The cornea is a highly transparent tissue covering one sixth of the anterior section of the eye with a natural barrier function that plays a vital role in the physical, biological, and immunological protection of underlying tissue from the external environment, and its structure is comprised of 3 primary cell layers, that is, the anterior epithelial layer, the middle stromal layer, and the posterior endothelial layer. In recent years, stem cell (SC)–based therapy has risen to the forefront to become a very promising and highly advanced research topic in the field of regenerative medicine. SCs are responsible for tissue homeostasis and the regeneration of organs throughout life and remain quiescent and undifferentiated for long time periods until they become activated in response to tissue damage and subsequent repair. Thus, SC research has captured the spotlight as state-of-the-art technology emerges in regenerative medicine for the treatment of devastating corneal diseases and vision-threatening ocular surface disorders. In this review, we present current advancements in successful cell-based therapeutic approaches and “SC theory” concepts for the treatment of severe corneal epithelial and endothelial diseases (Fig. 1), and provide insights on the future pathways of corneal regenerative medicine.
Research and Development of Surgical Pathways for the Treatment of Corneal and Ocular Surface Diseases
In general, research on surgical reconstruction of the cornea can be broadly divided into 2 main categories. One is the novel development of artificially constructed corneas using cutting-edge materials (eg, medical-grade polymethyl methacrylate), of which some are currently used in the clinical setting, such as the Boston keratoprosthesis (types 1 and 2) and KPro (Massachusetts Eye and Ear Infirmary, Boston, MA),1,2 an osteo-odonto keratoprosthesis surgery,3 and even more recently, artificial corneas developed via bioprinting technologies.4 An artificial corneal stroma for the treatment of keratoconus and corneal scarring,5 as well as an artificial posterior corneal surface layer, have also been experimentally applied in a few cases as substitutes for seriously damaged corneal stroma or endothelium,6 although debate remains as to the clinical relevance.
The other category involves corneal cell–based methods to reconstruct the cornea via layer-specific differentiating and proliferating corneal epithelial cells (CEpCs), corneal endothelial cells (CECs), and stromal cells, which are currently being made and have proven to be successful equivalents of fully-functional human corneal layers and the restoration of vision.7–9 In recent years, those methods have received considerable attention with the advancement and further development of cell culture systems.10 Cell-based therapies for the treatment of severe ocular diseases such as limbal SC deficiency,10,11 corneal endothelial failure (CEF) formerly termed as bullous keratopathy,12–14 and keratoconus15 have now become widely accepted throughout the world. To that end, regenerative medicine using cell-based tissue engineering techniques for the treatment of corneal diseases requires a deep understanding of SC biology and the physiology of cells.
Corneal Epithelial, Endothelial, and Stromal SCs
To fully reconstruct and regenerate a functional cornea using tissue engineering techniques, it is necessary to understand the properties of SCs and where they exist. Corneal epithelium is the most superficial layer of the cornea, and it is a nonkeratinized multilayered squamous cell epithelium. CEpCs turn over every 5 to 7 days and are supplied from basal cells, which are thought to be derived from limbal epithelium located at the border region between the cornea and the conjunctiva. It has been demonstrated that functional CEpSCs are present in the limbus, as the investigation has shown that corneal regeneration occurs when the corneal epithelium is scraped. However, when the entire corneal epithelium and bulbar conjunctival epithelium beyond the limbo-corneal junction was scraped, no reconstruction of the healthy corneal epithelium occurred.16 Furthermore, in a mouse model study, investigation revealed that cells not expressing keratin 3 were observed only in the basal cells of the limbus17 and that those cells were slow-cycling cells, which is one of the hallmarks of SCs.18 However, one opposing view is that the cells with a high capacity for proliferation can be distributed through the entire ocular surface, including even the central cornea.19 Thus, no definitive conclusions have been reached.20
The corneal stroma is mainly composed of collagen and proteoglycans. In the stromal layer, keratocytes, corneal fibroblasts, fibrocytes, or nonmyelinating Schwann cells, are significant specialized cells, as they are quiescent neural crest-derived cells that comprise the extracellular matrix in that layer. Findings from a few previous reports have demonstrated the presence of a small resident subpopulation of adult stromal SCs, primarily in the limbus.21,22 Current studies have reported that mesenchymal SCs possess the unique properties of anti-inflammation, antiangiogenesis, and immunomodulation, thus promoting wound healing and prevention of corneal scarring via paracrine effects as well as direct cell replacement.23,24 Moreover, it has been reported that bone marrow–derived SCs circulate in the corneal stroma, and are involved in immune surveillance and tissue repair.25,26
CECs are located at the posterior surface of the cornea and have limited capacity for proliferation in vivo, primarily due to cell-to-cell contact inhibition and high concentrations of transforming growth factor-beta 227 in the aqueous humor. Recent evidence demonstrates the possibility that the subpopulation of adult corneal endothelial stem cells (CESCs) or progenitor cells may reside at the peripheral region of the cornea.28,29 However, there is still ongoing debate as to whether those cells possess the property of SCs.
REGENERATIVE MEDICINE CORNEAL CELL–BASED THERAPY
In Vivo Expansion of Corneal Epithelium
Severe ocular surface diseases, such as Stevens-Johnson syndrome, ocular cicatricial pemphigoid, and thermal/chemical burns, etc, may result in CEpSC deficiency and depletion. In such cases, the lack of CEpSCs prompts the development of regenerating conjunctival epithelial cells, thus resulting in abnormal wound healing following the conjunctival invasion with corneal superficial neovascularization and scarring, which sometimes induces persistent epithelial defects, corneal ulcers, and corneal infection.30,31 The concept of treating CEpSC deficiency is to replenish the corneal epithelial cell source (ie, CEpSCs, ideally), however, mucosal epithelial SCs are possibly acceptable. To the best of our knowledge, the first successful reconstruction of the ocular surface epithelium was performed using 4 pieces of autologous contralateral bulbar conjunctival tissue as a substitute for corneal epithelium in patients afflicted with unilateral chemical burns.32 Keratoepithelioplasty, a surgical procedure involving the transplantation of allogeneic corneal superficial stromal and epithelial lenticules from donor eye, was found to result in successful corneal wound healing and recovery of vision in patients with bilateral ocular surface disorders.33 Moreover, the existence of CEpSCs in the limbus was later proposed,17 and the therapeutic concept of in vivo SC transplantation was subsequently established as limbal transplantation (LT) containing CEpSCs.34 Following those advancements, preserved human amniotic membrane (AM) was proposed as a healthy epithelial substrate, as it works to facilitate epithelial maintenance and growth by suppressing subepithelial scaring,35 thus suggesting that surgical manipulation of an epithelial-stromal combination can assist in maintaining optimal CEpSC differentiation.36 Therefore, the combination of AM and LT has enabled better visual acuity and corneal epithelial layer homeostasis to be achieved when treating refractory ocular surface disease cases,37 thus suggesting that it is optimal to reconstruct the ocular surface with an epithelial cell source as the “seeds” combined with AM as the “soil” scaffold. The various surgical transplantation procedures applied for ocular surface reconstruction have previously been extensively summarized in detail.38
Ex Vivo Expansion of Corneal Epithelium
As described above, both keratoepithelioplasty and LT can provide positive surgical outcomes in the treatment of a variety of severe ocular surface diseases. However, in autologous transplantation procedures, a significant portion of the limbal tissue of the contralateral healthy eye is required. Thus, ex vivo expansion in the format of regenerative medicine cell-based therapy is needed to generate the cultivated limbal epithelial cell sheet using tissue engineering techniques. For example, CEpSCs are cultivated from a small biopsy sample taken from the limbus of the patient’s contralateral healthy eye. Then, an autologous cultured CEpC sheet is generated and surgically transplanted onto the damaged cornea after superficial keratectomy, termed “cultivated limbal epithelial transplantation.”39,40 However, it has been found that even when host-graft rejection does not occur postsurgery, the cultured CEpC sheets often do not survive for an extended period of time. In such cases, the transplanted CEpCs were gradually replaced over time by surrounding conjunctival epithelial cells, probably due to the inability of CEpCs to sufficiently proliferate. Interestingly, a higher percentage of p63+ cells, which are assumed to be CEpSCs, reportedly contributed to the long-term survival of the cultivated CEpC sheet,41 thus suggesting clinical evidence supporting the theory of CEpSCs. At present, viable autologous human CEpC products in the band name of “Holoclar” (Holostem Terapie Avanzate srl, Modena, Italy) and “Nepic” (Nidek Co Ltd, Gamagori, Japan), respectively, are commercially available in Europe and Japan.
When corneal epithelial transplantation is performed, it is vital to always consider the source of the cells or tissues that are used, ie, autologous or allogeneic, because in cases of allogeneic transplantation, the transplanted cells/grafts can often fail due to immunological rejection. In addition, the transplantation of grafts involving the use of CEpSCs is essential when treating difficult bilateral ocular surface disorder cases, especially in patients afflicted with severe ocular surface diseases such as Stevens-Johnson syndrome, toxic epidermal necrolysis, ocular cicatricial pemphigoid, and chemical injuries.42 Moreover, in such cases, it is vital that effective immunosuppressive agents be administered over the long-term period postsurgery. Furthermore, the gene manipulation of allogeneic CEpCs that are not responding as an alloantigen (eg, human leukemia antigen) using the clustered regularly interspaced short palindromic repeats and clustered regularly interspaced short palindromic repeats–associated protein 9 system, needs to be deeply investigated in future studies.
It should be noted that oral mucosal epithelial cells have reportedly been utilized as an alternative cell source to autologous CEpCs.43–45 Interestingly, when directly transplanted, in vivo oral mucosal epithelium fails to survive with transparency on the ocular surface due to the fact that the biological characteristics of oral mucosal epithelial cells differ from that of CEpCs.46 However, cultured oral mucosal epithelial cells mimic the biological characteristics of in vivo corneal epithelium,43 and oral mucosal epithelial cell sheets cultured on AM44 or temperature-responsive dishes47 have been found to function properly posttransplantation in patients afflicted with severe bilateral ocular surface disease (Fig. 2). Long-term clinical data has revealed that cultivated oral mucosal epithelial transplantation (COMET) results in improved best-corrected visual acuity,48,49 even in end-stage severe ocular surface disease cases with complete limbal SC deficiency and severe dry eye (Fig. 3).51,52 Moreover, post-COMET, an additional penetrating keratoplasty operation can result in a better visual outcome due to the remaining autologous oral mucosal epithelium helping to maintain the ocular surface.53 In addition to visual recovery, COMET substitutes corneal epithelium and acts as a nonkeratinized epithelial layer to protect the ocular surface against the subsequent scarring and the symblepharon/fornix shortening over the long-term postoperative period.49 Of interest, a recent report has proposed a more direct method for addressing limbal SC deficiency and reconstruction of the ocular surface, termed “simple limbal epithelial transplantation” (SLET), which involves the transplantation of a small amount of autologous limbal epithelial tissue containing SCs obtained from the contralateral healthy eye on the exposed corneal stroma.54 SLET does not require the use of a highly sterile cell culture room in the cell processing center unit to create the cultured epithelial sheet used in the procedure. Thus, costs are saved when producing the cultured epithelial sheet used for SLET, which makes the procedure more widely applicable. However, it should be noted that SLET is based on autologous transplantation, and although challenging, allogeneic SLET has been performed.
Another cell source is human CEpCs differentiated from human induced pluripotent stem cells (iPSCs),55 which resemble the character of embryonic SCs. iPSCs are capable of inducing autologous cell sources from patients or allogeneic cell sources from donors. When taking into consideration the time and cost that is required to differentiate autologous human CEpCs from iPSCs derived from the patients, it might be feasible that allograft epithelial transplants could be matched at the human leukocyte antigen class to reduce the risk of allograft rejection.56 Another concept is to directly reprogram the CEpCs from other ocular surface cell sources, such as conjunctival cells or fibroblastic cells, by gene manipulation using the cocktails of Yamanaka factors and other transcription factors,57,58 as shown in the skin.59 Furthermore, nonkeratinized epithelial cells with mucosal functions, such as nasal epithelial cells, could be applicable for making mucosal epithelial sheets for use in the clinical setting for improvement of ocular surface wettability.60
Corneal Endothelial Reconstruction
Unlike corneal epithelium, which is a multilayered cell structure, the corneal endothelium is a more simplified layer of single cells that lines the posterior surface of the cornea. Moreover, unlike CEpCs, in vivo CECs are arrested in the G1 phase of the cell cycle and rarely proliferate due to cell-to-cell contact inhibition and a high concentration of transforming growth factor-beta 2 in the aqueous humor.27,61–64 Thus, cell migration and cell enlargement more likely occur in response to wounding repair than cell division.65 In corneal endothelium, the density of CECs slowly decreases with age, even in healthy subjects, and corneal endothelial dysfunction occurs due to an impairment of pump function and barrier function due to the abnormality of CECs and/or a decrease of CEC density in corneal endothelial dystrophies such as Fuchs endothelial corneal dystrophy, intraocular surgery–induced corneal endothelial trauma, etc, eventually resulting in complete CEF. It has been proposed that human CESCs may be present primarily in the peripheral cornea near the limbus, yet there is no definitive evidence supporting the presence of SCs. Furthermore, since corneal endothelial transplantation using a central cornea donor graft can sufficiently restore corneal transparency in cases of CEF for a substantial period of time, and since the transplantation in vivo CECs does not require constant cell proliferation, surgical strategies for the treatment of corneal endothelial dysfunction must be considered differently from those used for the treatment of limbal deficiency with CEpSC depletion.
With the development of corneal endothelial transplantation, the surgical strategy used for the treatment of corneal endothelial dysfunction has shifted from penetrating corneal transplantation to corneal endothelial transplantation, which restores only the corneal endothelial tissue that is damaged. These transplantation procedures are posterior lamellar keratoplasty,66 deep lamellar endothelial keratoplasty,67 Descemetorhexis,68 Descemet stripping with endothelial keratoplasty,69 Descemet stripping automated endothelial keratoplasty,70,71 Descemet membrane endothelial keratoplasty,72 and more recently, Descemet membrane transplantation73 or Descemetorhexis without endothelial keratoplasty/Descemet stripping only,74,75 which strips away the diseased CECs along with the Descemet membrane with cornea guttae in mild Fuchs endothelial corneal dystrophy cases. These concepts are based on the replenishment of CECs that are lost.
Recently, a great amount of interest in corneal endothelial research has focused on whether human corneal endothelial cells (hCECs) can expand in cell culture from in vivo CECs. The concept of CEC culture was first attempted in the late 1970s using rabbit corneas,76 which later expanded to human corneas.77 The use of an animal-derived extracellular matrix promoted the proliferative activity of hCECs in culture, however, the amount of proliferation was limited.78–82 In 2009, it was discovered that inhibition of Rho-associated protein kinase (ROCK) signaling enhances the adhesion and proliferation of cultured CECs and suppresses their apoptosis in cynomolgus monkeys,83 which subsequently led to the development of proper procedures for the culture of hCECs.
With a concept similar to that used for the production of the cultivated epithelial cell sheet, monkey endothelial cells were cultured on a type 1 collagen sheet, which was then transplanted into bullous keratopathy monkey-model eyes, and the successful recovery of corneal clarity84 showed that the cell therapy works properly in the corneal endothelium of nonhuman primates. More interestingly, the injection of cultured CECs with Y-27632, a selective ROCK inhibitor, eliminates the requirement of using any type of cell carrier, as studies revealed that the CEC suspension with ROCK inhibitor used for CEC injection therapy reconstructs the corneal endothelial layer in both rabbit and nonhuman primate models.85,86 Thus, those important findings led to subsequent human clinical trial (Fig. 4A–F),87 with the cultured hCEC injection therapy being found both safe and effective for the treatment of CEF and restoration of vision in the initial 11 cases87 for up to 5 years postoperative (Fig. 5A, B).88 In recent years, the concept of magnetizing CECs with nanoparticles has been proposed as an alternative way to attach them to the posterior surface of the cornea89 and some studies have reported the development of other types of cell expansion90–92 and non–cell-expansion delivery methods.93
For the treatment of CEF, the use of mature-differentiated cultured hCECs when performing hCEC injection therapy is considered important for obtaining successful clinical results, and the findings in the initial clinical trial confirmed this hypothesis. The mature-differentiated cultured hCECs express distinct cell surface markers such as CD166+, CD44−/dull, CD24−, CD26−, and CD105−/dull, which mimic those of in vivo CECs in the healthy human cornea.94 Peroxiredoxin 6 also seems to be specific in this regard.95 Mature-differentiated cultured hCECs possess better physiological function than immature-cultured hCECs, which often develop cell-state transition, including endothelial mesenchymal transition.94,96,97 To that regard, it was discovered that mature-cultured hCECs produced from young-age donor corneas tend to prevent senescence phenotype and cell-state transition cells, which may be an additional factor for successful hCEC injection therapy. ROCK inhibition also alleviates senescence-associated phenotype98 and promotes the maturity of hCECs,94 which supports the recent findings99 suggesting that the inhibition of ROCK signaling after a procedure (eg, Descemet stripping only or the acellular Descemet membrane transplantation) restores functionally to some extent in the corneal endothelium of Fuchs endothelial corneal dystrophy patients12 and that cultured hCEC injection with ROCK inhibitor results in a successful surgical outcome.87,88
It should be noted that cultured hCECs in a heterogeneous cell size differentially express surface markers from mature hCECs,96,97 which results in complete reprogramming of cellular metabolism.100,101 Reportedly, distinct metabolic changes lead to an inefficient method of energy production in the mitochondria, thus resulting in a low endothelial pump function.102 In fact, mitochondrial dysfunction and imbalance of oxidant-antioxidant are implicated in the pathogenesis of Fuchs endothelial corneal dystrophy.103,104 In other words, the mitochondrial respiratory function in low-mature cultured hCECs is altered, and the intracellular metabolic profile is reprogrammed toward mitochondrial glycolysis, thus resulting in the CECs producing adenosine triphosphate in an inefficient way that can impact the longevity and function of the cells.101,102
In previous studies, human embryonic SCs or iPSC-derived CECs have also been used as an alternative endothelial cell source,105,106 however, the question remains unanswered as to whether of not that produces cells of equal quality. Since Descemet membrane endothelial keratoplasty has less probability of allograft rejection than Descemet stripping automated endothelial keratoplasty and penetrating keratoplasty due to the low burden of graft cell sources, and since allogeneic CECs introduced into the anterior chamber are supposedly not recognized as an alloantigen from the host,107 the risk of allograft rejection in patients undergoing cultured hCEC injection therapy, in theory, is considered very low.
Different Concepts Applied for the Treatment of Corneal Epithelium and Endothelium
Although regenerative medicine cell therapy applied for the treatment of the damaged tissue works via the regeneration and reorganization of the healthy tissue being functionally restored, it can be assumed that the cell-therapy strategies used for the treatment of severe ocular surface diseases and corneal endothelial dysfunction differ. In severe ocular surface diseases, the loss of CEpSCs induces rather poor cell proliferation and abnormal cell differentiation, thus resulting in abnormal ocular surface homeostasis and wound healing. In other words, constant cell proliferation is essential for maintaining healthy ocular surface epithelium. This idea supports the concept that the use of CEpC sheets containing more CEpSCs, or their equivalents, contribute to long-term graft survival.41 In contrast, corneal endothelial dysfunction results from the loss of the physiological function of the CEC layer due to depletion or malfunction of the CECs themselves, not due to CESC deficiency. Thus, and as the findings in several in vivo studies have indicated, there is no need for CESCs or progenitor cells once cultured hCECs are introduced into the posterior corneal surface. In fact, the extended longevity and good cellular function of CECs, not a proliferative activity, is more likely to contribute to sufficient long-term graft survival even in standard corneal endothelial transplantations.108,109
Corneal regenerative medicine using cell-based therapy for refractory corneal diseases has long been one of the significant challenges for ophthalmologists worldwide. With the progress of basic research in tissue engineering, including regenerative medicine cell therapy, the understanding and development of new therapies such as cultivated limbal epithelial transplantation and COMET has significantly advanced. As for corneal endothelium, ROCK inhibitor has opened a new chapter in the culture of hCECs, and recent findings now illustrate that there is a heterogeneous population in cultured hCECs, which display different cell morphologies, cell surface markers, and cellular metabolism and function. Among those aspects, mature-differentiated cultured hCECs are key for successful cultured hCEC injection therapy.
In closing, unlike hematopoietic SCs, it still remains unclear as to what is responsible for the longevity of CEpSCs and CECs, so a deeper understanding of cell biology is increasingly essential for the further development of corneal cell therapies with a reasonably cost-effective approach.
1. Ahmad S, Mathews PM, Lindsley K, et al. Boston type 1 keratoprosthesis versus repeat donor keratoplasty for corneal graft failure: a systematic review and meta-analysis. Ophthalmology. 2016;123:165–177.
2. Srikumaran D, Munoz B, Aldave AJ, et al. Long-term outcomes of boston type 1 keratoprosthesis implantation: a retrospective multicenter cohort. Ophthalmology. 2014;121:2159–2164.
3. Falcinelli G, Falsini B, Taloni M, et al. Modified osteo-odonto-keratoprosthesis for treatment of corneal blindness: long-term anatomical and functional outcomes in 181 cases. Arch Ophthalmol. 2005;123:1319–1329.
4. Isaacson A, Swioklo S, Connon CJ. 3D bioprinting of a corneal stroma equivalent. Exp Eye Res. 2018;173:188–193.
5. Fagerholm P, Lagali NS, Ong JA, et al. Stable corneal regeneration four years after implantation of a cell-free recombinant human collagen scaffold. Biomaterials. 2014;35:2420–2427.
6. Auffarth GU, Son HS, Koch M, et al. Implantation of an artificial endothelial layer for treatment of chronic corneal edema. Cornea. 2021;40:1633–1638.
7. Griffith M, Osborne R, Munger R, et al. Functional human corneal equivalents constructed from cell lines. Science. 1999;286:2169–2172.
8. Zieske JD, Mason VS, Wasson ME, et al. Basement membrane assembly and differentiation of cultured corneal cells: importance of culture environment and endothelial cell interaction. Exp Cell Res. 1994;214:621–633.
9. McKay TB, Hutcheon AEK, Guo X, et al. Modeling the cornea in 3-dimensions: current and future perspectives. Exp Eye Res. 2020;197:108127.
10. Nakamura T, Inatomi T, Sotozono C, et al. Ocular surface reconstruction using stem cell and tissue engineering. Prog Retin Eye Res. 2016;51:187–207.
11. Nakamura T, Inatomi T, Sotozono C, et al. Recent advances and future challenges in ocular surface reconstruction: on the road to translational medicine. Asia Pac J Ophthalmol (Phila). 2012;1:28–34.
12. Kinoshita S, Colby KA, Kruse FE. A close look at the clinical efficacy of Rho-associated protein kinase inhibitor eye drops for fuchs endothelial corneal dystrophy. Cornea. 2021;40:1225–1228.
13. Faye PA, Poumeaud F, Chazelas P, et al. Focus on cell therapy to treat corneal endothelial diseases. Exp Eye Res. 2021;204:108462.
14. Catala P, Thuret G, Skottman H, et al. Approaches for corneal endothelium regenerative medicine. Prog Retin Eye Res. 2021;87:100987.
15. El Zarif M, Alio JL, Alio Del Barrio JL, et al. Corneal stromal regeneration therapy for advanced keratoconus: long-term outcomes at 3 years. Cornea. 2021;40:741–754.
16. Kinoshita S, Kiorpes TC, Friend J, et al. Limbal epithelium in ocular surface wound healing. Invest Ophthalmol Vis Sci. 1982;23:73–80.
17. Schermer A, Galvin S, Sun TT. Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells. J Cell Biol. 1986;103:49–62.
18. Cotsarelis G, Cheng SZ, Dong G, et al. Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: implications on epithelial stem cells. Cell. 1989;57:201–209.
19. Majo F, Rochat A, Nicolas M, et al. Oligopotent stem cells are distributed throughout the mammalian ocular surface. Nature. 2008;456:250–254.
20. Kinoshita S. The corneal epithelial stem cell puzzle: what future discoveries lie on the horizon? Arch Ophthalmol. 2008;126:725–726.
21. Du Y, Funderburgh ML, Mann MM, et al. Multipotent stem cells in human corneal stroma. Stem Cells. 2005;23:1266–1275.
22. Du Y, Sundarraj N, Funderburgh ML, et al. Secretion and organization of a cornea-like tissue in vitro by stem cells from human corneal stroma. Invest Ophthalmol Vis Sci. 2007;48:5038–5045.
23. Basu S, Hertsenberg AJ, Funderburgh ML, et al. Human limbal biopsy-derived stromal stem cells prevent corneal scarring. Sci Transl Med. 2014;6:266ra172.
24. Branch MJ, Hashmani K, Dhillon P, et al. Mesenchymal stem cells in the human corneal limbal stroma. Invest Ophthalmol Vis Sci. 2012;53:5109–5116.
25. Sosnova M, Bradl M, Forrester JV. CD34+ corneal stromal cells are bone marrow-derived and express hemopoietic stem cell markers. Stem Cells. 2005;23:507–515.
26. Nakamura T, Ishikawa F, Sonoda KH, et al. Characterization and distribution of bone marrow-derived cells in mouse cornea. Invest Ophthalmol Vis Sci. 2005;46:497–503.
27. Joyce N. Proliferative capacity of the corneal endothelium. Prog Retin Eye Res. 2003;22:359–389.
28. Hirata-Tominaga K, Nakamura T, Okumura N, et al. Corneal endothelial cell fate is maintained by LGR5 through the regulation of hedgehog and Wnt pathway. Stem Cells. 2013;31:1396–1407.
29. Yam GH, Seah X, Yusoff N, et al. Characterization of human transition zone reveals a putative progenitor-enriched niche of corneal endothelium. Cells. 2019:8.
30. Deng SX, Borderie V, Chan CC, et al. Global consensus on definition, classification, diagnosis, and staging of limbal stem cell deficiency. Cornea. 2019;38:364–375.
31. Deng SX, Kruse F, Gomes JAP, et al. Global consensus on the management of limbal stem cell deficiency. Cornea. 2020;39:1291–1302.
32. Thoft RA. Conjunctival transplantation. Arch Ophthalmol. 1977;95:1425–1427.
33. Thoft RA. Keratoepithelioplasty. Am J Ophthalmol. 1984;97:1–6.
34. Kenyon KR, Tseng SC. Limbal autograft transplantation for ocular surface disorders. Ophthalmology. 1989;96:709–722; discussion 722–723.
35. Kim JC, Tseng SC. Transplantation of preserved human amniotic membrane for surface reconstruction in severely damaged rabbit corneas. Cornea. 1995;14:473–484.
36. Friend J, Kinoshita S, Thoft RA, et al. Corneal epithelial cell cultures on stromal carriers. Invest Ophthalmol Vis Sci. 1982;23:41–49.
37. Tsubota K, Satake Y, Kaido M, et al. Treatment of severe ocular-surface disorders with corneal epithelial stem-cell transplantation. N Engl J Med. 1999;340:1697–1703.
38. Daya SM, Chan CC, Holland EJ, et al. Cornea Society nomenclature for ocular surface rehabilitative procedures. Cornea. 2011;30:1115–1119.
39. Pellegrini G, Traverso CE, Franzi AT, et al. Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium. Lancet. 1997;349:990–993.
40. Koizumi N, Inatomi T, Suzuki T, et al. Cultivated corneal epithelial stem cell transplantation in ocular surface disorders. Ophthalmology. 2001;108:1569–1574.
41. Rama P, Matuska S, Paganoni G, et al. Limbal stem-cell therapy and long-term corneal regeneration. N Engl J Med. 2010;363:147–155.
42. Sotozono C, Inatomi T, Nakamura T, et al. Oral mucosal epithelial transplantation and limbal-rigid contact lens: a therapeutic modality for the treatment of severe ocular surface disorders. Cornea. 2020;39(suppl 1):S19–S27.
43. Nakamura T, Endo K, Cooper LJ, et al. The successful culture and autologous transplantation of rabbit oral mucosal epithelial cells on amniotic membrane. Invest Ophthalmol Vis Sci. 2003;44:106–116.
44. Nakamura T, Inatomi T, Sotozono C, et al. Transplantation of cultivated autologous oral mucosal epithelial cells in patients with severe ocular surface disorders. Br J Ophthalmol. 2004;88:1280–1284.
45. Inatomi T, Nakamura T, Koizumi N, et al. Midterm results on ocular surface reconstruction using cultivated autologous oral mucosal epithelial transplantation. Am J Ophthalmol. 2006;141:267–275.
46. Gipson IK, Geggel HS, Spurr-Michaud SJ. Transplant of oral mucosal epithelium to rabbit ocular surface wounds in vivo. Arch Ophthalmol. 1986;104:1529–1533.
47. Nishida K, Yamato M, Hayashida Y, et al. Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium. N Engl J Med. 2004;351:1187–1196.
48. Nakamura T, Takeda K, Inatomi T, et al. Long-term results of autologous cultivated oral mucosal epithelial transplantation in the scar phase of severe ocular surface disorders. Br J Ophthalmol. 2011;95:942–946.
49. Komai S, Inatomi T, Nakamura T, et al. Long-term outcome of cultivated oral mucosal epithelial transplantation for fornix reconstruction in chronic cicatrising diseases. Br J Ophthalmol. 2021. [Epub ahead of print].
50. Sotozono C, Ang LP, Koizumi N, et al. New grading system for the evaluation of chronic ocular manifestations in patients with Stevens-Johnson syndrome. Ophthalmology. 2007;114:1294–1302.
51. Sotozono C, Inatomi T, Nakamura T, et al. Visual improvement after cultivated oral mucosal epithelial transplantation. Ophthalmology. 2013;120:193–200.
52. Sotozono C, Inatomi T, Nakamura T, et al. Cultivated oral mucosal epithelial transplantation for persistent epithelial defect in severe ocular surface diseases with acute inflammatory activity. Acta Ophthalmol. 2014;92:e447–e453.
53. Inatomi T, Nakamura T, Kojyo M, et al. Ocular surface reconstruction with combination of cultivated autologous oral mucosal epithelial transplantation and penetrating keratoplasty. Am J Ophthalmol. 2006;142:757–764.
54. Basu S, Sureka SP, Shanbhag SS, et al. Simple limbal epithelial transplantation: long-term clinical outcomes in 125 cases of unilateral chronic ocular surface burns. Ophthalmology. 2016;123:1000–1010.
55. Hayashi R, Ishikawa Y, Sasamoto Y, et al. Co-ordinated ocular development from human iPS cells and recovery of corneal function. Nature. 2016;531:376–380.
56. Khaireddin R, Wachtlin J, Hopfenmuller W, et al. HLA-A, HLA-B and HLA-DR matching reduces the rate of corneal allograft rejection. Graefes Arch Clin Exp Ophthalmol. 2003;241:1020–1028.
57. Kitazawa K, Hikichi T, Nakamura T, et al. OVOL2 maintains the transcriptional program of human corneal epithelium by suppressing epithelial-to-mesenchymal transition. Cell Rep. 2016;15:1359–1368.
58. Ouyang H, Xue Y, Lin Y, et al. WNT7A and PAX6 define corneal epithelium homeostasis and pathogenesis. Nature. 2014;511:358–361.
59. Kurita M, Araoka T, Hishida T, et al. In vivo reprogramming of wound-resident cells generates skin epithelial tissue. Nature. 2018;561:243–247.
60. Kobayashi M, Nakamura T, Yasuda M, et al. Ocular surface reconstruction with a tissue-engineered nasal mucosal epithelial cell sheet for the treatment of severe ocular surface diseases. Stem Cells Transl Med. 2015;4:99–109.
61. Chen KH, Harris DL, Joyce NC. TGF-beta2 in aqueous humor suppresses S-phase entry in cultured corneal endothelial cells. Invest Ophthalmol Vis Sci. 1999;40:2513–2519.
62. Joyce NC, Navon SE, Roy S, et al. Expression of cell cycle-associated proteins in human and rabbit corneal endothelium in situ. Invest Ophthalmol Vis Sci. 1996;37:1566–1575.
63. Joyce NC, Meklir B, Joyce SJ, et al. Cell cycle protein expression and proliferative status in human corneal cells. Invest Ophthalmol Vis Sci. 1996;37:645–655.
64. Joyce NC, Harris DL, Mello DM. Mechanisms of mitotic inhibition in corneal endothelium: contact inhibition and TGF-beta2. Invest Ophthalmol Vis Sci. 2002;43:2152–2159.
65. Laing RA, Sanstrom MM, Berrospi AR, et al. Changes in the corneal endothelium as a function of age. Exp Eye Res. 1976;22:587–594.
66. Melles GR, Eggink FA, Lander F, et al. A surgical technique for posterior lamellar keratoplasty. Cornea. 1998;17:618–626.
67. Terry MA, Ousley PJ. Deep lamellar endothelial keratoplasty in the first United States patients: early clinical results. Cornea. 2001;20:239–243.
68. Melles GR, Wijdh RH, Nieuwendaal CP. A technique to excise the descemet membrane from a recipient cornea (descemetorhexis). Cornea. 2004;23:286–288.
69. Price FW Jr, Price MO. Descemet’s stripping with endothelial keratoplasty in 50 eyes: a refractive neutral corneal transplant. J Refract Surg. 2005;21:339–345.
70. Gorovoy MS. Descemet-stripping automated endothelial keratoplasty. Cornea. 2006;25:886–889.
71. Melles GR. Posterior lamellar keratoplasty: DLEK to DSEK to DMEK. Cornea. 2006;25:879–881.
72. Melles GR, Ong TS, Ververs B, et al. Descemet membrane endothelial keratoplasty (DMEK). Cornea. 2006;25:987–990.
73. Soh YQ, Mehta JS. Regenerative therapy for Fuchs endothelial corneal dystrophy. Cornea. 2018;37:523–527.
74. Kaufman AR, Nose RM, Pineda R II. Descemetorhexis without endothelial keratoplasty (DWEK): proposal for nomenclature standardization. Cornea. 2018;37:e20–e21.
75. Borkar DS, Veldman P, Colby KA. Treatment of Fuchs endothelial dystrophy by Descemet stripping without endothelial keratoplasty. Cornea. 2016;35:1267–1273.
76. Jumblatt MM, Maurice DM, McCulley JP. Transplantation of tissue-cultured corneal endothelium. Invest Ophthalmol Vis Sci. 1978;17:1135–1141.
77. Baum JL, Niedra R, Davis C, et al. Mass culture of human corneal endothelial cells. Arch Ophthalmol. 1979;97:1136–1140.
78. Miyata K, Drake J, Osakabe Y, et al. Effect of donor age on morphologic variation of cultured human corneal endothelial cells. Cornea. 2001;20:59–63.
79. Engelmann K, Bohnke M, Friedl P. Isolation and long-term cultivation of human corneal endothelial cells. Invest Ophthalmol Vis Sci. 1988;29:1656–1662.
80. Zhu C, Joyce NC. Proliferative response of corneal endothelial cells from young and older donors. Invest Ophthalmol Vis Sci. 2004;45:1743–1751.
81. Wilson SE, Lloyd SA. Epidermal growth factor and its receptor, basic fibroblast growth factor, transforming growth factor beta-1, and interleukin-1 alpha messenger RNA production in human corneal endothelial cells. Invest Ophthalmol Vis Sci. 1991;32:2747–2756.
82. Blake DA, Yu H, Young DL, et al. Matrix stimulates the proliferation of human corneal endothelial cells in culture. Invest Ophthalmol Vis Sci. 1997;38:1119–1129.
83. Okumura N, Ueno M, Koizumi N, et al. Enhancement on primate corneal endothelial cell survival in vitro by a ROCK inhibitor. Invest Ophthalmol Vis Sci. 2009;50:3680–3687.
84. Koizumi N, Sakamoto Y, Okumura N, et al. Cultivated corneal endothelial cell sheet transplantation in a primate model. Invest Ophthalmol Vis Sci. 2007;48:4519–4526.
85. Okumura N, Koizumi N, Ueno M, et al. ROCK inhibitor converts corneal endothelial cells into a phenotype capable of regenerating in vivo endothelial tissue. Am J Pathol. 2012;181:268–277.
86. Okumura N, Sakamoto Y, Fujii K, et al. Rho kinase inhibitor enables cell-based therapy for corneal endothelial dysfunction. Sci Rep. 2016;6:26113.
87. Kinoshita S, Koizumi N, Ueno M, et al. Injection of cultured cells with a rock inhibitor for bullous keratopathy. N Engl J Med. 2018;378:995–1003.
88. Numa K, Imai K, Ueno M, et al. Five-year follow-up of first eleven cases undergoing injection of cultured corneal endothelial cells for corneal endothelial failure
. Ophthalmology. 2020.
89. Xia X, Atkins M, Dalal R, et al. Magnetic human corneal endothelial cell transplant: delivery, retention, and short-term efficacy. Invest Ophthalmol Vis Sci. 2019;60:2438–2448.
90. Peh GSL, Ong HS, Adnan K, et al. Functional evaluation of two corneal endothelial cell-based therapies: tissue-engineered construct and cell injection. Sci Rep. 2019;9:6087.
91. Frausto RF, Swamy VS, Peh GSL, et al. Phenotypic and functional characterization of corneal endothelial cells during in vitro expansion. Sci Rep. 2020;10:7402.
92. Arnalich-Montiel F, Moratilla A, Fuentes-Julian S, et al. Treatment of corneal endothelial damage in a rabbit model with a bioengineered graft using human decellularized corneal lamina and cultured human corneal endothelium. PLoS One. 2019;14:e0225480.
93. Ong HS, Peh G, Neo DJH, et al. A novel approach of harvesting viable single cells from donor corneal endothelium for cell-injection therapy. Cells. 2020:9.
94. Hamuro J, Toda M, Asada K, et al. Cell homogeneity indispensable for regenerative medicine by cultured human corneal endothelial cells. Invest Ophthalmol Vis Sci. 2016;57:4749–4761.
95. Dorfmueller S, Tan HC, Ngoh ZX, et al. Isolation of a recombinant antibody specific for a surface marker of the corneal endothelium by phage display. Sci Rep. 2016;6:21661.
96. Toda M, Ueno M, Yamada J, et al. The different binding properties of cultured human corneal endothelial cell subpopulations to Descemet’s membrane components. Invest Ophthalmol Vis Sci. 2016;57:4599–4605.
97. Toda M, Ueno M, Hiraga A, et al. Production of homogeneous cultured human corneal endothelial cells indispensable for innovative cell therapy. Invest Ophthalmol Vis Sci. 2017;58:2011–2020.
98. Park JT, Kang HT, Park CH, et al. A crucial role of ROCK for alleviation of senescence-associated phenotype. Exp Gerontol. 2018;106:8–15.
99. Schlotzer-Schrehardt U, Zenkel M, Strunz M, et al. Potential functional restoration of corneal endothelial cells in fuchs endothelial corneal dystrophy by ROCK inhibitor (ripasudil). Am J Ophthalmol. 2021;224:185–199.
100. Hamuro J, Ueno M, Asada K, et al. Metabolic plasticity in cell state homeostasis and differentiation of cultured human corneal endothelial cells. Invest Ophthalmol Vis Sci. 2016;57:4452–4463.
101. Hamuro J, Numa K, Fujita T, et al. Metabolites interrogation in cell fate decision of cultured human corneal endothelial cells. Invest Ophthalmol Vis Sci. 2020;61:10.
102. Numa K, Ueno M, Fujita T, et al. Mitochondria as a platform for dictating the cell fate of cultured human corneal endothelial cells. Invest Ophthalmol Vis Sci. 2020;61:10.
103. Liu C, Miyajima T, Melangath G, et al. Ultraviolet a light induces DNA damage and estrogen-DNA adducts in Fuchs endothelial corneal dystrophy causing females to be more affected. Proc Natl Acad Sci USA. 2020;117:573–583.
104. Zhu AY, Eberhart CG, Jun AS. Fuchs endothelial corneal dystrophy: a neurodegenerative disorder? JAMA Ophthalmol. 2014;132:377–378.
105. Hatou S, Sayano T, Higa K, et al. Transplantation of iPSC-derived corneal endothelial substitutes in a monkey corneal edema model. Stem Cell Res. 2021;55:102497.
106. Ali M, Khan SY, Gottsch JD, et al. Pluripotent stem cell-derived corneal endothelial cells as an alternative to donor corneal endothelium in keratoplasty. Stem Cell Reports. 2021;16:2320–2335.
107. Yamada J, Ueno M, Toda M, et al. Allogeneic sensitization and tolerance induction after corneal endothelial cell transplantation in mice. Invest Ophthalmol Vis Sci. 2016;57:4572–4580.
108. Baydoun L, Muller T, Lavy I, et al. Ten-year clinical outcome of the first patient undergoing descemet membrane endothelial keratoplasty. Cornea. 2017;36:379–381.
109. Price MO, Calhoun P, Kollman C, et al. Descemet stripping endothelial keratoplasty: ten-year endothelial cell loss compared with penetrating keratoplasty. Ophthalmology. 2016;123:1421–1427.