Pluripotent stem cells are primitive cells that are able to be self-renewing, proliferating indefinitely in their undifferentiated state, and differentiate into different cell types[1-2]. They can efficiently differentiate into specific cell types under defined conditions. Therefore, stem cells have been regarded as unlimited source of cell transplantation.Over the last decade, stem or progenitor cells transplantation as a means of replacing tissue have evolved rapidly. There are distinct kinds of stem cells according to their differentiation potential. It has already been found that embryonic stem cells(ESCs) and mesenchymal stem cells (MSCs) can directly differentiate into specialized cells, which attracted considerable interest. However, the future use of these cells is still facing challenges, such as immune rejection and ethical debate for ESCs; the heterogeneity, difficulty of large scale harvest,insufficient source, reduced differentiation capability after multiple generations for MSCs, which limit their clinical application.
The discovery of induced pluripotent stem cells (iPSCs)is considered as a breakthrough. In 2006, Takahashi and Yamanaka[3]reported that mouse fibroblasts could be induced into ESC-like cells and named them “induced pluripotent stem cells”. iPSCs have self-renewing and multi-directional differentiation potential, which are similar to ESCs.Differentiated iPSCs generated from the same patient enable their use in autologous transplantation to avoid immune rejection and ethical debate[4]. Moreover cells or tissues for generating iPSCs can be obtained simply and noninvasively.Therefore, iPSCs have drawn particular attention to biological scientists and clinicians[5].
Nowadays the work on iPSCs culture focus on the retinal and retinal cells, which has been revealed a formerly underrated level of intrinsic cellular self-renewing[6]. In recent years there has been considerable progress in the study of iPSCs and corneal diseases. Here we will summarize the recent advancement in iPSCs-derived corneal epithelial cells technology and discuss its therapeutic potential for patients with limbal stem cell de ficiency (LSCD). The review will also highlight the limitations that need to deal with and plausible strategies for the application, the advantage of iPSCs-derived disease models to understand the pathophysiology of cornea disorders, as well as the future of clinical trial for iPSCs as cell sources will also be discussed.
The development of ocular surface is a dynamic procedure. In the next part of the article, we will discuss the development of ocular surface and particular markers, which make structures speci fic. The ocular surface is composed of corneal epithelium, conjunctival epithelium and tear film. The surface of the cornea is covered by corneal epithelial cells, which are crucial for maintaining normal eye function. Corneal epithelial cells are derived from the epidermal layer of the embryo. When the human embryo is about 9 mm, the lens bubble differentiates completely from the ectoderm, and then the corneal epithelium differentiates from the ectoderm afterwards. With the continuous development of the embryo,the corneal epithelial cells gradually differentiate and form complete structure[7-8]. The morphology of normal corneal epithelium is like paving stone. Under normal condition the morphological characteristics of corneal epithelium are stable.The dynamic balance of corneal epithelium is important for maintaining normal vision and the integrity of ocular surface.Infection, injury or lack of limbus stem cells can lead to corneal fibrosis[7]. Cell proliferation, morphological changes,and the increased expression of mesenchymal markers of the long-term culture corneal epithelial cells indicated epithelial to mesenchymal transition[9]. The shape of corneal epithelial cells was transformed from cubic into fusiform-like fibroblast.At the same time the connection between epithelial cells was lost[10]. Cytokeratin (CK) 3 and CK 12 were known as speci fic markers of corneal epithelial cells. It was found that there was connexin 43 in the corneal epithelial cells, which could connect them[11]. When the corneal epithelial cells are in a transient state of proliferation, expression of connexin 43 is negative[12-13]. CK4 and CK13 were markers of nonkeratinized epithelium[14]. CK15 was the marker of corneal progenitor cell[15]. In general, connexin 43, CK3, CK12, CK4,CK13 and CK15 were considered as indicator of corneal differentiation[16-17].
The current studies of stem cells related to not only laboratory situations, but also clinical situations. The corneal epithelial cells are constantly shedding or dying due to external environment or internal metabolism. Under the basal cell proliferation and the differentiation of limbal epithelium stem cells (LESCs), which is found in the corneal limbus located between the transparent cornea and opaque conjunctiva, the corneal epithelial cells are contantly repaired by itself[18].Severe ocular surface disease can damage the limbic region,leading to the reduction or loss of LESCs to some extent and the destruction of barrier structures. That may cause corneal hazing, corneal epithelium defect, or even blindness, which is clinically termed as LSCD[19-20]. Many disease can cause LSCD, like chemical burn or traumatic injury, inherited corneal dystrophy and numerous immune disorders, for example,Stevens-Johnson syndrome[20].
Corneal stem epithelial cells are crucial for repair of corneal surface. They express different markers during the process of proliferation. It is widely believed that the corneal epithelial stem cells belong to unipotent somatic adult stem cells, and the stem cells are exclusively located in a particular limbal structure, which is defined as palisades of Vogt[21]. Limbal epithelial basal layer lacks the expression of keratins CK3 and CK12, the terminal differentiation marker of corneal epithelial cells[22]. The study found that when the central corneal epithelium was damaged, the corneal limbus basal cells were more active than those in the central region of the epithelium.The direction of the cell migration was from the cornea limbus to the central. Hayashiet al[23]found that the corneal epithelial stem cells were gradually replaced by LESCs, which located at the base of the cornea limbus. Although it is hard for the corneal epithelial stem cells to be classified and separated by just one particular marker, in recent years, ABCG2 and P63 (ΔNp63α isoform) has been found expressed in LESCs.However, there is no expression of CK3 and CK12 in these cells[24-25]. P63 is a kind of nucleoprotein, which expressed in the limbus in the basal layer of the cornea. The expression of P63 in corneal limbic cells is regarded as a marker of proliferation ability for these cells[26].
The treatment of LSCD includes drug and surgical therapy.Drug therapy such as artificial tear, bandage contact lens and autologous serum eye drop are only applied as adjuvant therapy, which have been only used in very mild cases[27].Surgical therapy include amniotic membrane transplantation,autologous conjunctival limbal stem cell transplantation,allogeneic limbal stem cell transplantation[28-31]. Now, stem cell transplantation is the most optimal method in severe cases and in total LSCD to restore a healthy corneal surface.Amniotic membrane transplantation, coupled with conjunctival epitheliectomy, could be a therapy in partial LSCD[32]. At present, LESCs transplantation is the main treatment method,but the lack of LESCs donor has limited the application of LESCs transplantation[33]. Though somatic stem cells have been applied to recover the ocular surface, the long-term clinical results have indicated that it is not be overly encouraging[34-36].
Human pluripotent stem cells (hPSCs) could be an alternative application in many fields. They have a wider differentiation potential and limitless self-renew than tissue-specific stem cells and they providing an unlimited source of cells. Because the capability of pluripotent ESCs to generate multiple cell types and their unlimites expansion potential, the application of them for tissue engineering may provide advantages over traditional sources of progenitor cells. Recently the development of stem cell technology provides a new method for LESC transplantation[37]. There were a couple of approaches for ESCs to differentiate into corneal epithelial cells, such as Pax6 gene transfection, microenvironment simulation and induction factor interpolation[17,38]. Hommaet al[39]first reported epithelial progenitors were successfully induced in 8d by culturing mouse ESCs on type IV collagen and the reconstruction of mice corneal surface is also feasible. These progenitors expressed corneal epithelial cells speci fic gene, keratin (K) 12. More importantly, complete reepithelialization of the corneal surface occurred within 24h after transplantation into damaged cornea[39]. Later, mouse ESCs were found to be capable of differentiating into a monolayer of epithelium-like cells. These corneal epitheliumlike cells were induced by Pax6 gene expression of mouse ESCs[38]. Ahmadet al[40]first reported differentiation of human ESCs into corneal epithelial-like cells byin vitroreplication of the corneal epithelial stem cell niche. Since then, several other studies[41-43]have been published, all relying on different types of unde fined or animal-derived components, such as amniotic membrane, feeder cells, or conditioned medium, alone or in combinations. Most recently, a serum free- and Xeno-free protocol has been reported to initiate differentiation towards LESC-like cells with induction medium and hPSC medium modified by decreasing KO-SR concentration, increasing bFGF concentration, and adding transforming growth factor β (TGF-β) inhibitor SB-505124, Wnt inhibitor IWP-2. After the induction stage, cell aggregates were plated onto plates coated with human placental collagen IV, and maintained in a defined and serum-free corneal epithelium medium CnT-30. After a total of 30-35d, the proteomics of human PSCsderived LESCs are similar to native corneal epithelial cells[44].However, potential immune rejection and ethical issues have limited the application of ESCs. What’s more, more researches about MSCs arrests our attention. MSCs have been found to differentiate into corneal epithelioid cells when they were co-cultured with corneal stromal cells and transplanted into ocular surface[45]. MSC could also inhibit postoperative corneal inflammation and angiogenesis. However, the reduction of differentiation ability after multiple generations limits their clinical application[37,46-47].
As previously mentioned, in 2006, Takahashi and Yamanaka[3]developed iPSCs through reprogramming several types of somatic cells by introducing four transcription factors (Oct3/4,Sox2, c-Myc, and Klf4) into these somatic cells under ESC culture conditions. For the first time, they demonstrated induction of pluripotent stem cells from mouse embryonic or adult fibroblasts by reprogramming. Since then, more academic researches are conducted profoundly. The finding allows the creation of pluripotent cells from homograft somatic cells of patient straightforward. With the development of iPSCs research technology in the last decade, a great progress have been made in the following aspects, such as cell source diversity used for reprogramming, function of transcription factors during the reprogramming, safety of vectors used for the delivery of transcription factors, advances in differentiation ef ficiency. iPSCs can provide new approaches for autologous cell-based therapy, possibly organ replacement treatments and iPSCs generated from patients offer profound insight into the mechanisms of disease. The study will impact human disease modeling, drug discovery and testing[48]. Studies have con firmed that iPSCs can differentiate into multiple cell types:islet cells, cardiomyocytes, nerve cells, retinal cells, and so on,which offered new therapy strategy for the patients of diabetes,myocardial infarction, Parkinson’s disease, and retinal disease.Besides that it represents a promising resource for new and ongoing studies of ocular morphogenesis.
Over the past few years, the research on iPSCs used as a source of regenerative medicine developed rapidly. Over the past decade, progress in stem cell researches for blinding diseases is now being applied to patients with retinal degenerative diseases. iPSCs technology has opened a new avenue for treating various diseases by using patient specific cells,eliminating the risk of immune rejection after transplantation of the patient’s autologous iPSCs-derived cells, such as retinal pigment epithelial (RPE) cells[49]. The use of iPSCs-derived corneal epithelial cell may provide an unlimited source of cells and circumvent the issues, which exist in human ESCsderived corneal epithelial cell. Recently, by mimicking the environmental niche of limbal stem cells, several protocols have been developed, aimed at the differentiation of iPSCs into the corneal epithelial lineage[50-51]. More recently, small molecule driven protocols have become available resulting in generation of corneal epithelial-like cells within 6wk[15].
Great achievements have been made in deriving iPSCs into corneal epithelial cells, especially remarkable researches of differentiation methods. In 2012, for the first time Hayashiet al[42]demonstrated a strategy for the differentiation of human iPSCs into corneal epithelial cells, and also noted that the epigenetic status was associated with the propensity of differentiation of iPSCs into corneal epithelial cells. In their study, they used stromal cell-derived inducing activity (SDIA)differentiation method[52-53]to induce corneal epithelial cells from the iPSCs, as Pax6+/K12+ corneal epithelial colonies were observed after prolonged differentiation culture (12wk or later) in both the human adult corneal limbal epithelial cells (HLEC)-derived iPSCs (L1B41) and human adult dermal fibroblast (HDF)-derived iPSCs (253G1). The corneal epithelial differentiation ef ficiency was higher in L1B41 than that in 253G1. There was no significant difference between L1B41 and 253G1 iPSCs was noted in methylation status of the corneal epithelium-related genes, such as K12, K3, and Pax6[42]. However another study found that when the gene methylation patterns of iPSCs in comparison to their parental cells, limbal-derived iPSCs had fewer unique methylation changes than fibroblast-derived iPSCs. Limbus-derived iPSCs cultured for 2wk on human amniotic membrane (HAM)developed markedly higher expression of putative LESC markers keratins 14, 15, and 17, ABCG2, ΔNp63α, N-cadherin,and TrkA than did fibroblast iPSCs[54]. The study also described a different approach, differentiating LESC-derived iPSCs without feeder cells but using biological supports that were identical (denuded organ-cultured corneas) or similar (denuded HAM) to their natural niche. They emphasized the importance of niche factors, including underlying BM and stromal cells[54-55].Then in 2013, Yuet al[56]co-cultured iPSCs in the presence of additional factors bFGF, EGF and NGF, activated keratin K12 expression (a marker of corneal epithelial cells) and downregulated Nanog with corneal limbal stroma, which is separated by a transwell membrane. They found that after 12d of differentiation procedure, by replication of a corneal epithelial stem cell niche, mouse iPSCs could differentiate into corneal epithelial-like cells. Through scanning electron microscopy they found differentiated iPSCs also had multiple microcilia, like normal mouse corneal epithelial cells.Differentiated iPSCs were smaller than cultured epithelial cells was the difference between the two types of culture. Besides that they found iPSCs-derived epithelial cells expressed K12,a speci fic marker of corneal epithelial cells, and pax6, which was necessary for early development of eyes. The expression of P63, a marker of corneal epithelial stem cells, was activated after co-culture. The expression of pluripotent gene, Nanog,decreased after differentiation[56]. These studies will help successor to explore more efficient, especial and handy methods for differentiation from iPSCs to corneal epithelial cells.
However, the above studies have a tissue-speci fic focus solely and fail to re flect the complicacy of whole eye development.In the past, we also have made progress at whole eye level.Hayashiet al[57]demonstrated the generation of ocular cells in a self-formed ectodermal autonomous multi-zone (SEAM)from iPSCs. In a way, the concentric SEAM mimicked wholeeye development. The approach also demonstrated the cells in the SEAM could be purified by fluorescence-activated cell sorting (FACS) and expandedin vitroto form a corneal epithelium that recovered function in an experimentally induced animal model of corneal blindness. In their protocol,they adopted a 2D culture system with the laminin 511 E8 fragment as a substrate for ocular cell growth, and they used a serum-free differentiation medium to promote autogenic eye-like differentiation of human iPSCs[57]. The new protocol for differentiation of iPSCs to corneal included human iPSCs autonomous differentiation, purification, and subsequent differentiation. Especially the culture techniques did not need feeder cells or fetal bovine serum. Besides, the protocol used a combination of antibodies to puri ficate iPSCs-derived corneal epithelial stem and progenitor cells by FACS-sorting. So that the final product was devoid of non-corneal iPSCs-derived epithelial cells, and of cellular impurities that might originate from feeder cells[58]. Recently a direct transdifferentiation approach was established to circumvent the intermediate state of pluripotency (iPS-stage). The resulting cells, which are obtained directly by transdifferentiation from fibroblasts to limbal cells, displaied corneal epithelial cell morphology and corneal epithelial markers experssion. They showed a direct transdifferentiation of human dermal fibroblasts into the corneal epithelial lineage that may serve as a source for corneal epithelial cells for transplantation approaches and avoided tumorigenic potential[50].
Above studies include how to harvest stem cells, the source of stem cells, their progeny, and the differentiation method of iPSCs. The following studies enhanced clinical success by discussing iPSCs-derived keratocytes to promote comprehending cornea disease mechanisms, establishing corneal disease organoid models or keratoconus (KC) model.iPSCs-derived keratocytes allow for novel approaches in disease modeling and drug development platforms.Keratocytes derived from iPSCs of patients offer an autologous source, which will be used as a research tool for understanding cornea disease mechanisms. One study indicated this iPSCsmodel system allowed for the identifying of miR-450b-5p as a molecular switch of Pax6, a major regulator of eye development. MiR-450b-5p and Pax6 were found reciprocally distributed at the presumptive epidermis and ocular surface,respectively. It suggests that by repressing Pax6, miR-450b-5p triggers epidermal specification of the ectoderm, while the absence of miR-450b-5p accompanied with Pax6 expression allows ocular epithelial development[51]. A recent study showed human iPSCs-derived organoids through sequential rounds of differentiation shared features of the developing cornea, appeared translucent with a clear or a dense center,and harbored three distinct types of cells with expression of key epithelial, endothelial and stromal cell markers. Cornea organoid cultures could be a powerful 3D model system for investigating the developmental process of corneal and their disruptions in pathological conditions[59]. Another study made a model of KC using iPSCs generated from fibroblasts of both normal human corneal stroma and KC with a viral vector. The author found that the inhibition FGFR2-Pi3-kinase pathway affected the AKT phosphorylation, and subsequently affecting the keratocytes survival signals, while the normal group did not affect the keratocytes survival signals. A potential mechanism for the KC-specific decreased cell survival and apoptosis of keratocytes could be the inhibition of the survival signals[60].Therefore, continuous development in this area will lead to a novel understanding of the multifactorial and complex corneal disease.
Stem cell technology is a promising approach to provide a limitless source for cell replacement therapy of LSCD.Increasing number of studies reported differentiation of iPSCs into corneal epithelial cells and successfully established reproducible protocols. Transplantation of the iPSCs-derived corneal epithelial cell could recover function in an animal model of corneal blindness[57]. However, the current method of differentiation is time-consuming, expensive, and inefficient,which has not yet reached the standard and level of clinical application. So the protocol of iPSCs differentiation into corneal epithelium should be improved continually. At present,iPSCs-derived corneal epithelial cell is only demonstrated to repair the corneal function in animal model. No clinical trial has been reported to confirm its efficacy and safety of cells post-transplantation in human. As we known, the undifferentiated iPSCs have limitless proliferation potentials and/or tumorigenic transformed cells, the tumorigenic potential of iPSCs-derived corneal epithelial cell has not yet been tested,which is attributable to contamination by undifferentiated iPSCs[61]. The risk of teratoma formation associated with the use of iPSCs hinders applications from lab into clinics. All of these prevent iPSCs-derived corneal epithelial cells from the clinical treatment of patients with corneal disease. Although in the past five years, the technology of iPSCs differentiation into corneal epithelial cells has made great progress, but there are some important obstacles that must be overcome. It still has a long way to go before iPSCs-derived corneal epithelial cells can be used in clinic.
Conflicts of Interest: Zhu J, None; Slevin M, None; Guo BQ, None; Zhu SR, None.
1 Nazari H, Zhang L, Zhu D, Chader GJ, Falabella P, Stefanini F,Rowland T, Clegg DO, Kashani AH, Hinton DR, Humayun MS. Stem cell based therapies for age-related macular degeneration: the promises and the challenges.Prog Retin Eye Res 2015;48:1-39.
2 Weissman IL. Stem cells: units of development, units of regeneration,and units in evolution.Cell2000;100(1):157-168.
3 Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell2006;126(4):663-676.
4 Fields M, Cai H, Gong J, Del Priore L. Potential of induced pluripotent stem cells (iPSCs) for treating age-related macular degeneration (AMD).Cells2016;5(4).pii: E44.
5 Tang Z, Zhang Y, Wang Y, Zhang D, Shen B, Luo M, Gu P. Progress of stem/progenitor cell-based therapy for retinal degeneration.J Transl Med2017;15(1):99.
6 Bracha P, Moore NA, Ciulla TA. Induced pluripotent stem cell-based therapy for age-related macular degeneration.Expert Opin Biol Ther2017;17(9):1113-1126.
7 Echevarria TJ, Di Girolamo N. Tissue-regenerating, vision-restoring corneal epithelial stem cells.Stem cell Rev2011;7(2):256-268.
8 Kitazawa K, Hikichi T, Nakamura T,et al. OVOL2 Maintains the transcriptional program of human corneal epithelium by suppressing epithelial-to-mesenchymal transition.Cell Rep2016;15(6):1359-1368.
9 Saburina IN, Kolokoltsova TD, Kopaev SY, Zurina IM, Borzenok SA.Experience of culturing anterior epithelial corneal cells from human eye ball.Patol Fiziol Eksp Ter2014;(4):120-126.
10 Park GB, Kim D, Kim YS, Kim S, Lee HK, Yang JW, Hur DY. The Epstein-Barr virus causes epithelial-mesenchymal transition in human corneal epithelial cells via Syk/src and Akt/Erk signaling pathways.Invest Ophthalmol Vis Sci2014;55(3):1770-1779.
11 Chen Z, Evans WH, Pflugfelder SC, Li DQ. Gap junction protein connexin 43 serves as a negative marker for a stem cell-containing population of human limbal epithelial cells.Stem Cells2006;24(5):1265-1273.
12 Li DQ, Wang Z, Yoon KC, Bian F. Characterization, isolation,expansion and clinical therapy of human corneal epithelial stem/progenitor cells.J Stem Cells2014;9(2):79-91.
13 Schlotzer-Schrehardt U, Kruse FE. Identi fication and characterization of limbal stem cells.Exp Eye Res2005;81(3):247-264.
14 Ramos T, Scott D, Ahmad S. An update on ocular surface epithelial stem cells: cornea and conjunctiva.Stem Cells Int2015;2015:601731.
15 Mikhailova A, Ilmarinen T, Uusitalo H, Skottman H. Small-molecule induction promotes corneal epithelial cell differentiation from human induced pluripotent stem cells.Stem Cell Reports2014;2(2):219-231.
16 Tseng SC, Chen SY, Shen YC, Chen WL, Hu FR. Critical appraisal of ex vivo expansion of human limbal epithelial stem cells.Curr Mol Med2010;10(9):841-850.
17 Zhang C, Du L, Pang K, Wu X. Differentiation of human embryonic stem cells into corneal epithelial progenitor cells under de fined conditions.PLoS One2017;12(8):e0183303.
18 Gonzalez G, Sasamoto Y, Ksander BR, Frank MH, Frank NY. Limbal stem cells: identity, developmental origin, and therapeutic potential.Wiley Interdiscip Rev Dev Biol2018;7(2).
19 Whitcher JP, Srinivasan M, Upadhyay MP. Corneal blindness: a global perspective.Bull World Health Organ2001;79(3):214-221.
20 Ljubimov AV, Saghizadeh M. Progress in corneal wound healing.Prog Retin Eye Res2015;49:17-45.
21 Li W, Hayashida Y, Chen YT, Tseng SC. Niche regulation of corneal epithelial stem cells at the limbus.Cell Res2007;17(1):26-36.
22 Kurpakus MA, Maniaci MT, Esco M. Expression of keratins K12, K4 and K14 during development of ocular surface epithelium.Curr Eye Res1994;13(11):805-814.
23 Hayashi Y, Watanabe N, Ohashi Y. The “replacement hypothesis”:corneal stem cell origin epithelia are replaced by limbal stem cell origin epithelia in mouse cornea during maturation.Cornea2012;31 Suppl 1:S68-S73.
24 Figueira EC, Di Girolamo N, Coroneo MT, Wakefield D. The phenotype of limbal epithelial stem cells.Invest Ophthalmol Vis Sci2007;48(1):144-156.
25 Nam SM, Maeng YS, Kim EK, Seo KY, Lew H. Ex vivo expansion of human limbal epithelial cells using human placenta-derived and umbilical cord-derived mesenchymal stem cells.Stem Cells Int2017;2017(3):4206187.
26 O’Sullivan F, Clynes M. Limbal stem cells, a review of their identi fication and culture for clinical use.Cytotechnology2007;53(1-3):101-106.
27 Wirostko B, Ra fii M, Sullivan DA, Morelli J, Ding J. Novel therapy to treat corneal epithelial defects: a hypothesis with growth hormone.Ocul Surf2015;13(3):204-12.e1.
28 Baradaran-Ra fii A, Ebrahimi M, Kanavi MR, Taghi-Abadi E, Aghdami N, Eslani M, Bakhtiari P, Einollahi B, Baharvand H, Javadi MA. Midterm outcomes of autologous cultivated limbal stem cell transplantation with or without penetrating keratoplasty.Cornea2010;29(5):502-509.
29 Pauklin M, Fuchsluger TA, Westekemper H, Steuhl KP, Meller D. Midterm results of cultivated autologous and allogeneic limbal epithelial transplantation in limbal stem cell de ficiency.Dev Ophthalmol2010;45:57-70.
30 Sabater AL, Perez VL. Amniotic membrane use for management of corneal limbal stem cell de ficiency.Curr Opin Ophthalmol2017;28(4):363-369.
31 Sangwan VS, Basu S, Vemuganti GK, Sejpal K, Subramaniam SV,Bandyopadhyay S, Krishnaiah S, Gaddipati S, Tiwari S, Balasubramanian D. Clinical outcomes of xeno-free autologous cultivated limbal epithelial transplantation: a 10-year study.Br J Ophthalmol2011;95(11):1525-1529.
32 Atallah MR, Palioura S, Perez VL, Amescua G. Limbal stem cell transplantation: current perspectives.Clin Ophthalmol2016;(10):593-602.
33 Sasamoto Y, Ksander BR, Frank MH, Frank NY. Repairing the corneal epithelium using limbal stem cells or alternative cell-based therapies.Expert Opin Biol Ther2018;18(5):505-513.
34 Nakamura T, Inatomi T, Sotozono C, Amemiya T, Kanamura N,Kinoshita S. Transplantation of cultivated autologous oral mucosal epithelial cells in patients with severe ocular surface disorders.Br J Ophthalmol2004;88(10):1280-1284.
35 Nishida K, Yamato M, Hayashida Y, Watanabe K, Maeda N, Watanabe H, Yamamoto K, Nagai S, Kikuchi A, Tano Y, Okano T. Functional bioengineered corneal epithelial sheet grafts from corneal stem cells expanded ex vivo on a temperature-responsive cell culture surface.Transplantation2004;77(3):379-385.
36 Pellegrini G, Traverso CE, Franzi AT, Zingirian M, Cancedda R,De Luca M. Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium.Lancet1997;349(9057):990-993.
37 Yuan S, Fan G. Stem cell-based therapy of corneal epithelial and endothelial diseases.Regen Med2015;10(4):495-504.
38 Ueno H, Kurokawa MS, Kayama M, Homma R, Kumagai Y, Masuda C,Takada E, Tsubota K, Ueno S, Suzuki N. Experimental transplantation of corneal epithelium-like cells induced by Pax6 gene transfection of mouse embryonic stem cells.Cornea2007;26(10):1220-1227.
39 Homma R, Yoshikawa H, Takeno M, Kurokawa MS, Masuda C, Takada E, Tsubota K, Ueno S, Suzuki N. Induction of epithelial progenitors in vitro from mouse embryonic stem cells and application for reconstruction of damaged cornea in mice.Invest Ophthalmol Vis Sci2004;45(12):4320-4326.
40 Ahmad S, Stewart R, Yung S, Kolli S, Armstrong L, Stojkovic M,Figueiredo F, Lako M. Differentiation of human embryonic stem cells into corneal epithelial-like cells by in vitro replication of the corneal epithelial stem cell niche.Stem Cells2007;25(5):1145-1155.
41 Hanson C, Hardarson T, Ellerstrom C, Nordberg M, Caisander G,Rao M, Hyllner J, Stenevi U. Transplantation of human embryonic stem cells onto a partially wounded human cornea in vitro.Acta Ophthalmol2013;91(2):127-130.
42 Hayashi R, Ishikawa Y, Ito M, Kageyama T, Takashiba K, Fujioka T,Tsujikawa M, Miyoshi H, Yamato M, Nakamura Y, Nishida K. Generation of corneal epithelial cells from induced pluripotent stem cells derived from human dermal fibroblast and corneal limbal epithelium.PLoS One2012;7(9):e45435.
43 Hewitt KJ, Shamis Y, Carlson MW, Aberdam E, Aberdam D, Garlick JA. Three-dimensional epithelial tissues generated from human embryonic stem cells.Tissue Eng Part A2009;15(11):3417-3426.
44 Mikhailova A, Jylhä A, Rieck J, Nättinen J, Ilmarinen T, Veréb Z, Aapola U, Beuerman R, Petrovski G, Uusitalo H, Skottman H.Comparative proteomics reveals human pluripotent stem cell-derived limbal epithelial stem cells are similar to native ocular surface epithelial cells.Sci Rep2015;5:14684.
45 Li F, Zhao SZ. Mesenchymal stem cells: potential role in corneal wound repair and transplantation.World J Stem Cells2014;6(3):296-304.46 Dulak J, Szade K, Szade A, Nowak W, Jozkowicz A. Adult stem cells: hopes and hypes of regenerative medicine.Acta Biochim Pol2015;62(3):329-337.
47 Kao WW, Coulson-Thomas VJ. Cell therapy of corneal diseases.Cornea2016;35(Suppl 1):S9-S19.
48 Scudellari M. How iPS cells changed the world.Nature2016;534(7607):310-312.
49 Bhattacharya S, Gangaraju R, Chaum E. Recent advances in retinal stem cell therapy.Curr Mol Biol Rep2017;3(3):172-182.
50 Cieślar-Pobuda A, Rafat M, Kno flach V, Skonieczna M, Hudecki A,Małecki A, Urasińska E, Ghavami S, Łos MJ. Human induced pluripotent stem cell differentiation and direct transdifferentiation into corneal epithelial-like cells.Oncotarget2016;7(27):42314-42329.
51 Shalom-Feuerstein R, Serror L, De La Forest Divonne S,et al. Pluripotent stem cell model reveals essential roles for miR-450b-5p and miR-184 in embryonic corneal lineage speci fication.Stem Cells2012;30(5):898-909.
52 Kawasaki H, Mizuseki K, Nishikawa S, Kaneko S, Kuwana Y,Nakanishi S, Nishikawa SI, Sasai Y. Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity.Neuron2000;28(1):31-40.
53 Kawasaki H, Suemori H, Mizuseki K,et al. Generation of dopaminergic neurons and pigmented epithelia from primate ES cells by stromal cell-derived inducing activity.Proc Natl Acad Sci U S A2002;99(3):1580-1585.
54 Sareen D, Saghizadeh M, Ornelas L, Winkler MA, Narwani K,Sahabian A, Funari VA, Tang J, Spurka L, Punj V, Maguen E, Rabinowitz YS, Svendsen CN, Ljubimov AV. Differentiation of human limbal-derived induced pluripotent stem cells into limbal-like epithelium.Stem Cells Transl Med2014;3(9):1002-1012.
55 Ordonez P, Di Girolamo N. Limbal epithelial stem cells: role of the niche microenvironment.Stem Cells2012;30(2):100-107.
56 Yu D, Chen M, Sun X, Ge J. Differentiation of mouse induced pluripotent stem cells into corneal epithelial-like cells.Cell Biol Int2013;37(1):87-94.
57 Hayashi R, Ishikawa Y, Sasamoto Y,et al. Co-ordinated ocular development from human iPS cells and recovery of corneal function.Nature2016;531(7594):376-380.
58 Hayashi R, Ishikawa Y, Katori R, Sasamoto Y, Taniwaki Y, Takayanagi H, Tsujikawa M, Sekiguchi K, Quantock AJ, Nishida K. Coordinated generation of multiple ocular-like cell lineages and fabrication of functional corneal epithelial cell sheets from human iPS cells.Nat Protoc2017;12(4):683-696.
59 Foster JW, Wahlin K, Adams SM, Birk DE, Zack DJ, Chakravarti S.Cornea organoids from human induced pluripotent stem cells.Sci Rep2017;7:41286.
60 Joseph R, Srivastava OP, Pfister RR. Modeling keratoconus using induced pluripotent stem cells.Invest Ophthalmol Vis Sci2016;57(8):3685-3697.
61 Kanemura H, Go MJ, Shikamura M, Nishishita N, Sakai N, Kamao H, Mandai M, Morinaga C, Takahashi M, Kawamata S. Tumorigenicity studies of induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) for the treatment of age-related macular degeneration.PLoS One2014;9(1):e85336.