Introduction of Stem Cells in Ophthalmology

Abstract

Purpose: In this article, we review main clinical trials reported in the past decade.
Methods: Stem cells are relatively undifferentiated, with unlimited proliferative ability; self-renewal capability and also they can differentiate into specialized cells. Somatic stem cells in adult organisms are responsible for regenerating and self-renewing tissue. Many different stem cell types reside in the eye.
Results: One of the most important stem cells is limbal stem cell that retains in an undifferentiated state and exists in an optimal microenvironment or “niche”. The importance of limbal stem cells in maintaining the corneal epithelium throughout life has long been recognized. Furthermore, stem cells in the retina have been suggested for treatment of the retinal degeneration, which generally results in constant visual disturbance or even blindness.
Conclusion: This review will briefly focus on the principal stem cells in the eye especially limbal stem cells.

Full text

Annex Publishers | www.annexpublishers.com
Volume 1 | Issue 1
Abstract
Purpose: In this article, we review main clinical trials reported in the past decade.
Introduction
Introduction of Stem Cells in Ophthalmology
Khoshzaban A, Jafari E, Soleimani M*, Tabatabaei SA, Nekoozadeh S and Ekbatani AAZ
Ocular Trauma & Emergency Department, Eye Research Center, Farabi Eye Hospital, Tehran University of Medical
Sciences
*Corresponding author: Soleimani M, Ophthalmologist, Anterior Segment Subspecialist, Assistant Professor
of Ophthalmology, Ocular Trauma & Emergency Department, Eye Research Center, Farabi Eye Hospital, Teh-
ran University of Medical Sciences, Tel: 0098 912 1096496, Email: Soleimani_md@yahoo.com
Citation: Khoshzaban A, Jafari E, Soleimani M, Tabatabaei SA, Nekoozadeh S, et al. (2018) Introduction of
Stem Cells in Ophthalmology. J Stem Cells Clin Pract 1(1): 104
Research Article Open Access
Volume 1 | Issue 1
Journal of Stem Cells and Clinical Practice
Keywords: Limbal stem cell; Limbal stem cell deciency; Transplantation; Retinal progenitor; Clinical trials
Stem cells are undierentiated cells with unlimited proliferative ability of self-renewal dierentiating into dierentiated cells.
Limbal stem cells are nested in an optimal microenvironment or “niche”. e importance of limbal stem cells in maintaining the
corneal epithelium throughout life has long been recognized. Limbal epithelium consists of a non-keratinized stratied squamous
epithelium and located between corneal and conjunctival epithelia [1-5]. It has been found to include a source of stem cells (SCs)
known as corneal epithelial SCs or limbal stem cells (LSCs). Limbal Stem Cell Deciency (LSCD) is a heterogeneous group of
diseases in which the limbal epithelial stem cells (LESCs) are depleted by excessive damage or disease in the limbus to replenish the
consistent corneal epithelial regeneration [6]. ere are several procedures to address LSCD; some of the most global procedures
are keratolimbal allogra (KLAL), conjunctival limbal autogra (CLAU) and simple limbal epithelial transplantation (SLET). In
this review, we want to focus on stem cell eld in the ophthalmology especially limbal stem cells.
Methods: Stem cells are relatively undierentiated, with unlimited proliferative ability; self-renewal capability and also they can
dierentiate into specialized cells. Somatic stem cells in adult organisms are responsible for regenerating and self-renewing tissue.
Many dierent stem cell types reside in the eye.
Results: One of the most important stem cells is limbal stem cell that retains in an undierentiated state and exists in an optimal
microenvironment or “niche”. e importance of limbal stem cells in maintaining the corneal epithelium throughout life has long been
recognized. Furthermore, stem cells in the retina have been suggested for treatment of the retinal degeneration, which generally results
in constant visual disturbance or even blindness.
Conclusion: is review will briey focus on the principal stem cells in the eye especially limbal stem cells.
We conducted a review of the literature using PubMed database to gather relevant English articles with the keywords “stem cell”
and “eye” or “ophthalmology”. Relevant articles during the past decade were selected.
Methods
Results
Limbal stem cells
Corneal epithelial stem cells: e ocular surface of the eye is covered with corneal epithelial surface, limbus, and conjunctiva [1].
Cornea has several roles such as: refraction, photo protection, transparency, and protection of internal ocular structures from the
external environment [2,3]. Mature corneal epithelium is ve or six layers of non-keratinizing stratied squamous epithelial cells
with about 0.05 mm thickness and derived from the head surface ectoderm overlying the lens aer invagination. e development
of the cornea is a terminal inductive event in eye formation [4]. Conjunctival epithelium is the signicant epithelium of the ocular

Annex Publishers | www.annexpublishers.com
Journal of Stem Cells and Clinical Practice
2
Volume 1 | Issue 1
surface expanding from the posterior margin of the eyelids, posterior surface to the peripheral edge of the tarsal plate, then folds
back to the sclera and then continues as the limbal epithelium [1-5]. Limbal epithelium consists of a non-keratinized stratied
squamous epithelium and located between corneal and conjunctival epithelia [6]. It has been found to include a source of SCs
known as corneal epithelial SCs or LSCs [7,8].
Concept of LSC: e repair and regeneration throughout the life of the adult cornea is responsible by LSC population [9]. In order to
replenish the SC population, only one of the daughter cells is divided asymmetrically and can re-enter the niche and become a stem
cell [6]. e characteristics of these stem cells are undierentiated, slow-cycling, self-renewal, to have high proliferative potential,
small size, and high nuclear to cytoplasm ratio [10-12]. e other cell is destined for dierentiation to transient amplifying cell
(TAC) which migrates to the corneal epithelium divides at an exponential rate and will nally dierentiate into a post-mitotic cell
(PMC) that can no longer multiply. e PMCs dierentiate and mature into terminally dierentiated cells (TDC) that represent
the nal phenotypic expression of the tissue type [6].
Limbal epithelial stem cell markers: e ability to identify SCs in dierent organs has been prevented by nonspecic and unreliable
markers. α-enolase is expressed in embryonic basal cells and localize to limbal basal epithelial cells without cytokeratin 3 (CK 3)
and it is suggested as a potential stem cell marker [21,22]. e relation of LESC [23] can identify the existence of associated markers
(e.g. ABCG2, vimentin, and cytokeratin 19) and the lack expression of dierentiation markers (e.g. CK 3/12, connexin 43, and
involucrin). Many of these markers are identied in early TACs. Consequently, small size of cell, high nucleus to cytoplasm ratio
and other morphological, phenotypic and functional characteristics of stem cells are used with stem cell markers [24]. In Table 1,
some important markers are described [25-34].
LSC niche: Stem cells in all renewable tissues are normally located in a unique and appropriate microenvironment called niche,
which supports self-renewal and multipotential activity and nurtures the stem cells. SCs organized in a ridge-like structure around
the circumference of the cornea, which have been suggested the ridge to be the rudimentary niche for LSC [13]. Some studies
indicate that the adult LSC niche exists within the basal interpalisade epithelial papillae of the Palisades of Vogt which contains
radially-oriented brovascular ridges. ese are concentrated along the superior and inferior limbus and found at the corneoscleral
limbus [14]. e function of normal SC depends more on their niche than their gene expression patterns. Interaction between
stem cells and their niches are critical for regulating SC function such as quiescence, apoptosis, division, or dierentiation of stem
cells [15,16]. SC behavior is regulated by wide variety of cells, including neighboring cells, signaling molecules (integrins, Wnt/β-
catenin,and Notch), local environmental factors such as extracellular matrix, and other intercellular contacts [17-19]. e precise
molecular mechanism by which the stromal niche regulates limbal stem cells is just beginning to be understood [20].
* DNp63α is one of 3 isoforms of p63 without an added transactivation domain (ere are
6 isoforms of p63) has been shown to be more specic for LESCs than the other isoforms.
[31] LSC: limbal stem cell. ATP: adenosine triphosphate
Table 1: Important stem cell markers
World Health Organization (WHO) estimates that 10 million of 45 million bilaterally blind people worldwide are the result of
corneal involvement [35]. LSCD is a heterogeneous group of diseases in which the LESCs are depleted by excessive damage or
disease in the limbus to replenish the consistent corneal epithelial regeneration. LSCD occurs in genetic or acquired disorders.
ere are hereditary or genetic causes, such as aniridia, keratitis associated with multiple endocrine deciencies, epidermal
dysplasia (ectrodactyly-ectodermal dysplasia-cleing syndrome, Keratitis-ichthyosis-deafness (KID) Syndrome) [36,37].
Aniridia(developmental dysgenesis of the anterior segment of the eye) caused by mutations in the pax6 gene. Pax6 is essential for
eye development (oculogenesis) and maintenance of LSC function.
Most commonly, LESC deciency is oen caused by acquired factors. ere are acquired causes, including ionizing and ultraviolet
radiation, extensive microbial infection, contact lens (CL) wear, industrial accidents, aer multiple resections of ocular surface
Limbal stem cell deciency: etiology and classication
Markers
ATP binding cassette transporter protein, in the limbal basal
epithelium, 0.3-0.5% of cells in the limbal epithelium exhibit the
side-population phenotype, to protect LSCs against oxidative
stress induced by toxins
ABCG2 [25-27]
a transcription factor, potential keratinocyte stem cell marker,
DNp63α* is thought to be a highly expressed in the limbus during
resting state, important for epithelial development
p63 [28-30]
CCAAT enhancer binding protein delta, a transcription factor,
induces G0/G1 cell cycle arrest in mammary gland epithelial cells,
C/EBPδ [30,32]
a repressor that to be expressed in the limbal epithelial side-
population, a Polycomb group repressor involved in the self-
renewal of various types of adult stem cell
Bmi1 [33]
a transmembrane receptor, maintaining cells in an
undierentiated state, localized to a small number of cells in the
limbal epithelial basal layer, co-expressed with ABCG2
Notch 1 [34]

3
Journal of Stem Cells and Clinical Practice
Volume 1 | Issue 1Annex Publishers | www.annexpublishers.com
tumors and Johnson syndrome (SJS) and Ocular cicatricial pemphigoid (OCP) that appears through dysfunction in mucous
membrane [38-40]. In other term, LSCD divided into primary or secondary. e primary, which does not present any identiable
external factors, is supported by an insucient microenvironment. Here, there are some dysfunction/poor regulation of stromal
microenvironment of limbal epithelial stem cells: congenital erythrokeratoderma, keratitis with multiple endocrine deciency
and poor nutritional or cytokine supply, neurotrophic keratopathy, peripheral inammation and sclerocornea [41,42]. Secondary
LSCD is acquired by external factors. Some causes of Secondary LSCD have also been described in acquired LSCD [40-42].
Clinical features of LSCD: Conjunctivalization of the cornea associated with goblet cells, supercial and deep vascularization,
chronic inammation, persistent epithelial defects and scarring are the essential clinical signs of LSCD [43-45]. e most impor-
tant clinical trait of LSCD is conjunctivalization of the cornea that results in depletion of LESCs or loss of function by trauma or
disease [46-48]. Tearing, reduced vision, chronic discomfort, chronic inammation, stromal scarring, blepharospasm, photopho-
bia, neovascularization, and persistent epithelial defects (PEDs) are clinical features of conjunctivalization [2,46]. It depends on the
size of limbal damage, which has a pattern of partial ingrowths (partial LSCD) or will aect the whole cornea (total LSCD) [49].
Also, a variance in the thickness and transparency of the corneal epithelium on slit lamp examination may be seen [50]. e cause
of the LSCD oen dictates whether the disease is aecting one eye or both (unilateral or bilateral) [6].
e aim of treatment for LSCD is to re-establish the physiologic and anatomic environment of the ocular surface by reconstruction
of the corneal and conjunctival epithelium [36]. Some techniques to replace limbal stem cells have been reported. Currently, the
main clinical procedures that are performed include CLAU, SLET, KLAL, and cultivated limbal stem cell transplantation (CLET)
[51]. e source of stem cell in unilateral disorders is the healthy contralateral eye [4,52]. Subsequently, transplantation of alloge-
neic limbal SCs that are dissected from cadaveric donors or living tissue-matched eyes to treat bilateral LSCD [53,54]. Limbal de-
ciency in the donor eye is a potential serious risk to this procedure. Both of auto or allogra limbal tissues in transplantation have
risks and benets. Lid pathology, dry eye, uncontrolled systemic disorders and other dierent factors aect the achievement of LSC
transplantation. Limbal transplantation is a denitive treatment for LSCD and may ameliorate the visual acuity of a patient with
ocular surface disease [55]. e second strategy to treat LSCD is ex vivo expansion of limbal stem cells from a single small biopsy to
transplant amniotic membrane (AMT), which forms the inner wall of the membranous sac surrounding the embryo during gesta-
tion. Anti-inammatory and anti-angiogenic properties of amnion cause to use it in ocular surface reconstruction [56]. A number
of clinical studies have shown transplantation of autologous, allogeneic limbal tissue or expanded cells in concomitance with AMT
to promote the rapid re-epithelization required to restore corneas with LSCD [57-59]. In unilateral cases with total LSCD, trans-
plantation of ex vivo cultured LSCs from the human eye (CLET) or a CLAU is utilized (conjunctival limbal auto-explant). Allo-
explant limbal transplant performed in patients with bilateral total LSCD and it is extracted from a cadaveric or a living relative
donor and then expanded in ex vivo. Recently, SLET has gained popularity in unilateral LSCD; in this technique, several pieces of
one clock hour of contralateral healthy limbus are spread on a layer of AMT on the involved ocular surface [57-59].
Limbal stem cells transplantation
Clinical trials of limbal stem cells transplantation: Variation of culture techniques, case selection between studies, performance
of both autologous, allogeneic transplants in studies, not including the corneal surface, visual improvement in some studies and
dierent follow-up periods limit the interpretation of clinical trial results from cultured LSC therapy [60-65]. Over the years, many
dierent methods have been developed. e rst stem cell autogra using conjunctival-limbal-corneal epithelium harvested from
the healthy fellow eyes of patients with unilateral chemical burns was reported by Barraquer in 1965 [60]. In 1997, Pellegrini et
al. [61] reported the rst experience of the clinical use of ex vivo cultured limbal epithelial stem cells (LESCs) for treating corneal
LESC deciency. In 2003,Sangwan and colleagues published a study demonstrated reconstruction of the ocular surface in a case
of severe bilateral partial LSCD with using autologous cultured conjunctival and limbal epithelium extracted from the healthy eye
[62]. In addition, in 2006, they reported the clinical outcome of autologous cultivated limbal epithelial transplantation between
March 2001 and May 2003.ere were 88 eyes of 86 patients with limbal stem cell deciency (LSCD). Sixty-four percent of patients
were due to alkali burns and 69% eyes had total LSCD. Finally results shown 73.1% (57 eyes) were successful outcome with a stable
ocular surface without conjunctivalization, 26.9% (21 eyes) had a considered failures and 10 patients were lost to follow-up [63].
e purpose of the study performed by Sangwan et al in 2011 was the ecacy of xeno-free autologous cell-based treatment with
unilateral total limbal stem cell deciency due to ocular surface burns treated between 2001 and 2010. A small limbal biopsy was
taken from the healthy eye and the limbal epithelial cells were expanded ex vivo on human amniotic membrane using a xeno-free
explant culture system. e resulting cultured epithelial monolayer and amniotic membrane substrate were transplanted on to the
patient’s aected eye. A completely epithelized, avascular and clinically stable corneal surface was seen in142 of 200 (71%) eyes in
this retrospective study. An improvement in visual acuity, without further surgical intervention, was seen in 60.5% of eyes. All do-
nor eyes remained healthy [64]. In 2005, Daya performed a study on 10 eyes of 10 patients with profound LSCD due to ectodermal
dysplasia (3 eyes), Stevens-Johnson syndrome (3 eyes), chemical injury (2 eyes), thermal injury (1 eye), and rosacea blepharocon-
junctivitis (1 eye) to investigate the outcome of ex vivo expanded stem cell allogra for LSCD. Seventy percent of eyes (7 of 10) had
improved parameters, including vascularization, conjunctivalization, inammation, epithelial defect, photophobia, and pain and
40% of eyes (4 cases of 7) had improved visual acuity [66]. A study by Nakamuraet al (2006) investigated 9 eyes from 9 patients
with total limbal stem cell deciency (2 eyes with Stevens-Johnson syndrome, 1 with chemical injury, 1 with ocular cicatricial pem-

Journal of Stem Cells and Clinical Practice
4
Annex Publishers | www.annexpublishers.com
Volume 1 | Issue 1
phigoid, 1 with Salzmann corneal dystrophy, 1 with aniridia, 1 with gra-versus-host disease, and 2 with idiopathic ocular surface
disease). e authors compared autologous serum (AS)-derived corneal epithelial equivalents with those derived from fetal bovine
serum (FBS)-supplemented medium, so cultivated corneal epithelial transplantation can be used for the treatment of severe ocular
surface disease. Allologous (7 cases) and autogenic (2 cases) AS-derived cultivated corneal epithelial equivalents were transplanted
onto the ocular surface. During the follow-up period, the corneal surface of all patients remained stable and transparent, without
signicant complications and visual acuity improvement was seen in all eyes [67]. In 2006, Javadi et al. published a more detailed
report on the early results of transplantation of autologous limbal stem cells cultivated on amniotic membrane (AM) in four eyes
of 4 patients with total unilateral LSCD. Aer 5-13-month follow-up, visual acuity, corneal opacication, and vascularization im-
proved in all cases [68]. In 2010, they performed keratolimbal allogra (KLAL) for treatment of 21 eyes of 20 patients with total
LSCD and adequate tear production were included. Mean visual acuity improved. Gra survival rate was 61.9% at 1 year and 31%
at 20 months [69]. Alex J. Shortt used a novel culture system without 3T3 feeder cells to determine the outcome of ex vivo cultured
LESC transplantation. Allogeneic (7 eyes) and autologous (3 eyes) corneal LESCs were cultured on human amniotic membrane.
Tissue was transplanted to the recipient eye aer supercial keratectomy. e success rate was 60% with a successful outcome ex-
perienced [70]. In 2014, Vazirani with the cooperation of Sangwan performed a study on seventy eyes of 70 patients with unilateral,
partial LSCD and reported the outcomes of autologous cultivated limbal epithelial transplantation using the healthy part of the af-
fected eye or the fellow eye as a source of limbal stem cells in patients. In 36 eyes, the limbal biopsy was harvested from the healthy
fellow eye (contralateral group) and in the remaining eyes from the healthy part of the limbus of the same eye (ipsilateral group).
Clinical success was achieved in 70.59% of eyes in the ipsilateral group and 75% of eyes in the contralateral group. Outcomes are
similar irrespective of whether the limbal biopsy is taken from the healthy part of the ipsilateral eye or the contralateral eye [65].
Stem cell therapy for retinal degenerative diseases
Introduction: Every year many people suer from visual disturbance or even blindness caused by retinal detachment. e
retina is a part of the central nervous system (CNS) consisting of neuronal cells (photoreceptors, horizontal cells, amacrine cells,
bipolar cells and retinal ganglion cells) and glial cells (Müller glia is a specialized type of glial cell only present in the retina) [71].
Epidemiologists have found that damage to the retina can occur the entire age spectrum. For instance, the pediatric and young
adult populations are aected by retinitis pigmentosa (RP) and middle-aged adults are aected by diabetic retinopathy (DR), and
the elderly are aected by age related macular degeneration (AMD) [72,73]. Retinal neurodegenerative disorders divided into
diseases aecting the inner retina, example glaucoma and can aect both bipolar cells and retinal ganglion cells (RGCs) [74]. ose
aecting the outer retina oen leads to the death of the photoreceptors. Retinal progenitor cells were identied as possible cell
candidates for an accepted potential treatment strategy for retinal injury. ese cells represent many of the properties associated
with stem cells: 1) proliferation and expression of Nestin, a neuroectodermal stem cell marker, 2) multipotential property, and 3)
self-renewal capacity [75]. Photoreceptors, intermediate neurons and Muller glia are dierentiated in vitro to form sphere colonies
of cell. Progenitor cells nd themselves in an inhibitory environment and this is the result of the failure of retinal progenitor cells
to renew retinal cells in the postnatal period [76]. Muller glia cells play a key role in the generation of multipotent precursor cells
from embryonic retinal cells and these have the potential to become neurogenic retinal progenitor cells [77]. In retinal disease such
as retinitis pigmentosa and age-related macular degeneration can use retinal progenitor cells for transplantation in the adult retina.
Main sources of progenitor cells are: embryo, the bone marrow, neuronal genesis region, and eye (ciliary body epithelium, the iris,
the ciliary marginal zone, and retina) [78].
Stem cells in retina: e ability of donor cells migrate into the desired location, to be alive aer transplantation, and to dierentiate
into retinal cells are the three causes successful of stem cell therapy. Recent researches have shown embryonic SC (ESC), adult SC
and induced pluripotent SC (iPSC) are three main types of stem cells being considered as the potential source for retinal repair and
regeneration. ese eye-derived PCs have the potential to dierentiate into retina-specic cells in an allowable environment. IPSCs
are somatic cells, which can be genetically reprogrammed to become ESC-like with the risk of tumor genesis. A sub-population of
Muller glia with SC characteristics has been founded in the adult human retina. e aim of transplantation of functional retinal
cells or stem cells is to restore vision by repopulating the degenerated retina via rescuing retinal neurons from further degeneration
[79-81].
Target cell types for retinal degeneration treatment
Retinal pigmented epithelial cells (RPE): Human embryonic stem cells (HESCs) and human induced pluripotent stem cells
(hiPSCs) can be dierentiated into all retinal cell types [82] RPE plays a key role in maintenance of neural retinal function that can
suggest retinal degeneration can be treated with sub-retinal injections of RPE cells. e improvement in stem cell dierentiation
techniques can make pluripotent stem cells dierentiation into RPE cells. Extended monolayers of RPE can be isolated and
transferred to a variety of substrates [83-85].
Photoreceptor: e retina includes highly specialized photoreceptors that capture the photons and transduce them into electrical
signals. e ability to produce true multipotent neuroretinal progenitor cells (NRPCs) and being renewable are distinct advantages
of human pluripotent stem cells (HPSCs) as sources of donor neuroretinal cell types [86]
Clinical studies: Recent studies on stem cell therapy in retinal diseases are summarized in Table 2.

Annex Publishers | www.annexpublishers.com
Volume 1 | Issue 1
5
Journal of Stem Cells and Clinical Practice
Table 2: Recent studies on stem cell therapy in retinal diseases. AMD: age related macular degeneration, RPE: retinal
pigment epithelium, hESC: human embryonic stem cell, RCS: Royal College of Surgeons
Discussion
e focus of this review was principal stem cells in the eye especially limbal stem cells. One of the most important ocular stem cells
is limbal stem cell that retains in an undierentiated state and exists in an optimal microenvironment or “niche”. e importance of
limbal stem cells in maintaining the corneal epithelium throughout life has long been recognized. Moreover, stem cells in the retina
as possible edge of the science, have been suggested for treatment of the retinal degeneration, which generally results in constant
OutcomesRetina diseaseStudy
2003
Postoperative vision with a 2-line increase in three
patients
AMD (clinical)Van Meurs JC et al. [87]
2004
TreatmentRetinal degenerationOtani A et al. [88]
2005
Survive and rescue photoreceptors using grasPhotoreceptor lossWang S et al. [89]
2006
Improvement in visual performance was 100%Retinal dystrophyLund RD et al. [90]
2007
Autologous RPE transplantation restores visionneovascular AMD (clinical)MacLaren RE et al. [91]
2008
Rod photoreceptor, bipolar and amacrine cell
markers were expressed by graed cells
Retinal degenerationCastanheira P et al. [92]
2009
iPSCs dierentiate into functional RPEs
Retinitis pigmentosa and
AMD
Buchholz DE et al. [84]
e cells sustained visual function
and photoreceptor integrity
Macular degenerationLu B et al. [93]
2010
BM-MSCs deliver neurotrophic factors and
neuroprotection
GlaucomaLevkovitch-Verbin H et al. [94]
Photoreceptors were present up to 12 months
post-transplantation
Neural retina repairWest EL et al. [95]
2011
Maintenance of visual acuityAMD Takeuchi K et al. [96]
Autologous RPE sheet, Maintenance of visual
acuity
AMDFalkner-Radler et al. [97]
2012
Protection of retina in macular degeneration by
replacement of the structural and trophic support
provided by retinal pigment epithelium
AMDSchwartz et al. [98]
Successful transplantation of hESC- RPE
gra patch
RCS ratHu Y et al. [99]
2013
Neural activity similar to native photoreceptors
AMD and retiniti
pigmentosa
K Homma et al. [100]
HiPSCs less ecient in comparison with hESCretinitis pigmentosaBuchholz D et al. [101]
2014
RPE cell sheets generated without any articial
cell sheets and performed subretinal injection into
RCS
AMDHiroyuki Kamao et al. [102]
rescue of retinal function and signicantly
delayed photoreceptor degeneration
retinal dystrophyAdi Tzameret et al. [103]
2015
hESCs and their dierentiation maintaining
into RPE using Xeno-Free derivation (a novel,
synthetic substrate)
AMDBritney O et al. [104]
increase in visual acuity
AMD and Stargardt’s
macular dystrophy
Chen ZG et al. [105]

Journal of Stem Cells and Clinical Practice
6
Annex Publishers | www.annexpublishers.com
Volume 1 | Issue 1
visual disturbance or even blindness. However, the limbal stem cells have been studied deeply during previous days. When the
patient is labeled as LSCD according to the clinical features, one of above-mentioned procedures could be done. In bilateral cases a
KLAL or CLET procedure may be needed. In unilateral involvement, a CLAU or SLET could be savior. It seems that we need more
studies in the eld of ophthalmic stem cells
1. Pellegrini G, De Luca M, Arsenijevic Y (2007) Towards therapeutic application of ocular stem cells. Semin Cell Dev Biol 18: 805-18.
References
2. Dua HS, Azuara-Blanco A (2000) Limbal stem cells of the corneal epithelium. Surv Ophthalmol 44: 415-25.
3. Shanbhag SS, Saeed HN, Paschalis EI, Chodosh J (2017) Keratolimbal allogra for limbal stem cell deciency aer severe corneal chemical injury: a systematic
review. Bjophthalmol 311249.
4. Dua HS, Azuara-Blanco A (2000) Autologous limbal transplantation in patients with unilateral corneal stem cell deciency. Br J Ophthalmol 84: 273-8.
5. Sepsakos L, Cheung AY, Holland EJ (2017) Outcomes of Keratoplasty aer Ocular Surface Stem Cell Transplantation. Cornea 36: 1025-30.
6. Ahmad S, Kolli S, Lako M, Figueiredo F, Daniels JT (2010) Stem cell therapies for ocular surface disease. Drug Discov Today 15: 306-13.
7. Ramos T, Scott D, Ahmad S (2015) An update on ocular surface epithelial stem cells: cornea and conjunctiva. Stem Cells Int 2015: 601731.
14. Shortt AJ, Secker GA, Munro PM, Khaw PT, Tu SJ (2007) Characterization of the limbal epithelial stem cell niche: novel imaging techniques permit in vivo
observation and targeted biopsy of limbal epithelial stem cells. Stem Cells 25: 1402–9.
13. Davies SB, Chui J, Madigan MC, Provis JM, Wakeeld D, et al. (2009) Stem cell activity in the developing human cornea. Stem Cells 27: 2781-92.
12. Basu S, Bahuguna C, Singh V (2018) Simple limbal epithelial transplantation: Impactful innovation. Indian J Ophthalmol 66: 53-4.
11. Romano AC, Espana EM, Yoo SH, Budak MT, Wolosin JM, et al. (2003) Dierent cell sizes in human limbal and central corneal basal epithelia measured by
confocal microscopy and ow cytometry. Invest Ophthalmol Vis Sci 44: 5125-9.
8. Le Q, Xu J, Deng SX (2018) e diagnosis of limbal stem cell deciency. Ocul Surf 16: 58-69.
9. Fuchs E, Tumbar T, Guasch G (2004) Socializing with the neighbors: stem cells and their niche. Cell 116: 769-78.
10. Chen SY, Han B, Zhu YT, Mahabole M, Huang J, et al. (2015) HC-HA/PTX3 Puried From Amniotic Membrane Promotes BMP Signaling in Limbal Niche Cells
to Maintain Quiescence of Limbal Epithelial Progenitor/Stem Cells. Stem Cells 33: 3341-55.
15. Tseng SC, He H, Zhang S, Chen SY (2016) Niche Regulation of Limbal Epithelial Stem Cells: Relationship between Inammation and Regeneration. Ocul Surf
14: 100-12.
16. Watt FM, Hogan BL (2000) Out of Eden: stem cells and their niches. Science 287: 1427-30.
17. Fuchs E, Tumbar T, Guasch G (2004) Socializing with the neighbors: stem cells and their niche. Cell 116: 769-78.
18. Cotsarelis G (2006) Epithelial stem cells: a folliculocentric view. J Invest Dermatol 126: 1459-68.
19. Blanpain C, Horsley V, Fuchs E (2007) Epithelial Stem Cells: Turning over New Leaves. Cell 128: 445-58.
20. Ahmad S, Stewart R, Yung S, Kolli S, Armstrong L, et al. (2007) Dierentiation of Human Embryonic Stem Cells into Corneal Epithelial‐Like Cells by In Vitro
Replication of the Corneal Epithelial Stem Cell Niche. Stem Cells 25: 1145-55.
21. Kim KH, Mian SI (2017) Diagnosis of corneal limbal stem cell deciency. Curr Opin Ophthalmol 28: 355-362.
22. Vazirani J, Nair D, Shanbhag S, Wurity S, Ranjan A (2018) Limbal Stem Cell Deciency - Demography And Underlying Causes. Am J Ophthalmol 9394: 30026-
6.
23. Schlötzer-Schrehardt U, Kruse FE (2005) Identication and characterization of limbal stem cells. Exp Eye Res 81: 247-64.
24. O’Sullivan F, Clynes M (2007) Limbal stem cells, a review of their identication and culture for clinical use. Cytotechnology 53: 101-6.
25. Kubota M, Shimmura S, Miyashita H, Kawashima M, Kawakita T, et al. (2010) e anti-oxidative role of ABCG2 in corneal epithelial cells. Invest Ophthalmol
Vis Sci 51: 5617-22.
27. Umemoto T, Yamato M, Nishida K, Yang J, Tano Y, et al. (2006) Limbal Epithelial Side‐Population Cells Have Stem Cell–Like Properties, Including Quiescent
State. Stem Cells 24: 86-94.
26. Zhou S, Schuetz JD, Bunting KD, Colapietro AM, Sampath J, et al. (2001) e ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and
is a molecular determinant of the side-population phenotype. Nat Med 7: 1028-34.
28. Lohrum MA, Vousden KH (2000) Regulation and function of the p53-related proteins: same family, dierent rules. Trends Cell Biol 10: 197-202.
29. Celli J, Duijf P, Hamel BC, Bamshad M, Kramer B, et al. (1999) Heterozygous germline mutations in the p53 homolog p63 are the cause of EEC syndrome. Cell
99: 143-53.
30. Barbaro V, Testa A, Di Iorio E, Mavilio F, Pellegrini G, et al. (2007) C/EBPδ regulates cell cycle and self-renewal of human limbal stem cells. J Cell Biol 177:
1037-49.
31. Ahmad S, Figueiredo F, Lako M (2006) Corneal epithelial stem cells: characterization, culture and transplantation. Regen Med 1: 29-44.
32. Nakamura T, Inatomi T, Sotozono C, Koizumi N, Kinoshita S (2016) Ocular surface reconstruction using stem cell and tissue engineering. Prog Retin Eye Res
51: 187-207.
33. Takács L, Tóth E, Berta A, Vereb G (2009) Stem cells of the adult cornea: from cytometric markers to therapeutic applications. Cytometry A 75: 54-66.
34. omas PB, Liu YH, Zhuang FF, Selvam S, Song SW, et al. (2007) Identication of Notch-1 expression in the limbal basal epithelium. Mol Vis 13: 337-44.
35. Whitcher JP, Srinivasan M, Upadhyay MP (2001) Corneal blindness: a global perspective. WHO 79: 214-21.
37. Li W, Chen YT, Hayashida Y, Blanco G, Kheirkah A, et al. (2008) Down‐regulation of Pax6 is associated with abnormal dierentiation of corneal epithelial cells
in severe ocular surface diseases. J Pathol 214: 114-22.
36. Bakhtiari P, Djalilian A (2010) Update on limbal stem cell transplantation. Middle East Afr J Ophthalmol 17: 9-14.
38. Dhamodaran K, Subramani M, Jeyabalan N, Ponnalagu M, Chevour P, et al. (2015) Characterization of ex vivo cultured limbal, conjunctival, and oral mucosal
cells: A comparative study with implications in transplantation medicine. Mol Vis 21: 828-45.

Annex Publishers | www.annexpublishers.com
Volume 1 | Issue 1
7
Journal of Stem Cells and Clinical Practice
39. Puangsricharern V, Tseng SC (1995) Cytologlogic evidence of corneal diseases with limbal stem cell deciency. Ophthalmol (10): 1476-85.
40. Kruse FE (1994) Stem cells and corneal epithelial regeneration. Eye 8: 170-83.
41. Pster RR (1994) Corneal stem cell disease: concepts, categorization, and treatment by auto-and homotransplantation of limbal stem cells. Eye & Contact Lens
20: 64-72.
42. Fujishima H, Shimazaki J, Tsubota K (1996) Temporary corneal stem cell dysfunction aer radiation therapy. Br J Ophthalmol 80: 911-4.
43. Dua HS, Joseph A, Shanmuganathan VA, Jones RE (2003) Stem cell dierentiation and the eects of deciency. Eye 17: 877-85.
44. Lavker RM, Tseng SC, Sun TT (2004) Corneal epithelial stem cells at the limbus: looking at some old problems from a new angle. Exp Eye Res 78: 433-46.
45. Tseng SC, Sun TT (1999) Stem cells: ocular surface maintenance. Corneal surgery theory, technique, tissue. St. Louis 9-18.
52. Tsai RJ, Tseng SC (1994) Human allogra limbal transplantation for corneal surface reconstruction. Cornea 13: 389-400.
51. Holland EJ, Schwartz GS (2004) e Paton lecture: ocular surface transplantation: 10 years’ experience. Cornea 23: 425-31.
50. Coster DJ, Aggarwal RK, Williams KA (1995) Surgical management of ocular surface disorders using conjunctival and stem cell allogras. Br J Ophthalmol
79: 977-82.
49. Shortt AJ, Secker GA, Notara MD, Limb GA, Khaw PT, et al. (2007) Transplantation of ex vivo cultured limbal epithelial stem cells: a review of techniques and
clinical results. Surv Ophthalmol. 52: 483-502.
46. Puangsricharern V, Tseng SC (1995) Cytologlogic evidence of corneal diseases with limbal stem cell deciency. Ophthalmol 102: 1476-85.
47. Tseng SC (1996) Regulation and clinical implications of corneal epithelial stem cells. Mol Biol Rep 23: 47-58.
48. Huang AJ, Tseng SC (1991) Corneal epithelial wound healing in the absence of limbal epithelium. Invest Ophthalmol Vis Sci 32: 96-105.
53. Dua HS, Azuara-Blanco A (2000) Limbal stem cells of the corneal epithelium. Surv Ophthalmol 44: 415-25.
54. Holland EJ (1996) Epithelial transplantation for the management of severe ocular surface disease. Trans Am Ophthalmol Soc 94: 677.
55. Nishiwaki-Dantas MC, Dantas PE, Reggi JR (2001) Ipsilateral limbal translocation for treatment of partial limbal deciency secondary to ocular alkali burn.
Br J Ophthalmol 85: 1031-3.
56. Tsai RJ, Li LM, Chen JK (2000) Reconstruction of damaged corneas by transplantation of autologous limbal epithelial cells. Am J Opthamol 4: 543.
57. Basu S, Sureka SP, Shanbhag SS, Kethiri AR, Singh V, et al. (2016) Simple Limbal Epithelial Transplantation: Long-Term Clinical Outcomes in 125 Cases of
Unilateral Chronic Ocular Surface Burns. Ophthalmology 123: 1000-10.
58. Tseng SC, Prabhasawat P, Barton K, Gray T, Meller D (1998) Amniotic membrane transplantation with or without limbal allogras for corneal surface recon-
struction in patients with limbal stem cell deciency. Ophthalmology 116: 431-41.
59. Mittal V, Jain R, Mittal R, Vashist U, Narang P (2016) Successful management of severe unilateral chemical burns in children using simple limbal epithelial
transplantation (SLET). Br J Ophthalmol 100: 1102-8.
60. Barraquer RI, de Toledo JP, Barraquer J (1997) Storage, surgery, outcome, complications, and new developments in corneal and conjunctival gras. Curr Opin
Ophthalmol 8: 31-40.
61. Pellegrini G, Traverso CE, Franzi AT, Zingirian M, Cancedda R, De Luca M (1997) Long-term restoration of damaged corneal surfaces with autologous cul-
tivated corneal epithelium. Lancet 349: 990-3.
62. Sangwan VS, Vemuganti GK, Iekhar G, Bansal AK, Rao GN (2003) Use of autologous cultured limbal and conjunctival epithelium in a patient with severe
bilateral ocular surface disease induced by acid injury: a case report of unique application. Cornea 22: 478-81.
63. Sangwan VS, Matalia HP, Vemuganti GK, Fatima A, Ihekar G, et al. Clinical outcome of autologous cultivated limbal epithelium transplantation. Indian J
Ophthalmol 54: 29-34.
65. Vazirani J, Basu S, Kenia H, Ali MH, Kacham S, et al. (2014) Unilateral partial limbal stem cell deciency: contralateral versus ipsilateral autologous cultivated
limbal epithelial transplantation. Am J Ophthalmol 157: 584-90.
64. Sangwan VS, Basu S, Vemuganti GK, Sejpal K, Subramaniam SV, et al. (2011) Clinical outcomes of xeno-free autologous cultivated limbal epithelial transplanta-
tion: a 10-year study. Br J Ophthalmol 95: 1525-9.
66. Daya SM, Watson A, Sharpe JR, Giledi O, Rowe A, et al. (2005) Outcomes and DNA analysis of ex vivo expanded stem cell allogra for ocular surface recon-
struction. Ophthalmology 112: 470-7.
67. Nakamura T, Inatomi T, Sotozono C, Ang LP, Koizumi N, et al. (2006) Transplantation of autologous serum-derived cultivated corneal epithelial equivalents for
the treatment of severe ocular surface disease. Ophthalmology 113: 1765-72.
68. Javadi MA, Einollahi B, Raei AB, Baharvand H, Ebrahimi M, et al (2006) Early results of autologous cultivated limbal stem cell transplantation in total limbal
stem cell deciency. Iranian J Ophthalmic Res 1: 71-80.
69. Baradaran-Rai AR, Abdolahi A, Jabarpoor Bonyadi MH, Mirdehghan SA, Javadi MA, et al. (2010) Keratolimbal allogra in total limbal stem cell deciency.
Bina J Ophthalmol 15: 167-76.
70. Shortt AJ, Secker GA, Rajan MS, Meligonis G, Dart JK, et al. (2008) Ex vivo expansion and transplantation of limbal epithelial stem cells. Ophthalmology 115:
1989-97.
71. Bull ND, Martin KR (2011) Concise review: Toward stem cell‐based therapies for retinal neurodegenerative diseases. Stem Cells 29: 1170-5.
72. Sohocki MM, Daiger SP, Bowne SJ, Rodriquez JA, Northrup H, et al. (2001) Prevalence of mutations causing retinitis pigmentosa and other inherited retinopa-
thies. Hum Mutat 17: 42-51.
73. Kobrin Klein BE (2007) Overview of epidemiologic studies of diabetic retinopathy. Ophthalmic Epidemiol 14: 179-83.
75. Fischer AJ, Reh TA (2003) Potential of Müller glia to become neurogenic retinal progenitor cells. Glia 43: 70-6.
74. Johnson TV, Bull ND, Hunt DP, Marina N, Tomarev SI, et al. (2010) Neuroprotective eects of intravitreal mesenchymal stem cell transplantation in experimen-
tal glaucoma. Investi Ophthalmo Vis Sci 51: 2051-9.
76. Mayer EJ, Hughes EH, Carter DA, Dick AD (2003) Nestin positive cells in adult human retina and in epiretinal membranes. Brit J Ophthalmol 87: 1154-8.
77. Layer PG, Rothermel A, Willbold E (2001) From stem cells towards neural layers: a lesson from re-aggregated embryonic retinal cells. Neuroreport 12: A39-46.
78. Bi YY, Feng DF, Pan DC (2009) Stem/progenitor cells: a potential source of retina-specic cells for retinal repair. Neuroscience research 65: 215-21.
79. Tropepe V, Coles BL, Chiasson BJ, Horsford DJ, Elia AJ, et al. (2000) Retinal stem cells in the adult mammalian eye. Science 287: 2032-6.

Journal of Stem Cells and Clinical Practice
8
Annex Publishers | www.annexpublishers.com
Volume 1 | Issue 1
Submit your next manuscript to Annex Publishers and
benet from:
Submit your manuscript at
http://www.annexpublishers.com/paper-submission.php
→ Easy online submission process
→ Rapid peer review process
→ Open access: articles available free online
→ Online article availability soon aer acceptance for Publication
→ Better discount on subsequent article submission
→ More accessibility of the articles to the readers/researchers within the eld
80. Coles BL, Angénieux B, Inoue T (2004) Facile isolation and the characterization of human retinal stem cells. Proc Natl Acad Sci U S A 101: 15772-7.
81. Chacko DM, Rogers JA, Turner JE, Ahmad I (2000) Survival and dierentiation of cultured retinal progenitors transplanted in the subretinal space of the rat.
Biochem Biophy Res Comm 268: 842-6.
82. Rowland TJ, Buchholz DE, Clegg DO (2012) Pluripotent human stem cells for the treatment of retinal disease. J Cell Physiol 227: 457-66.
83. Meyer JS, Howden SE, Wallace KA, Verhoeven AD, Wright LS, et al. (2011) Optic vesicle‐like structures derived from human pluripotent stem cells facilitate a
customized approach to retinal disease treatment. Stem cells 29: 1206-18.
84. Buchholz DE, Hikita ST, Rowland TJ, Friedrich AM, Hinman CR, et al. (2009) Derivation of functional retinal pigmented epithelium from induced pluripotent
stem cells. Stem cells 27: 2427-34.
85. Singh R, Shen W, Kuai D, Martin JM, Guo X, et al. (2012) iPS cell modeling of Best disease: insights into the pathophysiology of an inherited macular degenera-
tion. Human molecular genetics 22: 593-607.
92. Castanheira P, Torquetti L, Nehemy MB, Goes AM (2008) Retinal incorporation and dierentiation of mesenchymal stem cells intravitreally injected in the
injured retina of rats. Arq Bras Oalmol 71: 644-50.
91. MacLaren RE, Uppal GS, Balaggan KS, Tufail A, Munro PM, et al. (2007) Autologous transplantation of the retinal pigment epithelium and choroid in the treat-
ment of neovascular age-related macular degeneration. Ophthalmol 114: 561-70.
90. Lund RD, Wang S, Klimanskaya I, Holmes T, Ramos-Kelsey R, (2006) Human embryonic stem cell–derived cells rescue visual function in dystrophic RCS rats.
Cloning and stem cells 8: 189-99.
89. Wang S, Lu B, Wood P, Lund RD (2005) Graing of ARPE-19 and Schwann cells to the subretinal space in RCS rats. Invest Ophthalmo Vis Sci 46: 2552-60.
86. Borooah S, Phillips MJ, Bilican B (2013) Using human induced pluripotent stem cells to treat retinal disease. Progress in Retinal Eye Res 37: 163-81.
87. van Meurs JC, Van Den Biesen PR (2003) Autologous retinal pigment epithelium and choroid translocation in patients with exudative age-related macular
degeneration: short-term follow-up. Am J Ophthalmol 136: 688-95.
88. Otani A, Dorrell MI, Kinder K, Moreno SK, Nusinowitz S, et al. (2004) Rescue of retinal degeneration by intravitreally injected adult bone marrow–derived
lineage-negative hematopoietic stem cells. J Clin Invest 114: 765-774.
93. Lu B, Malcuit C, Wang S, Girman S, Francis P, et al. (2009) Long‐term safety and function of RPE from human embryonic stem cells in preclinical models of
macular degeneration. Stem cells 27: 2126-35.
94. Levkovitch-Verbin H, Sadan O, Vander S, et al. (2010) Intravitreal injections of neurotrophic factors secreting mesenchymal stem cells are neuroprotective in
rat eyes following optic nerve transection. Invest Ophthalmo Vis Sci 51: 6394-400.
95. West EL, Pearson RA, Barker SE, Luhmann UF, Maclaren RE, et al. (2010) Long‐term survival of photoreceptors transplanted into the adult murine neural retina
requires immune modulation. Stem cells 28: 1997-2007.
96. Takeuchi K, Kachi S, Iwata E, Ishikawa K, Terasaki H (2012) Visual function 5 years or more aer macular translocation surgery for myopic choroidal neovas-
cularisation and age-related macular degeneration. Eye 26: 51-60.
97. Falkner-Radler CI, Krebs I, Glittenberg C, Povazay B, Drexler W, et al. (2010) Human retinal pigment epithelium (RPE) transplantation: outcome aer autolo-
gous RPE-choroid sheet and RPE cell-suspension in a randomised clinical study. Br J Ophthalmol 95: 370-5.
98. Schwartz SD, Hubschman JP, Heilwell G, Franco-Cardenas V, Pan CK, et al. (2012) Embryonic stem cell trials for macular degeneration: a preliminary report.
e Lancet 379: 713-20.
99. Hu Y, Liu L, Lu B, Zhu D, Ribeiro R, et al. (2012) A novel approach for subretinal implantation of ultrathin substrates containing stem cell-derived retinal pig-
ment epithelium monolayer. Ophthalmic Res 48: 186-91.
100. Homma K, Okamoto S, Mandai M, Gotoh N, Rajasimha HK, et al. (2013) Developing Rods Transplanted into the Degenerating Retina of Crx‐Knockout Mice
Exhibit Neural Activity Similar to Native Photoreceptors. Stem Cells 31: 1149-59.
101. Buchholz DE, Pennington BO, Croze RH, Hinman CR, Coey PJ, et al. (2013) Rapid and ecient directed dierentiation of human pluripotent stem cells into
retinal pigmented epithelium. Stem Cells Transl Med 2: 384-93.
102. Kamao H, Mandai M, Okamoto S, Sakai N, Suga A, et al. (2014) Characterization of human induced pluripotent stem cell-derived retinal pigment epithelium
cell sheets aiming for clinical application. Stem Cell Rep 2: 205-18.
103. Tzameret A, Sher I, Belkin M, Treves AJ, Meir A, et al. (2014) Transplantation of human bone marrow mesenchymal stem cells as a thin subretinal layer ame-
liorates retinal degeneration in a rat model of retinal dystrophy. Exp Eye Res 118: 135-44.
105. Chen Z, Zhang YA (2015) Cell therapy for macular degeneration--rst phase I/II pluripotent stem cell-based clinical trial shows promise. Sci China Life Sci
58: 119.
104. Pennington BO, Clegg DO, Melkoumian ZK, Hikita ST (2015) Dened culture of human embryonic stem cells and xeno-free derivation of retinal pigmented
epithelial cells on a novel, synthetic substrate. Stem Cells Transl Med 4: 165-77.