Concept and Clinical Application of Cultivated Epithelial Transplantation for Ocular Surface Disorders

Clinical Science GARY N. FOULKS, MD, SECTION EDITOR

Concept and Clinical Application of Cultivated Epithelial Transplantation for Ocular Surface Disorders SHIGERU KINOSHITA, MD, PHD, NORIKO KOIZUMI, MD, PHD, CHIE SOTOZONO, MD, PHD, JUN YAMADA, MD, PHD, TAKAHIRO NAKAMURA, MD, PHD, TSUTOMU INATOMI, MD, PHD ABSTRACT Corneal epithelial replacement using a tissue engineering technique holds much promise for ocular surface reconstruction in cases of corneal epithelial stem cell deficiency. However, even though an autologous cultivated corneal epithelial stem cell sheet is the safest and most reliable form of sheet, bilaterally affected ocular surface disorders cannot be treated by this method. To treat bilateral cases, we must choose either an allogeneic cultivated corneal epithelial sheet or an autologous cultivated oral mucosal epithelial sheet. In the case of the former, immunological reaction is a threat. Thus, understanding of the immunological background of ocular surface reconstruction using allogeneic tissues is essential. In the case of the latter, the transplanted sheet is not exactly the same as corneal epithelium, and understanding ocular surface epithelial biology is important. In this review, we summarize and explain the concept and clinical application of cultivated mucosal epithelial transplantation for ocular surface disorders. KEY WORDS amniotic membrane, cultivated corneal epithelial sheet, ocular surface reconstruction, oral mucosal epithelium, stem cell

Accepted for publication December 2003. From the Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan. Supported by grants from the Japanese Ministry of Health and Welfare and the Japanese Ministry of Education, Tokyo, the Kyoto Foundation for the Promotion of Medical Science, the Intramural Research Fund of the Kyoto Prefectural University of Medicine. The authors have no proprietary interest in any product or concept discussed in this article. Single copy reprint requests to: Shigeru Kinoshita, MD, PhD (address below). Abbreviations are printed in boldface where they first appear with their definitions. Corresponding author: Shigeru Kinoshita, MD, PhD, Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kawaramachihirokoji, Kamigyo-ku, Kyoto 602-0841, Japan. Tel: 81-75-251-5577, Fax: 81-75-251-5663. e-mail:[email protected] ©2004 Ethis Communications, Inc. All rights reserved.

I. INTRODUCTION he concept of the ocular surface as an integrated unit, proposed by Thoft and Friend in 1977,1 has been accepted by cornea specialists and visual scientists who are endeavoring to understand the physiology, molecular and cellular biology of the ocular surface, and the immunopathology of ocular surface disorders. On the basis of several scientific discoveries over the past 20 years, such as the identification of corneal epithelial stem cells, the establishment of novel methods of epithelial culturing, and the understanding of extracellular matrices, ocular surface disorders can now be treated with surgical approaches that employ regenerative medicine. Regenerative medicine via tissue engineering2 uses somatic stem cells to generate biological substitutes and improve tissue functions. The success of this approach depends on two important factors: stem cells and the extracellular matrices. We are now able to produce cultivated corneal or cornea-like epithelial sheets in vitro and transplant them onto ocular surfaces that have been severely affected by disease. The process is called cultivated corneal epithelial stem cell transplantation, also known as ex vivo expansion of corneal epithelial stem cells. In this review, we summarize and explain the concept and clinical application of cultivated mucosal epithelial transplantation for ocular surface disorders.

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II. CONCEPT OF CELLULAR SURGERY FOR OCULAR SURFACE RECONSTRUCTION A. History and Biological Aspects

In 1977, Thoft described conjunctival transplantation for unilaterally affected chemical injuries. The surgery consisted of removing scarred tissue from the corneal surface and placing four pieces of conjunctival autograft taken from the patient’s contralateral eye at the limbus, in order to resurface the cornea by regenerating conjunctival epithelial cells from these autografts.3 To our knowledge, this was the first report regarding the modern concept of cellular surgery in ocular surface reconstruction. In 1984, Thoft described a similar surgical technique called keratoepithelioplasty, using a different tissue source—donor corneal lenticules—to regenerate corneal epithelial cells.4

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CULTIVATED EPITHELIAL TRANSPLANTATION / Kinoshita, et al OUTLINE I. Introduction II. Concept of cellular surgery in ocular surface reconstruction A. History and biological background B. Immunological aspects 1. Immunological differences between limbal allograft transplantation and penetrating keratoplasty 2. Basic mechanism of limbal allograft rejection 3. Use of immunosuppressive agents 4. Immunological strategy to promote limbal allograft survival C. What is clinical success? III. Transplantation of cultivated mucosal epithelial cell sheets A. History of creating mucosal epithelial sheets B. Transplantation of cultivated corneal epithelial stem cell sheets 1. Biological aspects 2. Immunological aspects 3. Clinical outcomes C. Transplantation of cultivated oral mucosal epithelial sheets 1. Animal experiments 2. Preliminary clinical experience IV. Conclusion and future goals

Although the concept of corneal epithelial stem cells had not been established at that time, the biological differences between regenerated corneal and conjunctival epithelia were known. Initially, great controversy surrounded keratoepithelioplasty because it was an epithelial allograft, which, at that time, was considered to rarely survive on the ocular surface. Furthermore, it was not known whether the donor corneal lenticules maintained the corneal surface any longer than it would be maintained via penetrating keratoplasty.5 Over time, however, the method was accepted, due to the progress made in corneal epithelial cell biology. Because keratoepithelioplasty supplies not only a regenerated corneal epithelium but also an appropriate corneal substrate for inhibiting conjunctival invasion onto the cornea, this procedure has proved to be dramatically effective for the treatment of peripheral corneal ulcers, including Mooren’s ulcer.6 In 1986, Schermer et al7 presented findings to suggest that corneal epithelial stem cells are located in the limbus; this had important implications for keratoepithelioplasty, and limbal autograft transplantation was proposed by Kenyon and Tseng in 1989.8 Shortly thereafter, Tsai and Tseng used human allograft limbal transplantation with the goal of achieving the permanent survival of regenerated corneal epithelium by stem cell transplantation, although intensive immunosuppressive therapy was necessary.9 An especially noteworthy finding, which was reported by Kim and Tseng in 1995, was 22

that amniotic membrane transplantation was capable of inhibiting subepithelial scarring in ocular surface reconstruction.10 Since then, amniotic membrane transplantation combined with limbal allografts has been used to treat severe ocular surface disorders, such as Stevens-Johnson syndrome and ocular cicatricial pemphigoid, conditions that were previously considered contradictions for corneal transplantation.11 A review of these procedures and clinical results has been published by Holland et al.12 B. Immunological Aspects

It is possible that limbal transplantation could be performed as an autograft, using contralateral limbocorneal tissue in unilaterally affected cases, although such cases are rare. Most ocular surface reconstructions use allogeneic limbo-corneal tissues taken from post-mortem donors or living related subjects. Therefore, basic knowledge of the immunological aspects of ocular surface reconstruction using allogeneic tissues is important. 1. Immunological Differences Between Limbal Allograft Transplantation and Penetrating Keratoplasty It is well documented that limbal allograft transplantation exhibits the highest rejection rate among all types of corneal transplantation.13-17 In fact, although 50% of corneal allografts are accepted indefinitely in mice penetrating keratoplasty (PKP) models,18 100% of limbal allografts are swiftly rejected (within 2 weeks) if immunosuppressive agents are not used; a similar pattern is displayed in heart allografts (Figure 1). The success of PKP can be attributed to various features of the normal cornea and anterior segment that together contribute to ”immuneprivileged” states.19 These features include a) the avascularity of the corneal stroma, b) the absence of corneal lymphatics, c) the rarity of indigenous professional antigenpresenting Langerhans cells or macrophages in the central

Figure 1. Rejection patterns of orthotopic allografts in BALB/c mice. In comparison with PKP (closed circle), LT shows frequent and swift rejection (open circle, MST = 9.5 ± 2.2 days, mean ± SE), similar to heart allografts (open square, MST = 8.1 ± 1.1 days).

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cornea, and d) the capacity to induce anterior chamberassociated immune deviation (ACAID) by the inoculation of antigens into the anterior chamber. It is worth noting that ACAID is not induced by the limbal allograft itself, as graft components are separated from the recipient’s anterior chamber. Another important point is that the limbal area and heart do not have immune privilege; therefore, they exhibit intense and frequent rejection. 2. Basic Mechanism of Limbal Allograft Rejection The high rate of limbal allograft rejection is associated with the presence of antigen presenting cells (APCs),20 vessels, and lymphatics in the limbal area. The donor-derived APCs can present alloantigens (major histocompatibility complex [MHC]) directly to recipient T cells by what is known as the direct pathway of allorecognition, allowing for greater host recognition of the graft. The recipientderived APCs can present peptides derived from the processing of alloantigens (minor histocompatibility [minor H] antigens) to recipient T cells by the indirect pathway of allorecognition. Since Yao et al21 reported that the mean onset times of limbal allograft rejection were at a frequency similar among MHC-only disparate grafts, multiple minor H-only disparate grafts, and both MHC and minor H disparate grafts, MHC and minor H antigens have both been related to corneal epithelial rejection. Essentially, this means that an allogeneic response induced through each pathway is capable of causing the swift rejection of a limbal graft. Moreover, the corneal limbus of the recipient contains blood vessels and lymphatics, as well as APCs, allowing for acute allosensitization and swift rejection. As described above, MHC matching did not improve limbal allograft-survival in mice.21 In MHC-compatible transplantations, donor MHC molecules play an efficient role in antigen presentation of minor H antigens; this is not the case with MHC-incompatible transplantation. Theoretically, when living related transplants are used, efficient antigen presentations occur both through MHCcompatible APCs and direct pathways through MHC-incompatible APCs. Then living related donors may show vigorous allograft rejection. It is a controversial fact, however, that, from a clinical point of view, transplants from living related donors survive well.22-27 Further immunological investigations are needed to understand this. 3. Use of Immunosuppressive Agents Comparatively long-term use of strong immunosuppressants is essential for the renewed allogeneic epithelium to remain viable, and cyclosporine A and corticosteroids are widely used clinically after limbal allograft transplantation.9,13 The local and/or systemic use of immunosuppressants is associated with significant complications,28,29 including opportunistic infection, steroid-induced glaucoma, and cataract formation, and, even with the use of immunosuppressants, intense postoperative corneal allograft rejection can occur.14 Recently, in limbal transplantation and also in kidney transplantation, lower concentrations of three different kinds of

immunosuppressants, including corticosteroid, cyclosporine A, and mycophenolate mofetil (an antimetabolite), have been used to enhance immunosuppression while minimizing adverse effects. Tacrolimus (FK506), a recently developed macrolide antibiotic, may contribute to improved future success in avoiding graft rejection in patients refractory to cyclosporine A.30,31 However, Mills et al,32 using a rat model of a limbal allograft, found that although clinical allograft survival was prolonged to a modest extent by immunosuppression, donor cell survival on the ocular surface was not improved. Development of immunological strategies to suppress allograft rejection are needed for improved success of allogenic donor ocular surface reconstruction. 4. Immunological Strategy to Promote Limbal Allograft Survival It is believed that allograft rejection in corneal transplantation develops via a T-cell-mediated immune response. In particular, the CD4 T-cell-mediated delayedtype hypersensitivity (DTH), rather than the CD8 T-cellmediated cytotoxic T lymphocyte, appears to play a dominant role in corneal epithelial rejection.33 Several strategies are reported to promote limbal allograft survival. Firstly, after an injection of allogeneic cells into the anterior chamber of the eye, the recipients acquire donorspecific DTH suppression (ACAID). Yao et al34 were able to promote multiple minor H antigen only disparate limbal allograft survival by the induction of ACAID. Secondly, DTH response is mediated by CD4+ T helper 1 (Th1) type cells, which are cross-regulated by T helper 2 (Th2) type responses. In their study, Maruyama et al35 induced a vigorous Th2 type response and were also able to promote minor H-only incompatible limbal allograft survival. Importantly, their strategy could not mediate MHC disparate graft survival. This highlights the importance of discovering a DTH-suppressing strategy, including anti-MHC suppression, in the near future in order to prevent immunological rejection. C. What is Clinical Success? Ideally, regenerated corneal epithelium would survive permanently after keratoepithelioplasty or limbal allografts. However, regenerated corneal epithelium may be replaced by surrounding host conjunctival epithelium, probably because of the limited life-span of transplanted donor cells or because of sudden donor cell loss caused by immunological reaction. In fact, analysis by fluorescence in situ hybridization (FISH) has demonstrated the slow replacement of donor superficial epithelial cells by host epithelial cells in most cases,36 implying that similar events also occur in basal epithelial cells. However, in some cases, the clinical outcomes of limbal allografts are successful, although cellular replacements can occur; this suggests that host regenerating conjunctival epithelial cells may transdifferentiate into corneal epithelial cells,37,38 though debate continues. Clinical outcomes fall into three categories: 1) complete success and full survival of the donor corneal epithelium; 2) partial success with replacement of some of

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the donor corneal epithelium by the host conjunctival epithelium; and 3) failure due to host conjunctival invasion with massive vascularization. Since the clinical phenomena causing these three possible outcomes are still unclear, one has to explore the divergence between clinical manifestations and donor/host cell phenotypes via careful observation of the postoperative host eyes and also by intensive examination of the origin of the epithelial cells taken from the corneal surface of host eyes. Tsubota’s group has reported their clinical experience regarding limbal allografts, suggesting that it is worthwhile to perform repeated allografts as long as the patient’s visual acuity is maintained.14 III. TRANSPLANTATION OF CULTIVATED MUCOSAL EPITHELIAL CELL SHEETS

There is no doubt that corneal epithelial transplantations, including limbal autografts and limbal allografts, have helped to improve the outcome of ocular surface reconstruction in a number of situations. However, limbal autograft transplantation requires a fairly large amount of limbal tissue from the healthy eye and is not an option if the disease or injury is bilateral. Allografts always carry the risk of rejection, and they require immunosuppressive treatment for the rest of the patient’s life. To improve the surgical results of ocular surface reconstruction for severe stem cell deficiencies, it is essential to develop cultivated corneal epithelial sheets in vitro from a small portion of limbal epithelium. Then, a similar method using limbal epithelial cell suspension would make it possible to prepare “ready-to-use” epithelial grafts containing a large number of corneal epithelial stem cells. A. History of Creating Mucosal Epithelial Sheets

In the 1980’s, the dawn of ocular surface epithelial surgery, at least two forms of investigation were carried out with the goal of creating mucosal epithelial sheets. One approach involved the direct sampling of either corneal or oral mucosal epithelial sheets using dispase and gentle mechanical treatment.39,40 Although the corneal epithelial sheet taken directly from in vivo cornea could be attached to the corneal stroma in experimental animals,39 lid movement easily peeled it off. On the other hand, the oral mucosal epithelial sheet remained on the cornea,40 but induced severe neovascularization. The other approach involved establishing a cultivated epithelial cell sheet. For this purpose, many investigators endeavored to reconstruct corneal epithelial sheets on carrier materials, such as collagen sheets41,42 and corneal stromal carriers,43 to create stratified corneal epithelial cell layers. Some groups tried to reconstruct not only the epithelial sheet, but also three layers of corneal tissue—a “corneal equivalent”— using cell-line cells supported by natural and synthetic polymers.44,45 This kind of corneal equivalent is now ready to be used for testing toxicity and drug efficacy, but it is not ready for clinical application because of the use of immortalized cells (which have been genetically modified to prolong their survival), and other factors related to carrier materials. 24

B. Transplantation of Cultivated Corneal Epithelial Stem Cell Sheets

1. Biological Aspects Despite the potential drawbacks of cultivated corneal epithelial transplantation, its first clinical application was demonstrated by Pellegrini and colleagues in Italy in 1997.46 They developed a method to reconstruct stratified corneal epithelial cell sheets on petrolatum gauze or a soft contact lens as a carrier and treated two patients who had unilateral chemical burns by transplanting cultivated corneal epithelial cells taken from the limbus of the healthy contralateral eye. They adopted the well-established keratinocyte-culturing method of Rheinwald and Green,47 which involves the use of 3T3 feeder layers to help maintain epithelial stem cells, and this most likely contributed to the reported success of the treatment. After the report of Pellegrini et al, researchers realized the potential of amniotic membrane as a carrier for corneal epithelial cell culture. After all, this material had been used for several years in a range of ocular surgeries with or without limbal transplantation,48 and had proven to be useful for the treatment of thermal and chemical injuries,10,49 severe pterygium,50,51 persistent or deep corneal ulcers,52,53 ocular cicatricial pemphigoid, Stevens-Johnson syndrome, and other limbal stem cell deficiencies.11,54 Amniotic membrane is the innermost layer of the fetal membrane, and it is composed of a monolayer of amniotic epithelial cells, a thick basement membrane, and an avascular stroma. It is known to have many unique characteristics that are beneficial to ocular surface reconstruction. Notably, amniotic membrane inhibits conjunctival fibrosis by suppressing the transforming growth factor beta signaling system, and it also prevents myofibroblastic differentiation of normal fibroblasts.55,56 Furthermore, the normal differentiation of conjunctival epithelial cells is encouraged after amniotic membrane transplantation.57 The basement membrane of amniotic membrane is reported to resemble that of the conjunctival epithelium,58 and, interestingly, we have found that it closely resembles the basement membrane of the corneal epithelium (unpublished data). In addition, growth factors, such as epidermal growth factor (EGF), keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF), detected in amniotic membrane59 may also play a role in accelerated epithelialization after amniotic membrane transplantation. A high and therapeutic level of nerve growth factor (NGF) is also reported to be present in amniotic membrane.60 Recently, it has been reported that amniotic membrane has anti-inflammatory effects by inducing the suppression of interleukin 1α and interleukin 1β in limbal epithelial cells,61 and also by trapping and preventing polymorphonuclear cells infiltrating into the corneal stroma.62 Based on these interesting clinical and laboratory findings, preserved amniotic membrane has been considered to be one of the appropriate carrier materials for the transplantation of cultivated corneal epithelial cells. Although there is still debate regarding its use, including the merits

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and demerits of the denuding process for amniotic epithelial cells, and controversy regarding methods, the short-term results of cultivated corneal epithelial transplantation using an amniotic membrane carrier are encouraging. Tsai reported the successful transplantation of autologous limbal epithelial cells cultured and expanded on intact (cellular) amniotic membrane for severe unilateral corneal diseases.63 Our group has reported the successful culture and transplantation of corneal epithelial stem cells cultivated on EDTA-denuded amniotic membrane with the presence of an inactivated 3T3 feeder layer.64-66 Schwab transplanted corneal epithelial cells passaged on denuded amniotic membrane.67 Tseng’s group has carried out extensive studies to show the usefulness of intact amniotic membrane as a carrier for limbal epithelial cell culture; Meller et al68 and Grueterich et al69 demonstrated that limbal epithelial cells cultivated on intact amniotic membrane contain slow-cycling and labelretaining characteristics of cells that do not express K3 and K12 keratins and connexin 43. These characteristics resemble the limbal basal epithelium, which is considered to be the location of corneal stem cells in vivo.7,70 We have reported the usefulness of denuded amniotic membrane to promote the prompt migration of corneal epithelial cells in vitro (Figure 2) and the potential of amniotic membrane to make wellstratified and differentiated corneal epithelial cell layers that express corneal epithelium specific keratins, K3 and K12 (Figure 3).71,72 In our clinical experience, we have found that the ocular surface condition of candidates for cultivated corneal epithelial transplantation is very severe, often accompanied by complications, such as severe aqueous-deficient dry eye and eyelid abnormality. For these patients, we consider it essential to transplant well-stratified epithelial cell layers that have developed barrier functions with well-controlled proliferative activity in basal cells and differentiated in superficial cells. For this purpose, we have developed a culture system using an air-lifting method to promote epithelial cells via tight junction formation. By air-lifting, we have obtained cultivated epithelial cell sheets with smaller intercellular spaces in the superficial cells and with an epithelial barrier function.73 We have also attempted to transplant cultivated corneal epithelial cells, including limbal stem cells, and have developed a cell-suspension culture system capable of supplying cultivated corneal epithelial sheets that are well developed, potentially allowing the transplantation of more corneal epithelial stem cells (Figure 3).72 Since 2000, we have transplanted cultivated corneal epithelial sheets created by the cell-suspension method on more than 15 eyes, and the early postoperative results are excellent. Examination with fluorescein 48 hours after transplantation demonstrated that in more than 95% of the eyes, the graft attached successfully. We will continue to carefully monitor the long-term survival of the transplanted corneal epithelium.74 Another group of investigators is also attempting to obtain cultivated corneal epithelial sheets, using a temperature-sensitive culture dish.75 2. Immunological Aspects In ocular surface reconstruction, donor-derived APCs,

Figure 2. The area of cellular outgrowth from limbal explants on intact and EDTA denuded amniotic membrane measured from five separate cultures in each case. Limbal epithelial cells can migrate rapidly on denuded amniotic membrane. Only limited migration was detected on intact amniotic membrane (*P<0.01). (Modified from Koizumi et al.71)

including dendritic cells (DCs) and ocular Langerhans cells, offer the strongest immune reaction. In the normal eye, APCs are located in the corneal limbus, where corneal epithelial stem cells exist. Hence, as both corneal stem cells and APCs are transplanted together in limbal allografts, they show intense and frequent rejection. On the other hand, very few or almost no APCs are contained in cultivated corneal epithelial sheets, since the culture medium for this procedure is not for bone marrow-derived cells, but for epithelial cell growth. In fact, no bone marrow-derived cells are detected immunohistologically in cultivated corneal epithelial cell sheets (unpublished data). Furthermore, resident corneal Langerhans celltype DCs are present in the anterior corneal stroma, both in humans76 and mice,77 and they undergo maturation and function as APCs during inflammation. Because we can avoid transplantation of the corneal stromal layer in cultivated corneal epithelial sheets, it is possible to minimize the donorderived APCs given to the recipient. For permanent survival of cultivated corneal epithelial sheets, it is important to suppress postoperative inflammation, as well as immunological rejection. Amniotic membrane, which has recently been shown to be an immune-privilegelike tissue, may possess anti-inflammatory properties, including a) immunoregulatory secretion factors,78 such as IL-1 receptor antagonist, and b) HLA-G and Fas ligand expression in the mesenchymal cells of amniotic stroma.78 Such properties may result in the reduction of corneal stromal inflammation and ulceration in HSV-1 keratitis models,79 and in the suppression of bFGF-induced corneal neovascular-

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Figure 3. Light micrograph showing a cross-section of cell-suspension-cultured epithelial cells on EDTA-denuded amniotic membrane (A). It shows a healthy, well-stratified epithelium that is firmly attached to the denuded amniotic membrane. Immunohistochemical staining showed the expression of K3 (B) and K12 (C) in cell-suspension-cultured epithelial cells. Transmission electron micrographs of cellsuspension-cultured epithelial cells (D-E) on denuded amniotic membrane. Cell-suspension-culture produced a healthy and well-differentiated cell layer very similar in appearance to a normal corneal epithelium (D). The basement membrane was evident and the basal cells were securely attached to the denuded amniotic membrane by hemidesmosomes (E). *=amniotic membrane. (Modified from Koizumi et al.72)

ization by the supernatant of amniotic membrane.80 These properties have an effect on surrounding cells, such as suppression of IL-1α and IL-1β gene expression, but upregulation of IL-1 receptor antagonists, in the case of cultured human corneal and limbal epithelial cells on amniotic membrane.61 In addition, alloreactive T cell proliferation is suppressed in co-culture on amniotic membrane.81 These data may explain, in part, the effect of amniotic membrane in reducing ocular surface inflammation (including that which occurs in cultivated corneal epithelial transplantation), underscoring the unique feature of amniotic membrane as a substrate for tissue engineering. Investigations are now in progress to establish whether or not acellular amniotic membrane also possesses similar properties. 3. Clinical Outcomes Based on the success of human corneal epithelial stem cell culture on amniotic membrane in vitro and cultivated corneal epithelial sheet transplantation in rabbit’s eyes in vivo, we embarked upon clinical trials of cultivated corneal epithelial transplantation. The Institutional Review Board of Kyoto Prefectural University of Medicine approved the trans26

plantation of cultivated corneal epithelial stem cell sheets in 1999. The use of cultivated corneal epithelial transplantation was restricted to those patients who had a poor visual prognosis with conventional corneal epithelial transplantation, such as limbal allografts or keratoepithelioplasty. Thus, we performed cultivated corneal epithelial transplantation on 33 eyes of 30 patients with severe ocular surface disease: severe chemical injury, Stevens-Johnson syndrome, and ocular cicatricial pemphigoid. While the acute-phase eyes with persistent epithelial defects received cultivated corneal epithelial transplantation for the purpose of covering the corneal surface, alleviating intensive inflammation, and avoiding complications that accompany persistent epithelial defects, the chronic phase eyes received cultivated corneal epithelial transplantation to obtain better visual function. During surgery, we removed scarred conjunctival tissue overlying the ocular surface from the cornea up to approximately 3 mm outside the limbus. After removing subconjunctival tissue, we placed the small tips of several microsponges containing 0.04% mitomycin C in the subconjunctival space adjacent to the cornea for 5 minutes and performed vigorous saline washing to prevent the development of sub-

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Figure 4. Cultivated corneal epithelial transplantation for acute phase (A, B) and chronic phase (C, D) chemical injuries. In the acute phase eyes, the corneal epithelial cells, including the limbal area, were totally destroyed (total limbal deficiency) and epithelial defect had persisted more than 60 days before surgery (A). Severe inflammation was detected. Two months after surgery, the ocular surface was covered with the transplanted cultivated corneal epithelial stem cell sheet (B). It is noteworthy that the severe inflammation that was detected before surgery had completely subsided after transplantation. In the chronic phase eyes (C), the corneal surface was covered with conjunctival epithelium accompanied by neovascularization. Two years after surgery, the corneal surface was covered with the transplanted cultivated corneal epithelial stem cell sheet (D).

conjunctival fibrosis after surgery. A cultivated corneal epithelial stem cell sheet on amniotic membrane was transferred onto the corneal surface and sutured by 10-0 nylon. We then applied a therapeutic soft contact lens. For the chronic phase eyes with corneal stromal scarring, lamellar keratoplasty was first performed with use of preserved donor grafts to replace the scarred corneal stroma, followed by cultivated corneal epithelial transplantation. Postoperatively, 0.1% dexamethasone, 0.3% ofloxacin and 0.05% cyclosporine-A were instilled topically. Dry eye patients received artificial tears. Systemic corticosteroid (betamethasone, 1 mg/day), cyclosporine A (150 mg/day) and cyclophosphamide (1mg/day), as well as mycophenolate mofetil (1g/day) in some cases, were used to prevent postoperative inflammation and immunological rejection. Systemic immunosuppression as described above was used for at least 6 months, after which it was gradually reduced, depending on clinical characteristics. In many cases, a low dosage of cyclosporine A (50 mg/day) continued to be administered for up to at least 1 year. The epithelial integrity was satisfactory in all cases, as evidenced by the fact that the transplanted corneal epithelium did not stain with sodium fluorescein just after

being transferred onto the ocular surface. In addition, there was no epithelial damage to the transplanted corneal epithelium 48 hours after transplantation. The transplanted amniotic membrane did not disturb the visual acuity, and clarity increased day by day. Surprisingly, the preoperative ocular surface inflammation, which had not been controlled by conventional treatment, including corticosteroid, cyclosporine A, therapeutic soft contact lenses, and amniotic membrane patch, decreased rapidly after surgery in all of the acute-phase patients (Figure 4). In the chronic-phase eyes, the long-term visual prognosis and epithelial stability were varied in the three kinds of diseases discussed below. In the case of severe chemical injury, the transplanted corneal epithelium was clear and stable up to 4 years after transplantation, and only a small amount or, indeed, no conjunctival inflammation was present during the entire postoperative period (Figure 4). On the other hand, in patients with Stevens-Johnson syndrome, mild to moderate ocular surface inflammation occurred several months after cultivated corneal epithelial transplantation, and then decreased within 18 months postoperatively (Figure 5). Whereas subconjunctival fibrosis had not progressed in the eyes with

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Stevens-Johnson syndrome, conjunctival scarring such as symblepharon and shortening of the fornix had progressed in the eyes with ocular cicatricial pemphigoid. In most of the chronic phase patients with immunological abnormalities, such as Stevens-Johnson syndrome and ocular cicatricial pemphigoid, the phenotypes of ocular surface cells on amniotic membrane gradually changed from donor to host epithelial cells over a couple of years; however, subepithelial scarring and neovascularization did not progress. In other words, host conjunctival epithelium replacement on amniotic membrane occurred without scarring. This phenomenon is considered to be partly due to a mild rejection of the transplanted corneal epithelial cells. Although graft survival was not very long in some eyes in these chronic cases, the ocular surface maintained its transparency and the patients obtained a better visual function than that which had existed before cultivated corneal epithelial transplantation. It is unknown whether or not replacement occurs only in severe forms of Stevens-Johnson syndrome, ocular cicatricial pemphigoid, or other conditions, and thorough investigation of this phenomenon is needed. We have successfully performed regrafting of cultivated corneal epithelium on 6 eyes from our series of 33 eyes in which the severity of epithelial opacity progressed after an episode of rejection or persistent conjunctival inflammation with unknown cause.82 It was possible to easily separate the amniotic membrane from the underlying corneal stroma and, in every case, the bare corneal stroma maintained its transparency. Corneal stromal transparency was good enough for phacoemulsification and successful intraocular lens implantation to be performed in those cases with cataract progression. Although methicillin-resistant Staphylococcus aureus or methicillin-resistant Staphylococcus epidemidis infection occurred with higher frequency in eyes with Stevens-Johnson syndrome compared to others, most cases healed with little corneal scarring.83 We found that persistent epithelial defects occurred in some eyes with chronic cases of ocular cicatricial pemphigoid, but corneal perforation did not occur, and we were successful in achieving epithelialization via conjunctivalization. Other reports have described the transplantation of autologous cultivated corneal epithelial stem cells from uninjured eyes as an effective therapy for patients with unilateral ocular surface diseases.63,67 Our clinical experience has been similar. We have found that there was less damage to the contralateral eye than has been the case with limbal autografts, and the cultivated corneal epithelial sheets formed well-stratified epithelial layers from the tiny corneal limbal tissue. After a substantial follow-up period, the transplanted epithelium remained transparent and stable, and the patient achieved good visual acuity with no complications in the healthy contralateral eye. Because biopsy examination, even when performed with impression cytology, is invasive for patients who receive epithelial transplantation, it has been impossible for us to investigate the survival of donor corneal epithelial cells. However, we have been able to assess the outcomes of cultivated corneal epithelial transplantation by studying the appearance of 28

the corneal surface with regard to epithelial transparency, vascularization, conjunctival invasion with subepithelial fibrous tissue, the formation of symblepharon, etc. The shortterm results were satisfactory in all cases; however, in most cases of Stevens-Johnson syndrome and ocular cicatricial pemphigoid, ocular surface inflammation was prolonged for several months after the operation, affecting the long-term outcomes. The long-term outcomes of the first 20 eyes (mean post-operative period is 42 months) can be summarized as follows: 6 out of the 9 eyes of the acute phase patients and 4 out of the 11 eyes of the chronic phase patients maintained epithelial transparency. Conjunctival invasion occurred gradually in 3 eyes of the acute-phase patients; however, the primary purpose of this surgery was to cover the corneal surface and reduce the intense inflammation, and this goal was achieved. Although transplantations on 5 eyes of the chronicphase patients were partially successful and 2 eyes were failures based on their appearances, 7 out of 11 of the chronicphase patients obtained visual improvement following this surgery. The long-term outcomes are satisfactory, considering the fact that we were treating only difficult cases. Other ocular surface diseases such as aniridia and conjunctival intraepithelial neoplasia, which have a good prognosis after conventional limbal allografts, might be good candidates for cultivated corneal epithelial transplantation. C. Transplantation of Cultivated Oral Mucosal Epithelial Sheets

1. Animal Experiments Because severe ocular surface diseases are usually bilateral, we normally perform allogeneic corneal epithelial transplantation (either limbal allograft or cultivated corneal epithelial transplantation). However, these procedures not only require sufficient donor tissue, but they are accompanied by the risk of rejection, so prolonged immunosuppression is required, severely affecting clinical results. With these drawbacks in mind, we have attempted to overcome immunological rejection complications by using autologous mucosal epithelium of nonocular surface origin. Early on, several researchers investigated the possibility of using oral mucosa for ocular surface reconstruction. In 1963, Ballen et al reported that oral mucosal grafts, which included both epithelium and subepithelial tissues, became heavily vascularized with early fibrosis.84 In 1986, Gipson et al reported that the in vivo oral epithelium freed of underlying connective tissue was not maintained in central avascular corneal regions.40 Based on these considerations, we carried out the culture of rabbit oral mucosal epithelial cells on amniotic membrane as a carrier, using a modified version of our previously established culture method.72 Small oral biopsies (approximately 2–3mm 85) were obtained from the oral cavity under local anesthesia. The biopsy specimens were then incubated with enzymatic reagents, such as dispase and trypsin-EDTA,to separate the cells from the underlying connective tissue. The resultant single-cell suspensions of oral mucosal epithelial cells were co-cultured for 2–3 weeks on a denuded amniotic membrane carrier, with inactivated 3T3

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fibroblasts. Toward the end of the culture period, an air-lifting technique was used to facilitate epithelial differentiation and stratification. The oral mucosal epithelial cells cultivated on amniotic membrane showed 5–6 layers of stratification and appeared very similar to in vivo normal corneal epithelium (Figure 6). We detected the presence of nonkeratinized, mucosal-specific keratins 4, keratin 13, and cornea-specific keratin 3 in the cultivated oral mucosal epithelial sheets via immunohistochemical analysis; however, keratinization-related keratin 1 or keratin 10 was not detected. Electron microscopic examination revealed that the cultivated oral mucosal epithelial sheet held anatomical junctional structures, such as desmosome, hemi-desmosome, and tight junctions that were almost identical to those of normal corneal epithelial cells. The apical surface of this sheet was also covered with numerous microvilli, indicating normal epithelial differentiation. From these experimental results, we have concluded that under appropriate culture conditions, oral mucosal epithelial cells cultivated on amniotic membrane have the potential ability to differentiate into cornea-like epithelial cells. After the successful culturing of oral mucosal epithelial cells on amniotic membrane, we tried, via animal experimentation, to reconstruct damaged ocular surfaces, using a rabbit model mimicking a severe ocular surface disease by superficial keratectomy. We then reconstructed the ocular surface with an autologous cultivated oral mucosal epithelial sheet. At 48 hours after surgery, most of the area of the transplanted cultivated oral mucosal epithelial sheet possessed an intact epithelium. At 10 days after transplantation, the ocular surface, covered by the transplanted epithelium, was intact and without defects; this suggests that the autologous transplantation of cultivated oral mucosal epithelia is a viable procedure for ocular surface reconstruction (Figure 7). 2. Preliminary Clinical Experience Encouraged by the success of the animal study model using cultivated oral mucosal epithelial sheet transplantation, we have applied this to human cases. Using this novel tissue engineering procedure generated from autologous oral mucosa, we can eliminate the intensive medication required for immunosuppression, thus reducing the risk of postoperative complications, such as infections and steroid-induced glaucoma and cataracts. In our initial clinical trials, we applied cultivated oral mucosal epithelial sheets in two different forms of surgery. One was reconstruction of the corneal surface of a severe bilateral corneal stem cell deficiency, using a cultivated oral mucosal epithelial sheet instead of allogeneic corneal epithelium. The other was reconstruction of the conjunctival fornix in patients with severe symblepharon formation associated with ocular cicatricial pemphigoid, Stevens-Johnson syndrome, and chemical or thermal burns. In order to prepare the cultivated oral mucosal epithelial sheet, 2–3 mm 2 oral mucosal biopsy specimens were obtained from our patients 3 weeks prior to transplantation. Single-cell suspensions of oral mucosal epithelial cells were cultured on acellular amniotic membrane, using the

same method carried out on the animal model. As in the case of the animal model, the cultivated oral mucosal epithelium consisted of 5–6 nonkeratinized epithelial layers with normal transparency just prior to surgery. The surgical procedure is almost the same as that for cultivated corneal epithelial sheet transplantation . After completely removing damaged tissues on the corneal surface and subconjunctival fibroblasts, residual subconjunctival tissue was treated for 5 min with 0.04% mitomycin C, followed by vigorous repeated washing with saline in order to suppress the excessive postoperative inflammation and subconjunctival fibrosis. Then, the cultivated oral mucosal epithelial sheet on amniotic membrane was transplanted onto the corneal surface and secured with 10-0 nylon sutures at the limbus. The integrity of the cultivated oral mucosal epithelium was confirmed via intraoperative fluorescein staining, and a therapeutic soft contact lens was applied. Forty-eight hours after surgery, the survival of the transplanted epithelium was confirmed, using slitlamp examination with fluorescein staining. An epithelial phenotype of transplanted cultivated oral mucosal epithelium was somewhat distinguishable from the conjunctival epithelium by fluorescein staining. Our preliminary data show the successful survival of autologous cultivated oral mucosal epithelium on the ocular surface without returning to an in vivo oral tissue phenotype, as was previously the case with oral mucosal transplantation. This major difference can be explained by the elimination of subepithelial fibrous tissue and vascular components in oral mucosa during the tissue culturing system. It is possible that amniotic membrane has some effect on this phenomenon as well. One adverse effect of this procedure was that the transplanted cultivated oral mucosal epithelium sometimes showed some extent of neovascularization in the peripheral cornea with epithelial thickening. Clinical investigations are now under way to understand this phenomenon. We also used cultivated oral mucosal sheets on amniotic membrane to reconstruct the conjunctival fornix (Figure 8); this form of surgery was successful in cases of cicatricial ocular surface diseases, including ocular cicatricial pemphigoid, chemical injury, etc. However, we have to be aware of abnormal postoperative fibrovascular proliferation caused by primary diseases, which is still critical to the long-term prognosis. The tissue engineering process described here represents the application of non-ocular mucosal epithelium in order to reconstruct an ocular surface that can provide an acceptable level of comfort and visual rehabilitation for visually-threatened patients. IV. CONCLUSIONS AND FUTURE GOALS The use of tissue-engineered corneal epithelial replacements holds much promise for ocular surface reconstruction in cases of corneal epithelial stem cell deficiencies. However, even though an autologous cultivated corneal epithelial stem cell sheet is the safest and most reliable form of sheet, bilaterally affected ocular surface disorders can not be treated by this method. To treat these disor-

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CULTIVATED EPITHELIAL TRANSPLANTATION / Kinoshita, et al

Figure 5. Eyes with chronic phase Stevens-Johnson syndrome that had undergone cultivated corneal epithelial transplantation. Before (A) and 1 month (B), 3 months (C) and 18 months (D) after cultivated corneal epithelial transplantation. The inflammation subsided just after the surgery (B), but it reoccurred later (C). Finally, the inflammation subsided again at 18 months (D). This is a representative postoperative clinical course that is often seen in eyes with Stevens-Johnson syndrome.

ders, one must choose either an allogeneic cultivated corneal epithelial sheet or an autologous cultivated oral mucosal epithelial sheet. In the case of the former, allogeneic immunological reaction is a threat, and in the case of the latter, one must remember that the sheet is not exactly the same as a corneal epithelium. The cultivated corneal epi-

thelial transplantation technique promises many future possibilities. Firstly, when a selected donor-recipient combination is required, such as in major histocompatibility matching, the cultivated corneal epithelial transplantation method will allow us to perform the matching, as there is sufficient time to select a recipient for the donor during

Figure 6. Histological sections of rabbit normal cornea (A) and cultivated corneal epithelial cells on amniotic membrane (B), stained with hematoxylin and eosin. *=amniotic membrane. Original magnification: X400.

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Figure 7. Representative slit lamp photographs of the rabbit eye taken before transplantation (A) and 10 days after transplantation (B). Before transplantation, the eye showed total limbal stem cell deficiency. Ten days after surgery, the corneal surface was completely covered.

Figure 8. An eye with severe chemical injury with a formation of symblepharon (A) underwent reconstruction with use of an auto cultivated oral mucosal sheet. Note the re-epithelialization by transplanted oral mucosal epithelium and reconstructed conjunctival fornix 6 months after operation (B).

the cell culture period. Furthermore, master stem cell banking may be possible. Secondly, a more sophisticated way of culturing oral mucosa or conjunctiva may provide us with a better quality epithelial sheet for autologous transplantation. It is our belief that more advanced strategies will be developed in the near future. ACKNOWLEDGEMENT The authors thank Ms Joanna Connon for reviewing the manuscript.

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