (NiDAs) were further validated
for expression of MAP2, DAT, synaptophysin, and synapsin I.
We next aim to model the nonviral gene delivery and
reprogramming processes in an organoid model. A virus-free
strategy to produce NiDAs from human astrocytes may
facilitate a safe and effective translation of direct reprogram-
ming of resident glia to dopaminergic neurons.
Keywords: Cell Reprogramming; Neurodegenerative Disease;
Nonviral Gene Delivery
98
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Poster Abstracts
167
HUMAN IPSC COLONY MORPHOLOGY VARIATIONS
ASSOCIATED WITH DISTINCT PROTEOMIC
SIGNATURES
Bjorlykke, Yngvild, Ghila, Luiza, Søviknes, Anne Mette, Vethe,
Heidun, Hoareau, Laurence, Chera, Simona, Ræder, Helge
Department of Clinical Science, University of Bergen, Norway
Human induced pluripotent stem cells (hiPSC) are of high
interest for both scientists and clinicians due to its capacity
to differentiate into a whole range of different cell types. In
theory, after a successful reprograming hiPSC has the
capacity for long term culturing in a healthy undifferentiated
state. However, some hiPSC lines spontaneously differenti-
ate and change cellular phenotype and colony morphology.
The variation is unpredictable, and not fully understood. In
our study, we assess the molecular differences on the
proteome level between 20 hiPSC lines classied in 3
distinct colony morphology groups (classic, intermediate
and monolayer). All 20 hiPSC lines were analysed with
global label-free proteomics, which yielded ~5000 identied
and quantied proteins for each samples. We nd distinct
proteomic signature for the different colony morphology
groups and our results indicate an EMT event induced by
TGFB signalling for one of the colony morphology groups.
Funding Source
The Western Norway Regional Health Authority; Bergen
Research Foundation.
Keywords: HiPSC; Colony morphology; Proteomics
168
TESTES-ON-CHIP MODEL FOR IN VITRO SPER-
MATOGENESIS
Sharma, Swati1, Venzac, Bastien2, Le Gac, Severine2, Schlatt,
Stefan1
1Center for Reproductive Medicine and Andrology, University
of Muenster, Germany, 2Applied Microuidics for Bioengineer-
ing Research, University of Twente, Enschede, Netherlands
Testes evolved as a bifunctional (steroid and gamete
producing) organ, consisting of basal, intraepithelial and
adluminal compartments. Endocrine regulation of the testes
is controlled by the HPG-axis via gonadotropins. Spermato-
genesis occurs within the long convoluted structures known
as seminiferous tubules. The tubular compartment compris-
es primarily of germ cells and somatic Sertoli cells. Sertoli
cells anchor the germinal epithelium, creating a blood-tes-
tis-barrier (BTB) separating the basal and the adluminal
compartments. Undifferentiated mitotic germ cells lie in
the basal region, while the differentiating meiotic cells are
intraepithelial and the spermatids are in the adluminal
region. Conventional culture approaches failed to recapitu-
late the in vivo testicular structure and complex stem-cell
niche microenvironment of the human testes. Niche-driven
fundamental mechanisms guiding testicular stem cells to
active or inactive (quiescent) states are largely unknown. To
address this research gap, we aimed to create a controlled
in vitro environment mimicking in vivo physiological and
biological conditions in testes, and developed a tested-on-
a-chip platform. Intact tubules were cultured to preserve the
complex cellular (and clonal) arrangements, testicular stem
cell niches and maintain the structural and functional
integrity of seminiferous tubules. Our microuidic device
consisted of a chamber surrounded by perfusion channels.
In contrast to classical approaches, our platform allowed
continuous shear-free medium renewal while locally main-
taining the tissue microenvironment with the microgrooves
placed between the culture chamber and the perfusion
channels. Devices were fabricated from PDMS using
soft-lithography and a 3D-printed mold, and bonded to glass
slides. Functionally regressed adult human testicular tissue
(from Gender dysphoria patients) was loaded in the culture
chamber and medium perfused at 90 μL/h for up to 11 days.
On-chip live-cell imaging revealed maintenance of the tubule
structural integrity, and active spermatogenesis during
culture. Off-chip live-dead assays showed the tissues remain
viable during the same period. This model will be further
exploited to understand spermatogenesis regulation,
spermatogonial stem cell expansion & differentiation.
Keywords: Testes; Spermatogenesis; Microuidics
170
A TUBULAR ORGANOID-DERIVED GUT-ON-A-CHIP
MODEL BY PRESERVING THE STEM CELL NICHE
OF LGR5+ INTESTINAL EPITHELIA
Kurek, Dorota1, Puschhof, Jens2, Naumovska, Elena1,
Kosim, Kinga1, Nicolas, Arnaud3, Lanz, Henriëtte1, Trietsch,
Sebastiaan J.3, Clevers, Hans C.2, Joore, Jos4, Vulto, Paul4
1Model Development, MIMETAS B.V., Leiden, Netherlands,
2Hubrecht Institute, Utrecht, Netherlands, 3Hardware Develop-
ment, MIMETAS B.V., Leiden, Netherlands, 4MIMETAS B.V.,
Leiden, Netherlands
Microuidic techniques are increasingly recognized as an
important toolbox to add physiologically relevant cues to
traditional cell culture. These cues include long term
gradient stability and continuous perfusion. Microuidic
technology allows patterning of cell layers as stratied
co-cultures that are devoid of articial membranes, in order
to capture complex tissue architectures found in vivo.
Previously, we have introduced the OrganoPlate® platform
for growing human intestinal gut tubules in a membrane-free
manner. Although suitable for toxicity studies, this model
uses human intestinal cell lines, such as adenocarcinoma
line Caco-2, which has limited differentiation capabilities and
99
AMSTERDAM NETHERLANDS
Poster Abstracts
harbors multiple gene mutations. In contrast, Lgr5+ intesti-
nal organoids can develop crypt-villi morphology and form
an epithelial barrier – features associated with gut epitheli-
um. These organoids are usually grown as a polarized
ball-like structures embedded in an ECM, with limited apical
access. Here we show a human organoid gut-on-a-chip
model which is composed of Lgr5+ gut epithelial cells
grown inside of the microuidic channels of the Organo-
Plate®. We established a tubular shaped epithelial barrier
model of the intestinal tract showing rapid cell polarization,
tight junction formation and proper expression of intestinal
markers. These gut tubules are suitable for high-throughput
screening of compound effects through real time imaging of
transport and barrier integrity. Moreover, the OrganoPlate®
facilitates development of complex models of gut epithelial
tubules co-cultured with endothelial vessels. These complex
gut-on-a-chip models allow mimicking disease phenotypes
such as inammatory bowel diseases (IBD) and support
screening for potential drug targets. Protocols have been
established that allow automated readout of the barrier
integrity, followed by image analysis and quantication. The
combination of Lgr5+ gut organoids with the OrganoPlate®
technology are a powerful combination to study physiology
and disease mechanisms in patient specic gut models.
Keywords: Microuidics; Gut-on-a-chip; Intestinal organoids
171
TOWARDS ENGINEERING HUMAN EXTRAHEPATIC
BILE DUCTS FROM DECELLULARIZED BILE DUCT
SCAFFOLDS AND BILE DUCT-DERIVED ORGAN-
OIDS
Willemse, Jorke, Voogt, Iris, Burka, Ksenia, Roos, Floris, de
Jonge, Jeroen van der Laan, Luc J.W., Verstegen, Monique
M.A.
Department of Surgery, Erasmus Medical Center, Rotterdam,
Netherlands
Bile duct-related complications are a common case of graft
failure after liver transplantation and may require retrans-
plantation. Tissue engineered extra hepatic bile duct (EBD)
constructs might help to solve this problem. Using decellu-
larization techniques, all cells can be removed from the
EBD-tissue to generate bile duct extracellular matrix (ECM)
which can be used as a bio-scaffold for tissue engineering
purposes. The aim of this study is to establish a reproduc-
ible protocol for the decellularization of EBD tissue and to
explore recellularization using human LGR5+ bile duct-de-
rived organoids. EBD tissue is obtained from human livers,
which are unsuitable for transplantation due to inferior
quality (N=10). EBD tissue was treated with Trypsin-EDTA for
30 minutes and 10 times 30 minutes 4% Triton-x-100 + 1%
NH4 to remove all cells. After decellularization, circular discs
(12,5 mm^2) were created. Bile duct derived organoids were
initiated from healthy EBD tissue and made single cells. The
cell suspension (1×〖10〗^5 cells per 10μl) was incubated on
the luminal side of the ECM surface and kept in culture for
up to 3 weeks. EBD tissue was completely decellularized,
while maintaining ECM proteins and architecture. After seed-
ing with organoid-derived cells, a conuent epithelial layer
was formed on the luminal surface of the EBD ECM. Further
analysis showed that cells polarize similar to large cholangio-
cytes and that cholangiocyte-markers, such a cytokeratin-7
and 19, were detectable at genetic and protein level. In
conclusion this study shows that decellularization and
recellularization of EBD tissue with bile duct derived organ-
oids is feasible. The complete surface of the ECM could be
covered with a conuent epithelial layer, potentially restoring
the barrier function. Furthermore, key cholangiocyte markers
were present suggesting differentiation towards functional
bile duct lining cells. Further study is required whether this
bile duct engineering is suitable for clinical application after
liver transplantation.
Keywords: Bile duct tissue engineering; Bile duct-derived
organoids; Extrahepatic bile duct
172
DIRECT GENERATION OF HUMAN NAÏVE INDUCED
PLURIPOTENT STEM CELLS FROM SOMATIC CELLS
IN MICROFLUIDICS
Pellegrini, Marco1, Giulitti, Stefano1, Zorzan, Irene1,
Martini, Paolo3, Gagliano, Onelia4, Mutarelli, Margherita5, Ziller,
Michael J.6, Cacchiarelli, Davide5, Romualdi, Chiara3,
Elvassore, Nicola4, Martello, Graziano1
1Department of Molecular Medicine, University of Padova, Italy,
3Department of Biology, University of Padua, Padova, Italy,
4Department of Industrial Engineering, University of Padua,
Padova, Italy, 5Telethon Institute of Genetics and Medicine,
Pozzuoli, Italy, 6Department of Translational Psychiatry, Max
Planck Institute of Psychiatry, Munich, Germany,
Induced pluripotent stem cells (iPSCs) are generated by
expression of transcription factors OCT4, SOX2, KLF4 and
cMYC (OSKM) in somatic cells. In contrast to murine naïve
iPSCs, conventional human iPSCs are in a more developmen-
tally advanced state called primed pluripotency. Here we
report that human naïve iPSCs (niPSCs) can be generated
directly from less than 1000 primary human somatic cells
without stable genetic manipulation by delivery of modied
messenger RNAs with microuidics. Expression of OSKM
in combination with NANOG for 12 days generates niPSCs
free of transgenes, karyotypically normal, and display tran-
scriptional, epigenetic and metabolic features indicative of
the naïve state. Importantly, niPSCs efciently differentiate
into all three germ layers. While niPSCs could also be generat-
ed at low frequency under conventional conditions, our
microuidics approach will allow robust and cost-effective
production of patient-specic niPSCs for regenerative
medicine applications, including disease modelling and
drug screening.
Keywords: Human somatic cell reprogramming; Human naïve
iPSCs; Microuidics
100
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Poster Abstracts
173
LARGE-SCALE PRODUCTION OF HUMAN LIVER
ORGANOIDS WITH IMPROVED HEPATOCYTE
DIFFERENTIATION
Schneeberger, Kerstin1, Sánchez Romero, Natalia2, van
Steenbeek, Frank G3, Oosterhoff, Loes A.3, Pla Palacin, Iris2,
Chen, Chen3, van Wolferen, Monique3, van Tienderen, Gilles3,
Lieshout, Ruby5, Schene, Imre6, Verstegen, Monique M.A.7,
van der Laan, Luc J.W.7, Penning, Louis C.3, Clevers, Hans C.8,
Baptista, Pedro M.2, Spee, Bart3
1Clinical Sciences of Companion Animals, Utrecht University,
Utrecht, Netherlands, 2IIS Aragon, Instituto de Investigación
Sanitaria de Aragón , Zaragoza, Spain, 3Department of Clinical
Sciences of Companion Animals, Utrecht University, Utrecht,
Netherlands, , 5Department of Surgery, Erasmus Medical
Center, Utrecht, Netherlands, 6Division of Pediatric Gastroen-
terology, Wilhelmina Children’s Hospital, University Medical
Center, Utrecht, Netherlands, 7Department of Surgery,
Erasmus Medical Center, Rotterdam, Netherlands, 8Hubrecht
Institute, Utrecht, Netherlands
Human organoids represent an exciting cell source to treat
end-stage liver failure. However, establishing large numbers
of organoids with current protocols is tedious, since organ-
oids are cultured in droplets of Matrigel. Here we estab-
lished a method for the expansion of large quantities of
human liver organoids in a bioreactor. We observed rapid
proliferation of the organoids in the bioreactors reaching an
average of 40-fold cell expansion after two weeks. Despite
their increased proliferation, the organoids did not form
tumors in a xenograft model. When culturing the organoids
in differentiation medium, expression levels of mature
hepatocyte markers such as CYP3A4, MRP2, and ALB were
highly upregulated and albumin secretion and midazolam
metabolism reached levels equivalent to hepatocytes. Lastly,
we showed that organoids from bioreactors were able to
repopulate decellularized liver discs and formed liver-like
tissue including cholangiocyte-like cells and hepatocyte-like
cells on the extracellular matrices.In conclusion, we estab-
lished a novel method to culture billions of liver organoid
cells in a time-, work-, and cost-efcient way. This method
paves the way for the application of organoids for cell
therapy, in which large quantities of cells are needed.
Funding Source
This work was supported by the Dutch Research Council
NWO TTW (15498) to B.S., Dutch Research Council NWO
ZON/MW (116004121) to L.C.P. and B.S.; and K.F. Heinfonds
to K.S.
Keywords: Human liver organoids; Tissue engineering;
Bioreactor
175
THE CRITICAL ROLE OF AUTOPHAGY AND
MITOCHONDRIAL REMODELING IN ENDOTHELIAL
CELL DIRECTED DIFFERENTIATION
Hekman, Katherine1, He, Congcong2, Wertheim, Jason1
1Department of Surgery, Northwestern University, Chicago, IL,
USA, 2Department of Cell and Molecular Biology, Northwestern
University, Chicago, IL, USA
Vascular tissue derived from patient-specic induced
pluripotent stem cells (iPSCs) suffers from premature
replicative senescence, limiting clinical applications and
creating a signicant barrier to the advancement of organ
and tissue bio-engineering. Autophagy plays a critical role
in stem cell survival and directed differentiation, at least in
part through selective autophagy of the mitochondria, i.e.,
mitophagy. While mitophagy also contributes to mediating
cellular senescence, the mechanisms of premature senes-
cence in reprogrammed iPSC-derived endothelial cells
remain poorly understood. The iPSC lines ACS1028 and Y6
were subjected to directed differentiation over 6 days, and
cells were then puried by positive selection for VE-cadherin.
Autophagy marker microtubule-associated proteins 1A/1B
light chain 3B (LC3) and mitochondrial matrix protein Tom20
were quantied through Western blotting. Mitochondria were
evaluated with MitoTracker staining. Endothelial cell function
was assessed through immunouorescence of key markers
and quantication of nitric oxide production. During directed
differentiation from iPSCs to endothelial cells, mitochondrial
morphology evolved from globular to lamentous. LC3
expression decreased by 50% in the middle phase of differen-
tiation (p<0.05), correlating with signicant mitochondrial
remodeling, suggestive of uctuating mitophagy. Mature
iPSC-derived endothelial cells had minimal autophagy activity
5 days after purication, over which time nitric oxide produc-
tion also declined by 60% (p<0.05) and key cell surface
marker expression decreased. These iPSC-derived endotheli-
al cells senesced within 2 weeks after purication. Mature
iPSC-derived endothelial cells demonstrate a signicant
decline in autophagy activity that correlates with the loss of
mature endothelial cell function, represented by decreased
nitric oxide synthesis, which precedes the onset of premature
replicative senescence. This supports the role of mitophagy
in mediating cellular senescence of iPSC-derived endothelial
cells, and renders autophagy induction a target for attenuat-
ing senescence.
Funding Source
NIH 1F32HL137292; Association for Academic Surgery
Trainee Award in Basic Science.
Keywords: endothelial cell; autophagy; mitochondria
101
AMSTERDAM NETHERLANDS
Poster Abstracts
176
LYMPHATIC ENDOTHELIAL PROGENITOR CELLS
AND VEGF-C LOADED WITH SELF-ASSEMBLING
PEPTIDE NANOFIBERS PROMOTE LYMPHANGIO-
GENESIS IN INFARCTED MYOCARDIUM
Wang, Hai-Jie, Zhang, Hai-feng, Tan, Yu-zhen, Wang, Yong-li
Department of Anatomy, Histology and Embryology, Shanghai
Medical School of Fudan University, Shanghai, China
Lymphatic vessels play a crucial role in draining excess uid
and transporting macromolecular substances from extracel-
lular spaces. Disfunction of lymphatic vessels may cause
lymph edema and chronic inammation, leading to brosis
of the local tissue. This study investigated efciency of
transplantation of lymphatic endothelial progenitor cells
(LEPCs) and sustained release of VEGF-C from self-assem-
bling peptide (SAP) on promoting lymphangiogenesis after
myocardial infarction (MI). CD34+VEGFR-3+ EPCs were
isolated from rat bone marrow. Sustained release of VEGF-C
from SAP nanobers (SAPNs) was detected with ELISA.
Compatibility of SAPNs with the cells was accessed with
transmission electron microscopy and EB/AO staining. After
rat MI models were established with ligation of the anterior
descending branch of the left coronary artery, SAP carrying
the cells and VEGF-C was injected at the border of the
infarcted region. At four week after transplantation, the
survival and differentiation of the cells labeled with GFP
were examined, and repair of the infarcted myocardium was
evaluated. Under induction with VEGF-C, CD34+VEGFR-3+
EPCs could differentiate into lymphatic endothelial cells. The
cells spread well along SAPNs. SAPNs protected the cells
from apoptosis in the condition of hypoxia, and released
VEGF-C sustainedly. After transplantation, cardiac function
was improved signicantly. The number of the survived
LEPCs increased, and some cells differentiated into lymphat-
ic endothelial cells. Density of lymphatic vessels increased,
and cardiac edema was reduced. Moreover, angiogenesis
and myocardiac regeneration were enhanced. These results
suggest that SAPNs load LEPCs and release VEGF-C
effectively. VEGF-C released from SAPNs induces differenti-
ation of LEPCs towards lymphatic endothelial cells. Loading
stem cells and releasing growth factor with SAPNs is a
promised strategy for MI therapy.
Funding Source
This work was supported by National Natural Science
Foundation of China (81870215, 81770288, 81470385).
Keywords: Self-assembling peptide; Endothelial progenitor
cells; VEGF-C
177
COMBINATION OF THREE-DIMENSIONAL
SCAFFOLDS AND INTERMITTENT HYDROSTATIC
PRESSURE IN EX-VIVO EXPANSION OF
HEMATOPOIETIC STEM/PROGENITOR CELLS
Shin, Jung-woog1, Kim, Ji Eun1, Kang, Yun Gyeong1,
Wu, Yanru2, Lee, Eun Jin1, Kim, Min Ju1
1Department of Biomedical Engineering, Inje University,
Gimhae-si, Korea, 2Department of Health Science and
Technology, Inje University, Gimhae-si, Korea
Hematopoietic stem/progenitor cells (HSPCs) transplanta-
tion is used for the treatment of several hematologic or
immunological diseases. Since successful outcomes
required sufcient number of cells, many researchers have
focused on ex vivo expansion of HSPCs. HSPCs reside
complex niche including various factors such as secreted
factors, extracellular matrix, mechanical environment and so
on. It is well known that niches can modulate expansion,
homing, migration, differentiation and others of HSPCs. In
this study, we constructed an optimized 3-dimensional (3D)
culture system to mimic niche by treating vitronectin with 3D
hierarchical scaffolds for ex vivo expansion of HSPCs.
Lattice was made of three layers using rapid prototyping
technique and nanober was electrospun on each layer of
three layered lattice using electrospinning technique.
Intermittent hydrostatic pressure (IHP) was also applied to
represent the in-vivo mechanical environment (4h/day,
20kPa). The group to which the stimuli was applied was
divided into two pattern: P(1min/7min) and P(2min/13min).
Trypan blue staining was performed to evaluate expansion
of total cells and ow cytometry was conducted to conrm
phenotypes of HSPCs (CD34+, CD34+CD38-). And clonogen-
ic potential was also examined by colony forming cell(CFC)
assay and long term culture-initiating cell(LTC-IC) assay.
Total cell expansion was higher in vitronectin treated 3D
scaffold compare to 2D culture system and untreated 3D
scaffold. Moreover, the number of total cells was further
increased under IHP. Maintenance of phenotype (CD34+,
CD34+CD38-) was better under HP, especially
P(2min/13min), and that it also had a greater colony
formation ability by CFC assay and LTC-IC assay. As a result,
the combination of 3D hierarchical scaffold and mechanical
environment was superior to control the fate of HSPCs, and
could be an useful model system mimicking niche.
Funding Source
This work was supported by Priority Research Centers
Program (2010-0020224, the Ministry of Education, ROK)
and by the National Research Foundation of Korea (NRF)
Grant (NRF-2015M3A9B6073643).
Keywords: Hematopoietic stem/progenitor cells; ex vivo
expansion; 3D hierarchical scaffolds
102
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Poster Abstracts
178
A PULSATILE FLOW BIOREACTOR ENHANCES
MESENCHYMAL STEM CELLS DIFFERENTIATION
INTO ESOPHAGEAL CELL LINEAGES IN THE
ELECTROSPUN TUBULAR SCAFFOLD
Shin, Jung-woog1, Wu, Yanru2, Kang, Yun Gyeong1,
Lee, Eun Jin1, Kim, Ji Eun1, Kim, Min Ju1
1Department of Biomedical Engineering, Inje University,
Gimhae-si, Korea, 2Department of Health Science and
Technology, Inje University, Gimhae-si, Korea
Tissue-engineered esophagus serves a potential therapeutic
approach for esophageal replacement. We aimed to simulta-
neous differentiate human mesenchymal stem cells (hM-
SCs) into esophageal epithelial cells (EpCs) and smooth
muscle cells (SMCs) on the electrospun tubular scaffold by
adopting mechanical stimuli in vitro for esophageal tissue
engineering. Briey, a two-layered tubular scaffold was
fabricated by electro-spinning and hMSCs were seeded onto
the inner and outer layer of the scaffold. Then the constructs
were placed in a self-designed hollow organ pulsatile ow
bioreactor and stimulated with EpCs and SMCs induction
medium in the inner and outer chamber for two weeks.
Meanwhile, either 0.1dyne/cm2 of shear stress or pulsatile
shear together with 50mmHg radius pressure and 5%
circumferential stretch was engaged during the second
week. Finally, esophageal epithelial and muscular differentia-
tion level was tested by RT-PCR and immuno-uorescence
staining. As a result, electro-spun bers were found random-
ly and circumferentially oriented on the inner and outer layer
of the tubular scaffold. After 14 days of culture, scaffolds
supported hMSCs attachment and esophageal cell lineage
differentiation was found under specic chemical stimuli, i.e.
EpCs differentiation markers were signicantly increased in
the inner layer while SMC markers were signicantly
increased in the outer layer. Furthermore, physiologic
pulsatile ow which also resulted in shear stress further
promote the differentiation level of epithelial and smooth
muscle cells in vitro and the compliance of the construction
was also slightly increased in the pulsatile ow group. In
conclusion, we demonstrated a potential platform for
tissue-engineered esophagus based on differentiation of
hMSCs under chemical and mechanical stimuli in vitro.
Selection of optimal differentiation conditions and further in
vivo tests are required.
Funding Source
This work was supported by Priority Research Centers
Program (2010-0020224, the Ministry of Education, ROK)
and by the National Research Foundation of Korea (NRF)
Grant (NRF-2015M3A9B6073643).
Keywords: esophageal tissue engineering; mesenchymal
stem cells; pulsatile ow
179
EFFICIENT GENERATION OF ARTIFICIAL
VASCULARIZED LIVER USING MULTIPLE HUMAN
CELL TYPES
Kim, Da-Hyun, Kim, Jae-Jun, Choi, Soon Won, Kang,
Kyung-Sun
College of Veterinary Medicine, Seoul National University,
Seoul, Korea
Liver transplantation is a well-established therapeutic
strategy for end-stage liver disease. As the patients with
severe liver failure have increased gradually, the demand for
organs has also grown worldwide. However, due to donor
organ shortage, liver tissue engineering has currently been
considered as an alternative. Decellularized extracellular
matrix scaffold, obtained by removal of cells from a donor
tissue, has been known as a promising substrate for tissue
remodeling. Due to preservation of its own structural and
biological signals, the cells seeded into decellularized
scaffold can migrate to the appropriate microenvironment.
Especially, the liver is a very vascular organ, which receives
about 25% of total cardiac output. Therefore, efcient
re-endothelialization of the scaffolds is the key to successful
reconstruction of the articial liver. Based on this, we aimed
to efciently generate vascularized liver with multiple human
cell types using decellularized rat liver scaffold. First, the
scaffolds were coated with reagent A and subsequently
repopulated with human umbilical vein endothelial cells
(HUVECs). Injected endothelial cells were specically
captured by reagent A in an efcient manner, leading to the
formation of conuent endothelium along the vascular
channels. To evaluate the efciency of endothelialization of
the scaffold, we perfused the scaffolds with human blood
and measured platelet counts. The results showed signi-
cantly reduced platelet aggregation in the perfusate of
reagent A-coated scaffolds compared with non-coated
scaffolds, indicating improvement of endothelialization and
less thrombogenicity of the scaffolds. Moreover, we devel-
oped a new protocol for recellularization of liver parenchy-
mal cells and non-parenchymal cells simultaneously. With
this protocol, reagent A-coated scaffolds were successfully
repopulated with HepG2, LX2 (hepatic stellate cell), HUVEC
and umbilical cord blood-derived mesenchymal stem cell
(UCB-MSC) which were properly localized to their original
sites. To this end, our study provides a new strategy for fabri-
cation of tissue-engineered liver, suggesting the possibility
to reconstruct full-sized human articial liver retaining
elaborate histological architectures including endothelializa-
tion.
Keywords: Articial Liver; Endothelialization; Decellularized
extracellular matrix
103
AMSTERDAM NETHERLANDS
Poster Abstracts
180
FIBROUS NEURAL ORGANOID USING CELL
ENCAPSULATION TECHNIQUE
Nagata, Shogo, Takeuchi, Shoji
Institute of Industrial Science, The University of Tokyo,
Meguro, Japan
Organoid formation from stem cell aggregates holds great
promise for contributing development biology, regenerative
medicine, drug development, and pathological modeling.
Until now, various type of tissue including brain, intestine,
and lung has been reconstructed by using organoid forma-
tion from human induced pluripotent stem cells (hiPSCs) or
human embryonic stem cells (hESCs). The organoids
reproduce a part of the developmental cell dynamics and
three-dimensional (3D) structure of the developing tissues in
vitro, and contribute for disease modeling and drug screen-
ing. However, in current methods for the organoid formation,
most organoids only represent single or partial component
of tissues. This is because it is often difcult to control
architectures of the organoids, organization, cell-cell/
cell-extracellular matrix (ECM) interactions within the
methods. Recently, to overcome the limitation, engineering
stem cell organoids has been focused. Here we generate
brous neural organoids from hiPSCs using cell encapsula-
tion technique. By using the microuidic device system (cell
ber technology), singly dissociated hiPSCs were encapsu-
lated into core-shell microbers composed of Matrigel
(core) and alginate hydrogel (shell). In the ECM-rich microen-
vironment of the core region of the bers, the hiPSCs formed
brous aggregates for a few days (hiPSC bers). The hiPSC
bers were cultured N2B27 containing CHIR99021 and TGFβ
inhibitor for several days to induce neuroepithelium, and
then the bers were differentiated in neuronal medium
(neural organoid bers). By controlling the ow condition for
the cell encapsulation using the cell ber technology, the
neural organoid bers ranging from μm to cm scale lengths
was easily constructed. In the bers, hiPSCs differentiated
into neuroepithelial cells and formed 3D neural rosette
structure, and a cortical layer-like structure was observed.
Furthermore, we found that GSK3 signaling regulated neural
cell expansion in the organoids bers as well as in develop-
ing brains. These results indicate that the organoid engineer-
ing technique can be a powerful tool for steering the 3D
architecture of the organoids and cell dynamics and organi-
zation in the organoids.
Funding Source
This work was partly supported by JSPS KAKENHI (Grant
Number 16H06329), and the Japan Agency for Medical
Research and Development (AMED), Research Center
Network for Realization of Regenerative Medicine, Japan.
Keywords: Organoid engineering; Neural organoid; Cell
encapsulation
181
REGULATION OF MOUSE IPS CELL PROLIFERATION
AND CARDIOMYOGENIC DIFFERENTIATION ON
POLYPEPTIDE MULTILAYER FILMS
Chiu, Yun-Hsuan1, Chiu, Yun-Hsuan1, Lo, Chun-Min3,
Wang, Hwai-Shi1
1Anatomy and Cell Biology, Yang Ming University, Taipei,
Taiwan, 3Biomedical Engineering, Yang-Ming University,
Taipei, Taiwan
The mechanical properties of extracellular matrix play
important roles in controlling cell adhesion, cell proliferation
and cell differentiation. In this study, the thickness, stiffness
and adhesiveness of polyelectrolyte multilayer lms were
modulated, then used to study the proliferation and cardio-
myogenic differentiation of mouse induced pluripotent stem
cells (iPSCs). Polypeptide multilayer lms were built up by
alternate deposition of cationic poly-L-lysine (PLL) and
anionic poly-L-glutamic acid (PLGA), cross-linked with EDC/
sulfo-NHS, and coated with gelatin. Quartz crystal microbal-
ance and atomic force microscopy were used to certicate
polypeptides depositing process and the granular structure
of the lm surface. The stiffness of the lms were modulat-
ed by cross-linking with EDC/sulfo-NHS. The lms were then
coated with gelatin to enhance cell adhesion. The prolifera-
tion assay showed that iPSCs were growing well in six layers
multilayered lms coated with gelatin. Cardiomyogenic
differentiation was monitored with electric cell-substrate
impedance sensing (ECIS). Mouse iPSCs were cultured on
gelatin-terminated PLL/PLGA multilayer-coated electrodes
and cardiomyogenic differentiation was monitored with
ECIS. The occurrences of beating cardiomyocytes were
observed when iPSCs were cultured on gelatin-terminated
PLL/PLGA multilayer-coated electrodes. In addition, the
periodic uctuations of measured impedance caused by
cardiomyocyte beating were observed accordingly. These
results demonstrated that the mechanical properties of the
gelatin-coated PLL/PLGA multilayer lms can enhance the
efciency of iPSCs differentiation into cardiomyocytes.
Funding Source
This work is nancially supported by the Ministry of Science
and Technology of the Republic of China, Taiwan, under
project number MOST-104-2320-B-010-009-MY3.
Keywords: induced pluripotent stem cells; polypeptide
multilayer lm; cardiomyogenic differentiation
104
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Poster Abstracts
182
PROCESS DEVELOPMENT FOR DIFFERENTIATION
OF HUMAN PLURIPOTENT STEM CELLS TOWARDS
MESENCHYMAL STEM CELLS IN 3D SUSPENSION
CULTURE
Domev-Cohen, Hagit1, Axelman, Elena1, Verbuk, Mila2,
Roytblat, Mark1, Dvir, Shlomi1, Shariki, Kohava1, Amit, Michal1
1Accellta, Haifa, Israel, 2Accellta, Nesher, Israel
Mesenchymal stem cells (MSCs) are multipotent cells which
have the ability to differentiate in-vitro into mesodermal
derivatives; adipocytes, osteocytes, and chondrocytes, and
with a lesser extent into ectodermal or endodermal deriva-
tives; neurons or hepatocytes respectively. In addition, these
cells have special immunomodulatory properties and
therefore are attractive candidates to the regenerative
medicine and cell-based therapy elds, but only if the cell
number barrier can be crossed. In addition, MSC derivatives
can also serve as a model for tailored-made drug screening.
MSCs can be derived from various adult and fetal tissues.
However, the availability of tissues for the isolation of adult
or fetal MSCs remains limited and often requires invasive
procedures. Moreover, their expansion potential in-vitro is
nite. Therefore, human pluripotent stem cells (hPSCs),
either - embryonic or induced, can potentially provide
alternative, unlimited and reproducible source of MSCs.
Two-dimensional (2D) adherent culture has been traditional-
ly used as the standard technique for in-vitro expansion of
MSCs. Nevertheless, the number of cells that can be
obtained in 2D adherent culture is limited and requires high
costs and laborious work. Accellta has developed carri-
er-free suspension platform for culturing PSCs as cell
aggregates (MaxellsTM) or single cells (SinglesTM), at high
densities of 10 million cells per ml and above. This platform
allows directed differentiation of PSCs towards ectodermal,
endodermal and mesodermal progenitors in suspension.
Thus, Accellta’s unique technology provides the solution for
mass production of cells needed for cells-based therapy in
cost-effective manner. Here we present, novel, specic and
efcient process for the directed differentiation of hPSCs
towards MSCs in non-adherent, carrier-free suspension
culture along with designated media. Following few days in
differentiation media, our protocols generated bona de
MSCs with high-rate differentiation towards adipocytes,
chondrocytes and osteoblasts in-vitro. Therefore, Accellta’s
carrier- free suspension culture may serve as a base for
directed differentiation of PSCs into mini organelles, that can
be utilized to the eld of cell-based therapy as well as for a
humanized model for drug screening.
Keywords: Mesenchymal stem cells, in-vitro; human pluripo-
tent stem cells, drug screening; mini organelles, carrier-free
suspension
183
BEHAVIORS OF AXONS FROM MOTOR NEURON
SPHEROID IN THE ECM ENVIRONMENT AND
MICROCHANNEL
Ahn, Jinchul1, Yang, Jihoon2
1Mechanical Engineering, Korea University, Seoul, Korea,
2College of Animal Bioscience & Technology, Korea
University, Seoul, Korea
Motor neurons in the central nervous system (CNS) plays
role in downstream signaling to the effectors through their
axons. The axons interact with their extracellular environ-
ment and navigate to reach to the nal target. There are
number of studies about axons or neurons to elucidate the
mechanisms about the development of neural circuits or
neural diseases. But in many cases, it is hard to distinguish
the parts of neural circuit unit visually. To understand their
interaction and build model of complex network, it is
necessary to separate the cells and the axons in controllable
manner. Here, we used a PDMS device with a microchannel
and reservoirs to observe the behaviors of axons of motor
neurons. The motor neurons were differentiated from small
molecule neural progenitor cell(smNPC) spheroids, and the
spheroids move to the device with Extracellular-matrix(ECM)
environment. Axons were classied from the motor neu-
ron-differentiated spheroids and protrude in Matrigel area.
During protrusion, the axons were affected by guidance of
PDMS structures and other axons. Axons which interact with
other axons build the fascicles. And lastly, the protrusion
was seemed to be affected by other spheroids with different
manner. The cells of differentiated NPC spheroid and the
axons were evaluated by immunouorescence images and
quantied.
Keywords: motor neuron; axon; guidance
105
AMSTERDAM NETHERLANDS
Poster Abstracts
184
HUMAN PLURIPOTENT STEM CELL DERIVED
NEURAL 3D CULTURES – BUILDING NEURAL TISSUE
BLOCK FOR BODY-ON-A-CHIP PLATFORM
Joki, Tiina A.1,2, Harju, Venla3, Ylä-Outinen, Laura3, Koivisto,
Janne4, Karvinen, Jennika4, Kellomäki, Minna4, Narkilahti,
Susanna3
1Faculty of Medicine and Life Scienses, University of Tampere,
Finland, 2BioMediTech, Institute of Biosciences and Medical
Technology, Tampere, Finland, 3MED, University of Tampere,
Finland, 4Biomedical Sciences and Engineering, Tampere
University of Technology, Finland
The brain tissue, like many other tissues, is highly organized
3D structure. When aiming to build an in vivo-mimicking BoC
platform, it is necessary to take into account tissue specic
architecture and extracellular matrix (ECM). Traditional 2D
cell culturing conditions fail to offer cells in vivo mimicking
growth cues, leading cell adaptation to unnatural conditions
and creating bias to gene and protein expression of the
cells. Some of these problems could be overcome by 3D
culturing, which can offer more in vivo-like cell to cell and
cell to ECM interactions. Hydrogels as biomaterials are soft
tissue ECM mimicking. For this reason, hydrogels are very
prominent materials to be used as an articial ECM in BoC
platforms. Here we compared the properties of several
hydrogels to nd the best material to be used in brain tissue
block. In this work, we used human pluripotent stem cell
derived neural cells. Hydrogels use were PuraMatrix, Gellan
gum, hyaluronic acid – poly(vinyl)alcohol (HA-PVA) and
collagen 1. The main ndings were: 1. When several bioma-
terials were compared, the mechanical properties of
scaffold material did not correlate with neural cell growth
inside the scaffold. 2. For culturing human neural cells inside
hydrogel scaffold, it was benecial to use multicomponent
hydrogels by using collagen 1 as one component for making
interpenetrating network scaffolds. During recent years,
many different hydrogels have been promoted as the state
of art scaffold materials for neural tissue engineering both in
vivo and in vitro. Based on the literature it is difcult to
evaluate what hydrogel would be most prominent to be used
in BoC. Our results suggest that both the choice of cells and
the choice of analysis methods have effect on which
material has the best performance. Also, the usability of the
biomaterial and reproducibility of the cultures are important
aspects to take into account. It seems that some biomateri-
als are good for some cell lines but not for others, but at the
same time it is possible to nd materials that are able to
support many cell lines, like the combination of HA-PVA-
Collagen 1.
Funding Source
TEKES (the Finnish Funding Agency for Innovation)
Human Spare Parts project, the Finnish Cultural Foundation
#00140325 and #00150312, the Academy of Finland grant
number #286990, and Center of Excellence grants #312409
and #312414.
Keywords: 3D culture; Hydrogel; Neural network
185
TRANSPLANTATION OF CARDIAC PATCH
LOADING TΒ4-OVEREXPRESSED MSCS PROMOTES
REPAIR OF INFARCTED MYOCARDIUM BY
ACTIVATING EPICARDIUM
Tan, Yu-zhen, Wang, Qiang-li, Wang, Hai-jie, Wang, Yong-li,
Zhang, Hai-feng
Department of Anatomy, Histology and Embryology, Shanghai
Medical School of Fudan University, Shanghai, China
Recent studies suggest that the epicardium plays an
important role in cardiomyogenesis during development,
while it becomes quiescent in adult heart. Thymosin beta 4
(Tβ4) has an effect on activating the epicardium. However,
effectiveness of Tβ4 administration is unsatisfactory.
Therefore, this study prepared cardiac patch and investigat-
ed efciency of activating the epicardium and repairing
infarcted myocardium by Tβ4 released sustainedly from the
cardiac patch. Mesenchymal stem cells (MSCs) isolated
from bone marrow of rats and mice were transfected with
Tβ4. Tβ4 release from the cells was determined with an
acquity ultra-performance liquid chromatography system.
For preparing of cardiac patch, the cells transfected with
Tβ4 and Flag were seeded on PLACL/collagen membrane
formulated by electrospinning. The survival and proliferation
of the cells on the nanobers were examined after treatment
with hypoxia. In MI models of rats and W 1CreERT2/+,R26mT-
mG
mice, the patches were implanted on the epicardium of
the infarcted region. In rat models, differentiation of the
epicardium-derived cells (EPDCs) and the engrafted MSCs
towards cardiovascular cells was examined by Wt1 immu-
nostaining and Flag labelling. In transgenic mouse models,
the activated EPDCs expressed GFP, so GFP could be used
to trace fate of the EPDCs. At four week and six month after
implantation of the patches, cardiac function was improved
signicantly, scar area in the infarcted region was reduced
obviously. EPDCs increased in subepicardium and myocardi-
um, and some Wt1+ cells and GFP+ cells expressed CD31,
α-SMA or cTnT. Angiogenesis and lymphangiogenesis in the
para-infarcted region were enhanced. At six month after
implantation, lymphangiogenesis were obvious. Flag
labelling showed that some engrafted MSCs migrated into
subepicardium and myocardium. These results suggest that
Tβ4 released from the transfected MSCs in PLACL/collagen
nanobrous patches may effectively attenuate left ventricu-
lar remodelling and improve cardiac function by activating
the epicardial cells. Our nding provides a novel strategy for
myocardial regeneration by enhancing the endogenous
regenerative mechanisms.
Funding Source
This work was supported by National Natural Science
Foundation of China (81270200, 81470385, 81770288).
Keywords: Tβ4; Epicardium; Cardiac patch
106
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Poster Abstracts
186
THE ROLE OF NOTCH IN THE ACTIVATION OF A
LATENT NEUROGENIC PROGRAM IN REACTIVE
ASTROCYTES
Porcionatto, Marimelia1, Riberio, Tais1,2, Garcia, Lina1,2
1Biochemistry, Federal University of Sao Paulo, Brazil,
2Neurobiology Lab, Federal University of Sao Paulo, Brazil
Astrocytes respond to injuries to the central nervous system
(CNS) by reactivating and acquiring an undifferentiated
state, which is characteristic of neural stem cells. Under in
vitro conditions, these cells regain multipotency and long
term self-renew, however, the molecular factors involved in
astrocyte dedifferentiation are poorly understood. In this
context, we investigated the role of Notch signaling as well
as Galectin-3 (Gal3) in triggering a latent neurogenic
program in reactive astrocytes in vitro. Astrocytes extracted
from neonatal mouse cortices were cultivated in vitro and
reactivated by mechanical lesion. Three days post lesion,
cells were xed, immunolabeled for NICD (Notch intracellu-
lar domain), Jagged1 (Notch1 ligand), Gal3 and GFAP, and
analyzed by confocal microscopy and ow cytometry. Our
results show that reactive astrocytes overexpress Gal3,
which is a lectin related to the proliferative capacity of
reactive astrocytes, and their potential to express neural
stem cells markers in vitro. Astrocytes submitted to me-
chanical lesion also showed increased NICD in the nucleus,
indicating that Notch signaling pathway was activated in
reactive astrocytes. Co-localization analysis further revealed
that a subpopulation of reactive astrocytes located in the
border of the injury co-localized with Notch/Jagged (recep-
tor/ligand) signicantly more than astrocytes located far
from the border, suggesting that Notch signaling pathway is
mainly activated at the border of a lesion. We conclude that
Notch signaling pathway has a main role in astrocyte
reactivity, dedifferentiation response and activation of a
latent neurogenic program. The study was approved by the
Ethics Committee on Animal Use of UNIFESP (CEUA number
7740290318).
Funding Source
FAPESP (2018/05846-9 Fellowship to TNR; 2016/19084-8
Fellowship to LMDG; 2017/18765-4 Research Grant to MP);
CNPq (Research Grant 465656/2014-5 to MP); CAPES
(Finance Code 001).
Keywords: reactive astrocyte; neurogenic program; Notch
187
NOVEL INSIGHTS IN HUMAN AXONAL DEVELOPMENT
FROM IPSC-DERIVED NEURONS
Lindhout, Feline W.1, Portegies, Sybren1, Kooistra, Robbelien1,
Stucchi, Riccardo1, MacGillavry, Harold D.2, Hoogenraad,
Casper C.1
1Cell Biology, Utrecht University, Utrecht, Netherlands, 2Bio-
physics, Utrecht University, Utrecht, Netherlands
During neuronal development, a symmetry break occurs
when one of the unipolar processes shows extensive growth
and transforms into an axon. To date, most mechanistic
insights of axon initiation come from non-human studies.
However, it remains unknown to what extent these mecha-
nisms can be extrapolated to human neurons, and human
axonopathies (e.g. paraplegia, degenerative motor neuron
diseases, etc.) remain yet unresolved. Here, we have
mapped the molecular events during axonal development in
human iPSC-derived neuron cultures using immunouores-
cence and mass spectrometry analysis. We observed
successful initiation of axon development in hiPSC-derived
neurons, indicating that this event is driven by cell-autono-
mous processes. Consistent with human brain development
in vivo, axon formation in hiPSC-derived neuron cultures
occurred relatively slow (~7–14 days). Unexpectedly, we
found that during this process the axon initial segment (AIS)
proteins Trim46 and AnkyrinG rst appear distally in axons,
after which they accumulate at the proximal axon. Hence,
we propose a working model where human axon develop-
ment is accomplished by a two-stage process. First, axon
formation starts with AIS proteins accumulating distally,
possibly to promote accelerated outgrowth which may
involve microtubule stabilization. Secondly, AIS proteins
reorganize at the proximal axon to form the specialized AIS
structure. Together, these data provide novel insights in the
cell-autonomous processes driving human axonal develop-
ment, and highlight the relevance of studying these mecha-
nisms in a human model system in addition to the existing
non-human model systems.
Funding Source
This work was supported by the NWO (NWO-ALW-VICI,
CCH), the ZonMW (ZonMW-TOP, CCH), the ERC (ERC-
consolidator, CCH), and IvD Utrecht (3Rs Stimulus fund,
FWL and CCH).
Keywords: axon; development; human neurons
107
AMSTERDAM NETHERLANDS
Poster Abstracts
188
LONGITUDINAL DISSECTION IN BRAIN ORGANOIDS
AT SINGLE CELL RESOLUTION UNCOVERS THE
DEVELOPMENTAL ROLE OF GSK3 IN HUMAN
CORTICOGENESIS
Lopez Tobon, Alejandro1, Villa, Carlo Emanuele2, Cheroni,
Cristina2, Trattaro, Sebastiano2, Caporale, Nicolo2, Iennaco,
Rafaele3, Lachgar, Maria2, Tulio, Marco2, Marco de la Cruz,
Berta2, Lo Riso, Pietro2, Tenderini, Erika2, Troglio, Flavia2,
De Simone, Marco4, Liste Noya, Isabel5, Piccolo, Stefano6,
Macino, Giuseppe7, Testa, Giuseppe2
1Stem Cell Epigenetics, Istituto Europeo di Oncologia, Milan,
Italy, 2Experimental Oncology, Istituto Europeo di Oncologia,
Milano, Italy, 3Stem Cell Biology, Istituto Nazionale Genetica
Molecolare, Milano, Italy, 4Immunology, Istituto Nazionale
Genetica Molecolare, Milano, Italy, 5Unidad de Regeneración
Neural, Instituto de Salud Carlos III, Madrid, Spain, 6Depart-
ment of Medical Biotechnology and Translational Medicine,
University of Milan, Italy, 7Department of Cellular Biotechnolo-
gies and Hematology, La Sapienza University of Rome, Italy
The regulation of proliferation and polarity of neural progeni-
tors is crucial for the development of the brain cortex, with
modes and timings of cell division intimately related to the
stereotypical acquisition of layer-specic neuronal identities.
Animal studies have implicated glycogen synthase kinase 3
(GSK3) as a pivotal regulator of both proliferation and
polarity, yet the functional relevance of its signaling for the
unique features of human corticogenesis remain to be
elucidated. Here we harness human cortical brain organoids
to probe, at single cell resolution, the longitudinal impact of
GSK3 inhibition through multiple developmental stages. Our
results indicate that chronic GSK3 inhibition increases the
proliferation of neural progenitors and causes massive
derangement of cortical tissue architecture. Surprisingly,
single cell transcriptome proling revealed only a discrete
impact on early neurogenesis and uncovered GSK3-specic
neurogenic trajectories. Through this rst single cell-level
dissection of the GSK3 regulatory network in human
corticogenesis, our work uncovers a remarkably specic
conduit between the architecture of progenitor niches and
lineage specication.
Funding Source
Associazione Italiana per la Ricerca sul Cancro (AIRC).
EPIGEN Flagship Project of the Italian National Research
Council (CNR). European Research Council. Fondazione
Cariplo. Fondazione Italiana per la Ricerca sul Cancro (FIRC).
Keywords: Brain organoids; Corticogenesis; GSK3
189
HUMAN FETAL VENTRAL MIDBRAIN IN STANDARD
CULTURE VERSUS ORGANOIDS
Birtele, Marcella, Fiorenzano, Alessandro, Nelander
Wahlestedt, Jenny
Experimental Medical Science, Lund University, Lund, Sweden
Parkinson disease (PD) is characterized by a loss of dopami-
nergic neurons in the substantia nigra pars compacta
(SNpc). The selective loss of this cell population makes PD a
good candidate for cell-based therapies. Clinical trials using
cells derived from human fetal ventral midbrain (hVM) have
shown that dopamine release was restored to normal levels
and in some PD patients produced substantial long-term
clinical improvement. Although new sources of cells such as
human embryonic stem cells (hESCs) and pluripotent stem
cells (IPCs) are now been the focus of studies and new
clinical trials, the hVM tissue represent the gold standard for
cell based therapies and serve as important comparator for
new cell sources. Therefore there is a need of an increased
understanding and characterization of this tissue with new
technologies such as organoid culture conditions and single
cell sequencing. This study is designed to give a detailed
characterization of human fetal VM tissue in standard
culture conditions (2D) versus organoids (3D). We used
immunocytochemistry to study the presence of ventral
midbrain markers in 2D versus 3D condition together with
single cell RNA sequencing to transcriptionally compare
fetal VM tissue in standard or organoid cultures. Our
analysis conrmed that the 3D condition preserve the
neuronal composition of the tissue more than standard
culture where instead, over a long period of time, the
majority of the cells are non-neuronal. This suggest that 3D
culture represent a better condition to maintain and study
dopaminergic neurons in culture.
Funding Source
Marcella Birtele has been funded by European Union Horizon
2020 Program (H2020-MSCA-ITN-2015) under the Marie
Skłodowska-Curie Innovative Training Networks and Grant
Agreement No. 676408.
Keywords: Human Fetal Cells; Ventral Midbrain; Single Cell
Sequencing
108
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Author Index
PAG E
PRESENTER NAME NUMBER
Ahn, Jinchul ............................ 104
Ahluwalia, Arti .......................... 29
Almeida, Eduardo ..................... 74
Ameneiro, Cristina .................... 44
Andrews, Madeline G. ............. 88
Apáti, Ágota .............................. 54
Arlotta, Paola ............................ 22
Baetz, Nicholas ........................ 76
Bernotiene, Eiva ........................ 82
Bhaduri, Aparna ........................ 20
Bjorlykke, Yngvild .....................98
Blau, Helen ................................ 31
Brandao, Karina ........................ 59
Brennenstuhl, Heiko ................. 58
Birtele, Marcella .....................107
Buzanska, Leonora ...................97
Cagavi, Esra .............................. 57
Cao, Xu ...................................... 79
Cardo, Lucia F. ......................... 59
Cevher, İnci ...............................53
Chiu, Yun-Hsuan ..................... 103
Cho, Ann-Na .............................. 77
Claudio, Ribeiro ........................ 34
Clevers, Hans C. ....................... 20
Connor, Bronwen J. ................. 41
Davis, Richard ........................... 58
De Andrade E Silva,
Ana Catarina............................. 75
De Filippi, Giovanna ..................35
De Jong, Menno R. .................. 49
Depla, Josse ............................. 60
Dinnyes, Andras ........................89
Dmitriev, Ruslan I. .................... 39
Do, Hyosang ............................. 57
Domev-Cohen, Hagit .............. 104
Dooves, Stephanie ...................40
Drakhlis, Lika ............................ 92
Durovic, Tamara ....................... 72
Dziedzicka, Dominika ............... 86
Emani,
Maheswarareddy
........... 45
Eng, Shermaine ........................ 32
Erceg, Slaven ............................ 51
Erharter, Anita ........................... 77
Escudero, Adriana .................... 93
Fjodorova, Marija......................40
Fiorenzano, Alessandro ........... 84
Fosby, Anne Helene ..................80
Gagliardi, Giuliana .................... 67
Garcia-Outeiral, Vera ................93
Giacomelli, Elisa ....................... 29
Giger, Sonja ............................... 74
Girgin, Mehmet U. .................... 90
Gomes, Ana Rita........................44
Grapin-Botton, Anne ................. 20
Grigsby, Christopher L. ............ 97
Habib, Shukry J. ...................... 30
Hakes, Anna E. ........................ 62
Hansen, Stine Lind ................... 95
Hao, Hsiao-Nan ........................ 37
Heinz, Maria C. ........................ 52
Hekman, Katherine ...........75, 100
Hillen, Anne E.J. ...................... 56
Hoekstra, Stephanie D. ............61
Hollingsworth, Ethan W. ..........50
Hor, Catherine H. ..................... 63
Huch, Meritxell ......................... 23
Hudson, James ........................ 30
Hurley, Killian J. ....................... 22
Ikeda, Masa-Aki ........................ 91
Imitola, Jaime ........................... 50
Islam, Mazharul ........................ 40
Jez, Mateusz ............................ 73
Joana, Filipa ............................. 34
Joki, Tiina A. .......................... 105
Jung, Yong Hun ........................ 94
Kagawa, Harunobu ................... 33
Kaindl, Johanna ........................ 59
Kannan, Nagarajan ...................68
Kavyanifar, Atria ....................... 94
Kim, Bumsoo ............................ 56
Kim, Da-Hyun .......................... 102
Kim, Eunjee ............................... 78
Kim, Min-Soo ............................ 79
Kim, Min-Seon .......................... 92
Kim, Suran ................................77
Kime, Cody ............................... 85
Klatt, Annemarie K. .................. 90
Knoblich, Juergen A. ............... 31
Koehler, Karl R. ........................ 23
Koning, Marije ..........................23
Kopper, Oded ............................ 26
Koskuvi, Marja .......................... 57
Kouroupi, Georgia .....................47
Kretzschmar, Kai ......................51
Kriegstein, Arnold R. ................ 28
Krzisch, Marine A. ................... 65
Kuninger, David ......................... 35
Kuo, Calvin ................................ 29
Kurek, Dorota ............................ 98
Laha, Kurt.................................. 84
Lee, Jiyoon ................................ 83
Lee, Vivian M. .......................... 38
Lieshout, Ruby .......................... 45
Lindhout, Feline W. ................106
Little, Melissa H. ...................... 26
Lohmussaar, Kadi ....................60
PAG E
PRESENTER NAME NUMBER
PAG E
PRESENTER NAME NUMBER
109
AMSTERDAM NETHERLANDS
Author Index
Lohrer, Benjamin ......................96
Lopez Tobon, Alejandro ......... 107
Low, Jian Hui ............................ 67
Lukovic, Dunja ..........................64
Lutolf, Matthias P. .................... 30
Madhavan, Mayur C. ............... 84
Madl, Christopher M. ...............55
Mantalas, Gary L. .................... 45
Martins, Soraia ......................... 42
Martins Garcia, Tânia ............... 87
Mathieu, Noelle ........................ 69
McDevitt, Todd .........................25
Meier, Anna ............................... 62
Mertens, Jerome ...................... 26
Micsik, Roxana ......................... 43
Monzel, Anna S. ....................... 48
Na, Jie ...................................... 48
Nagata, Shogo ....................... 103
Nagy, Andras, ............................ 25
Nakano, Michitaka ................... 93
Ng, Carrie .................................. 64
Nilsson Hall, Gabriella .............. 76
Noorwali, Abdulwahab A. ....... 64
Nouri, Parivash ......................... 91
Ogundipe, Vivian ...................... 69
Orzechowska, Emilia J. ........... 81
Pain, Bertrand ........................... 39
Palano, Giorgia ......................... 60
Panova, Alexandra V. ............... 48
Pardon, Gaspard .......................51
Park, Hyun Sook ....................... 83
Paterson, Yasmin Z. ................ 34
Peking, Patricia.........................68
Pellegrini, Laura ........................ 78
Pellegrini, Marco ......................99
Peng, Weng Chuan ................... 24
Pereira, Bruno ........................... 89
Peteri, Ulla-Kaisa ...................... 66
Piras, Roberta ........................... 81
Porcionatto, Marimelia ..........106
Preeti Prasannan ......................61
Preisler, Livia............................. 54
Previdi, Sara .............................. 70
Pringle, Sarah ...........................38
Prior, Nicole ..............................22
Raad, Farah ............................... 88
Rai, Shahin .............................. 28
Rao-Bhatia, Abilasha ................ 95
Renner, Magdalena ..................85
Repina, Nicole A. ..................... 21
Rialdi, Alexander ....................... 27
Rocchi, Cecilia .......................... 69
Roccio, Marta ........................... 24
Roos, Floris ............................... 70
Roska, Botond .............................. 24
Saraiva Lopes, Claudia A. ....... 55
Sanosaka, Tsukasa .................. 65
Sato, Toshiro ............................27
Schafer, Simon T. ..................... 42
Schneeberger, Kerstin ............ 100
Schroeder, Olaf ......................... 65
Schwach, Verena ......................82
Seo, Yoojin ................................ 78
Shankar, Anusha S. ...........71, 72
Sharma, Swati .......................... 98
Shi, Yan ..................................... 63
Shim, Joong Hyun .................... 33
Shin, Jung-woog .....................101
Simonsson, Stina ..................... 74
Slaats, Rolf ................................ 53
Smith, Kevin S. .........................85
Snippert, Hugo .......................... 27
Steinhart, Matthew R. ............. 92
Stevenson, Matthew ................52
Sullivan, Gareth J. ....................73
Suzuki, Ikuro ............................. 36
Taguchi, Atsuhiro .....................86
Tan, Yu-zhen ........................... 105
Tedesco,
Francesco Saverio ....................47
Terrasso, Ana P. ....................... 43
Thiruvalluvan, Arun ..................61
Tsakmaki, Anastasia ................ 46
Urbach, Achia ........................... 36
Uzbas, Fatma ............................33
van de Leemput, Joyce ............ 66
van den Brink, Lettine ...............55
Vankelecom, Hugo ................... 49
Verstegen, Monique M.A. ....... 71
Villela, Roberta F. ..................... 79
Voelkner, Manuela .................... 80
Wagstaff, Philip ........................ 83
Wang, Hai-Jie .........................101
Wang, Tongguang ....................87
Wang, Xuelu .............................. 34
Weeratunga,
Muhandiram P. ......................... 32
Wells, James M. ...................... 21
Wen, Jinming ............................ 35
Wen, Zhexing ............................ 41
Willemse, Jorke ........................ 99
Yen, Yu-Ting .............................. 96
Zafeiriou, Maria Patapia ..........37
Zhou, Jie ................................... 46
Zorzi, Giulio A. ......................... 37
PAG E
PRESENTER NAME NUMBER
PAG E
PRESENTER NAME NUMBER
PAG E
PRESENTER NAME NUMBER
Stem Cells & Organoids in Development & Disease
AMSTERDAM NETHERLANDS
Notes
110
Explore new dimensions in
3D cell modeling
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Find out more about culturing and analyzing organoids
and spheroids at thermofi sher.com/organoid
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3D Cell Culture and Analysis
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