Molecular Vision 2003; 9:635-643 <http://www.molvis.org/molvis/v9/a77/> Received 11 August 2003 | Accepted 5 December 2003 | Published 8 December 2003

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Functional reconstruction of rabbit corneal epithelium by human limbal cells cultured on amniotic membrane

Yiqin Du,^1,2,3 Jing Chen,¹ James L. Funderburgh,³ Xiuan Zhu,² Lingsong Li¹
 
 

¹Peking University Stem Cell Research Center and ²Peking University Eye Center, Beijing China; ³Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PA

Correspondence to: Yiqin Du, Department of Ophthalmology, University of Pittsburgh, 1013 EEINS, 203 Lothrop Street, Pittsburgh, PA, 15213; Phone: (412) 647-3852; FAX: (412) 647-5880; email: yiqindu@pitt.edu

Abstract

Purpose: To investigate the phenotype of fetal and adult human limbal cells cultured on human amniotic membrane and the ability of cultured adult human limbal cells to repair limbal stem cell deficiency in a rabbit model.

Methods: Human adult and fetal limbal cells were isolated and cultured either on plastic plates or on human amniotic membrane. Connexin43, p63, and keratins 3 and 12 (K3 and K12) were detected by immunofluorescence and RT-PCR. Limbal stem cell deficiency was established in rabbits using chemical ablation and mechanical debridement. Cultured adult human limbal cells were transplanted onto rabbit corneas one month after injury, then fixed and imbedded in paraffin forty days later. Immunofluorescent staining of human-nuclear antigen, p63, K3, and connexin43 identified human-specific cells, progenitor cells, and differentiated corneal epithelial cells, respectively.

Results: Adult and fetal cultured limbal cells appeared similar in morphology. RT-PCR results showed that cells cultured from the human adult and fetal limbal area expressed both p63 and K12, whereas cells from central adult epithelium expressed K12 only. Immunofluorescent staining showed that more cells were p63 positive when cultured on human amniotic membrane than on plastic. Double staining for p63 and connexin43 showed some p63-positive cells co-expressing connexin43. After transplantation of adult human limbal cells cultured on human amniotic membrane, injured rabbit corneas were completely reconstructed exhibiting epithelial integrity, improved corneal clarity, and little or no neovascularization. The majority of repopulated epithelial cells expressed anti-human nuclear antibody. Cells expressing p63 occurred throughout the new epithelium.

Conclusions: During healing, expression of p63 is not limited to epithelial stem cells but may also mark transient amplifying progenitor cells. Culture on human amniotic membrane suppresses differentiation of limbal epithelial cells and promotes the proliferation of p63 expressing cells. Amniotic membrane-cultured human limbal cells fully reconstructed rabbit corneas having limbal stem cell deficiency, with human cells providing most of the cells of the new epithelium. Expression p63 is distributed throughout the reconstructed tissue.

Introduction

The corneal epithelium has a capacity for rapid regeneration that depends on the self-renewal ability of corneal stem cells. Corneal epithelium consists of corneal stem cells, transient amplifying cells (TAC), post mitotic cells, and terminally differentiated cells. Both stem cells and TAC exhibit an ability to proliferate, whereas post-mitotic cells have lost this ability. Stem cells are imbued with potential for self-renewal and can proliferate extensively. TAC, on the other hand, exhibit a reduced proliferation lifespan compared to stem cells and have lost the ability for self-renewal. Cornea stem cells are located at the basal layer of the limbus whereas TAC are located throughout the corneal basal epithelial layer. This layer is generated by asymmetrical mitosis and centripetal movement of the stem cells [1,2]. As stem cells migrate from the limbus into the center of the cornea, they differentiate into mature corneal epithelial cells. Limbal stem cells are essential for the integrity and function of corneal epithelium. Many pathological conditions can lead to limbal stem cell deficiency (LSCD) such as chemical or thermal injury, Stevens-Johnson syndrome, contact lens-induced keratopathy, genetic disease of aniridia, and multiple endocrine deficiency-associated keratitis. In a severely injured cornea, both limbal and central epithelia are absent and conjunctival epithelial cells invade the corneal surface, resulting in an abnormal conjunctiva covering the corneal surface. This process is accompanied by chronic inflammation, persistent epithelial defects, stromal scarring, and neovascularization [3,4], producing decreased visual acuity and photophobia. Several methods have been used to treat these disorders, however, each of them has its limitation. For instance, amniotic membrane transplantation (AMT) can provide a good substrate for limbal stem cell (LSC) proliferation [5-7] thereby promoting recovery of the damaged ocular surface. The success of AMT depends on the presence of LSCs remaining on the cornea surface. Thus the transplantation of AMT is ineffective in complete LSCD. Auto- or allo-limbal transplantation can provide a source of stem cells. However, an autograft obtained from the contralateral corneal limbus is not suitable in cases of bilateral damage. Moreover, the donor eye is at risk of surgery-related LSCD. In allografts, graft rejection occurs frequently [8]. Recently, it has been reported that corneal limbal epithelial cells cultured ex vivo on human amniotic membrane (HAM) can repair injured ocular surfaces[9-12]. It appears that there is no immune rejection when cells derived from the contralateral limbus or from the cornea of the patient's relative are cultured and transplanted. It is currently not established whether, after healing, corneal epithelial cells reconstituting the new epithelium were derived from the engrafted cells or from host stem cells that remained on the ocular surface. Culture of limbal cells on amniotic membranes appears to preserve the limbal stem cells and retain their in vivo properties [13-15] but it remains to be demonstrated if HAM acts only as a substrate or actually promotes LSC proliferation when limbal cells (LC) are cultured on it.

Because stem cells have the ability for self-renewal and epithelial cells have a limited lifespan, defining conditions under which stem cell populations can be preferentially expanded in vitro is an important goal for use of these cells in transplantation and for better characterization of limbal stem cell properties. A functionally healed ocular surface should retain stem cells and provide a constant source of new differentiated epithelium as in normal corneas. Demonstration of a stem cell population in reconstructed corneas has been difficult because of a lack of LSC specific surface markers. Keratins 3 and 12 (K3 and K12) are widely used to identify differentiated corneal epithelial cells and connexin43 is expressed in basal epithelial (TAC) cells but not basal limbal (stem) cells. Thus these antigens currently serve as negative markers to identify cells which are not LSC [1,3,15-17].

The p63 gene, a homologue of the tumor-suppressor p53, is highly expressed in the basal or progenitor layers of many epithelial tissues and is essential for regenerative proliferation [18,19]. Work by Pellegrini et al., localized p63 to the limbal basal cells of the corneal epithelium and then proposed p63 as a marker of corneal stem cells [20]. Currently, however, it is unknown if p63 expression continues to be limited to the LSC during in vitro expansion or in healing epithelial wounds.

In this study, we compared the morphology and phenotype of cultured fetal and adult limbal cells examining p63, K3, K12, and connexin43 expression. Limbal cells cultured on HAM and on tissue culture plastic plates were compared to investigate whether or not HAM can promote LSC proliferation and at the same time suppress epithelial differentiation. Finally, we investigated reconstruction of the rabbit corneal ocular surface with adult human limbal cells cultured on HAM. This model provides a novel ability to determine the source of cells repopulating the reconstructed epithelium. We found that HAM cultured human limbal cells were responsible for regeneration of the new rabbit epithelium. Expression of p63 during culture and regeneration of the epithelium, however, did not appear to be limited to stem cells as defined by tissue localization and by connexin43 expression.

Methods

Materials

Dulbecco's modified Eagle's medium/Ham's F-12 nutrient mixture, (1:1, DMEM/F-12) and fetal bovine serum (FBS) were purchased from HyClone (Logan, UT). Amphotericin B and cholera-toxin were purchased from GIBCO BRL (Invitrogen, Grand Island, NY). Triiodothyronine, hydrocortisone, insulin, transferrin and selenium were from Sigma (St. Louis, MO). Recombinant human EGF was produced by ligation of EGF cDNA into a pGEX2T vector (Promega, Madison, WI) in the correct reading frame, and expression in E.coli after induction with IPTG. The fusion protein was purified on glutathione-agarose beads and the GST carrier removed by thrombin cleavage of the agarose bound protein using 0.1% (wt/wt) thrombin. The released protein was recovered by washing the beads with buffer and then lyophilized. The recombinant EGF showed a single band on SDS-PAGE and western blot analysis. Dispase II was obtained from Boehringer-Mannheim (Indianopolis, IN). The mouse monoclonal anti-human nuclear antibody was from Chemicon (Temecula, CA) and the polyclonal antibody against connexin 43 (Cx43) was from Zymed (South San Francisco, CA). Anti-K3 monoclonal antibody was kindly presented by Dr. TT Sun (New York University) [1] and anti-p63 antibody was kindly presented by Dr. Franklin Meckon (Harvard University) [19]. The FITC- or TRITC-conjugated secondary antibodies were from Sigma. The RNeasy Mini kit was from Qiagen (Valencia, CA).

Preparation of human amniotic membrane

In accordance with the tenets of the Declaration of Helsinki and with proper informed consent, human amniotic membrane (HAM) was obtained at the time of cesarean section. Preparation of the HAM was similar to that described by Tseng [21]. The tissue was washed with phosphate-buffered saline (PBS) containing antibiotics (100 U/ml penicillin and 100 μg /ml streptomycin). The HAM, a thin sheet consisting of the epithelium, basement membrane, and some underlying compact stroma, was separated from the bulk of the tissue and stored in DMEM containing 50% glycerol at -70 °C for up to 3 months, pending testing of donor sera for disease. Immediately before use, HAM was thawed, washed with PBS and cut into pieces of approximately 2 cm in diameter. The pieces were then treated with 0.25% trypsin in 0.02% EDTA at 37 °C for 30 min. The digested HAM was gently scrubbed with a plastic spatula to remove the epithelium without breaking the basement membrane. Acellularity of the scrubbed HAM was confirmed by HE staining. The denuded membrane was washed with PBS and allowed to adhere onto a 30 mm diameter Millicell microporous membrane tissue culture insert (Millipore Corp, Bedford, MA) with the basement membrane side (from which epithelial cells had been removed) facing up.

Cultivation of limbal cells

Human tissue was handled according to the Declaration of Helsinki. Limbal epithelial cells were obtained with a procedure similar to that previously described [22]. Briefly, cornea-scleral rims of approx 14 mm diameter from the anterior of human eyes were obtained from Peking University Eye Center (Beijing, China) after the removal of 7 mm central corneal buttons for corneal transplantation. Fetal corneas were obtained from a naturally aborted fetus of 20-week gestation. Permission to use human fetal tissue was granted by Peking University Health Science Center's Ethical Committee. Tissues were rinsed three times with D-Hanks' solution containing 50 μg/ml gentamicin and 1.25 μg/ml amphotericin B. Aided by careful dissection under a microscope, remaining conjunctiva, sclera, and iris were removed. The remaining tissues (including limbal epithelium and the underlying stroma) were minced and digested with Dispase II at 37 °C for 5 min. Cells were washed with DMEM plus 10% FBS and then seeded into the plastic plate. Cells were cultured with DMEM/F12 containing 5% FBS, 20 ng/ml human EGF, 4 mM glutamine, 2 nM triiodothyronine, 5 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml selenium, 0.5 μg/ml hydrocortisone, 30 ng/ml cholera toxin, 0.18 mM adenine, 50 μg/ml gentamicin, and 1.25 μg/ml amphotericin B. Cells were incubated at 37 °C in 5% CO₂ and the medium was changed every 3 days.

At 80-90% confluence, cells were digested with 0.25% trypsin/0.02% EDTA and then replated into plastic plates or on the basement membrane side of HAM prepared as described above at a density of 1x10⁴ cells/cm². Cells were cultured for an additional 2-3 weeks and either transplanted onto the rabbit corneas or fixed with formalin for immufluorescent staining.

RT-PCR Analysis

Total RNA was isolated from cells using the RNeasy Mini Kit (Qiagen) and quantified using ultraviolet absorption at 260 nm. RT-PCR was performed using 0.25 μg total RNA in 25 μl reactions.

Primers used for human ΔN p63 are 5'-CAG ACT CAA TTT AGT GAG (sense) and 5'-AGC TCA TGG TTG GGG CAC (antisense) [19]. PCR was carried out at 94 °C for 2 min, 94 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s, for 40 cycles, then at 72 °C for 10 min.

Human primers for K12 are 5'-CTA CCT GGA TAA GGT GCG AGC T (sense) and 5'-TCT CGC ATT GTC AAT CTG CA (antisense). PCR was carried out at 94 °C for 2 min, 94 °C for 30 s, 58 °C for 45 s, 72 °C for 30 s, for 35 cycles, then at 72 °C for 10 min.

Primers for human β-actin are 5'-GAG GCG TAC AGG GAT AGC AC-3' (sense) and 5'-GTG GGC ATG GGT CAG AAG-3' (antisense). PCR was carried out at 94 °C for 2 min, 94 °C for 30 s, 60 °C for 30 s, 72 °C for 45 s, for 35 cycles, then at 72 °C for 10 min.

Animal model of limbal stem cell deficiency

All animals were handled according to the animal protocols approved by the Peking University Institutional Animal Care and Use Committee.

Twenty New Zealand white rabbits (weighing 2-2.5 kg) were anesthetized with intravenous injection of pentobarbital (20-30 mg/kg), complemented with peribulbar anaesthesia (laedocaine) and topical proparacaine. Ocular surface damage was generated in the right eye of each rabbit. A filter paper ring with an inner diameter of 11 mm and outer diameter of 15 mm, saturated in 1 N NaOH, was applied to the eye for 30 s. A surgical blade was then used to remove residual limbal and central epithelia followed by irrigation with PBS. Dexamathazone-gentamicin solution was applied to each treated eye twice a day for 14 days after epithelial debridement and limbal ablation.

Surgical technique

Limbal cells cultured on HAM for 2-3 weeks were transplanted onto the injured rabbit corneas one month after the initial injury. The damaged corneal surfaces, including a 5 mm zone of peripheral conjunctival and subconjunctival tissue, were carefully keratectomized under anesthesia. The HAM was secured, with the basement membrane (with- or without cultured cells) up, onto the corneal surface with 10-0 nylon sutures, then another HAM was secured to cover the first with the basement membrane face down (Figure 1).

For autologous limbal transplantation, the procedure was adapted from that described by Kenyon and Tseng [23]. Briefly, a 360° limbal lamellar keratectomy was performed on the contralateral cornea. This ring of limbal tissue was then placed on the damaged corneal surface of the other eye that was keratectomized at the same time. The limbal transplant was secured to the limbal area by episcleral sutures using interrupted 10-0 nylon sutures.

After surgery, 0.5% cyclosporin A and 0.05% dexamethasone-gentamicin solution were instilled as ocular drops three times a day during the first week and twice a day thereafter. Cyclosporin A (5 mg/kg/day) and ampicillin (200 mg/kg/day) were injected intramuscularly for the first week and then reduced to half dosage to prevent xenograft rejection and postoperative infection.

Ocular surface evaluation

Rabbits were examined using a hand-held light daily and a slit-lamp once a week. A sodium fluorescein solution was used to indicate whether the corneal epithelial cells were damaged or reepithelialized. If corneal epithelial cells are integrated (reepithelialization), the cornea will not stain with fluorescein. The corneal surface was also examined for smoothness, clarity, and vascularization by slit-lamp examination.

Immunofluorescent staining

Forty days after HAM surgical placement, the rabbits were sacrificed and their corneas were collected, fixed in paraformaldehyde, and embedded in paraffin for sectioning and staining. Paraffin was removed from slides in xylene and the samples were rehydrated. Slides were rinsed 3 times in 0.5% Triton-X100 in PBS for 15 min and then in 0.01 M citrate buffer (PH 6.0) at 98 °C for 5 min. Samples were rinsed 3 times in 0.5% Triton-X100 in PBS for 5 min and incubated with 2% bovine serum albumin in PBS for 30 min. Primary antibodies were applied to tissues and incubated in a moist chamber for 1 h. After 3 rinses in 0.1% Tween-20 for 5 min each, the fluorescein-conjugated anti-mouse or anti-rabbit IgG was applied and incubated in a moist chamber for 1 h. For double-staining, mouse IgG was applied for 30 min to block the remaining antigens before another antibody was added. Samples were mounted in glycerol-carbonate buffer (glycerol/carbonate buffer=9:1, 0.5 M carbonate buffer, pH 8.5) for visualization and photographed using an Olympus Cool-CD or Leica Confocal microscope. Negative controls were incubated in mouse IgG instead of the primary antibody.

Results

Cultured limbal cells express p63

Nuclear protein p63 has been suggested as a marker for epithelilal stem cells and K12 is known to be expressed exclusively by differentiated corneal epithelial cells. We initially investigated expression of these markers in primary cultures of cells from central human epithelium and from limbal regions of adult and fetal human corneas. Gene expression in the cultures was compared using RT-PCR. Stromal fibroblast cells were also used as a negative control. As shown in Figure 2, corneal epithelial cells express K12 but not p63, whereas primary cultures of cells from both fetal and adult limbal express both K12 and p63. The expression of both genes in the limbal cultures suggested that these cultures were heterogeneous, containing both stem cells and differentiated epithelial cells.

Amniotic membrane promotes p63 positive cell proliferation

To test whether or not HAM could promote LSC proliferation in vitro, we used immunofluorescent staining to identify the expression of p63, connexin43, and K3 proteins among cells cultured either on HAM or on plastic. The cultured cells formed a dense monolayer of uniform morphology (Figure 3A,B). Finger-like projections and microvillus structure on the cell surface, features typical of epithelial cells, were observed by electron microscopy (Figure 3A, inset). The proportion of p63-positive cells in the cells cultured on HAM (Figure 3D) is much higher than that of cells cultured on plastic (Figure 3C). We counted p63 positive cells in 30 fields from 3 specimens. The percentage of p63 positive cells was 71%±16% (mean±SD) on HAM, but was only 16%±13% for cells cultured on plastic (p<0.05). On the other hand, the proportion of connexin43 positive cells was much lower in cells cultured on HAM (Figure 3F) than that of cells on plastic plate (Figure 3E). In cells cultured on HAM (Figure 3H), the number of p63 positive cells was much higher than that of connexin43 positive cells. Some cells were positive for both p63 and connexin43 (Figure 3G) when cultured on plastic, but this was rarely observed in cells cultured on HAM (Figure 3H). On the contrary, the number of K3 positive cells cultured on plastic plate was higher (Figure 3I) than that of the cells cultured on HAM (Figure 3J).

Ocular surface evaluation

To test the proposed function of HAM in vivo, human limbal cells cultured on HAM were used to reconstruct rabbit corneal epithelium in a chemical burn model of LSCD. Four weeks after the injury, all eyes showed moderate to severe corneal vascularization with epithelial defects, detected by positive fluorescein staining (yellow-green, Figure 4A,C,E). Impression cytology examination was used to detect conjunctiva goblet cells on the corneal surface to confirm LSCD had occurred. All twenty eyes used in the experiment were positive for Periodic Acid Schiff staining, indicating the presence of goblet cells (data not shown).

After transplantation, we observed that transplanted HAM began to be absorbed two weeks later and was absorbed completely around 5 weeks after the transplantation. Evaluation of transplantation, success was determined by reepithelization (absence of fluorescein staining), decreased or absent neovascularization, improved corneal transparency, and negative Periodic Acid Schiff staining. If a cornea demonstrated any of the following: Fluorescein staining, severe neovascularization, decreased or no improved transparency, or positive Periodic Acid Schiff staining, it was considered a failure.

As summarized in Table 1, 8 of 10 eyes that received transplantation of limbal cells cultured on HAM (row A, HAM-LC transplantation) showed smooth surface, minimal vessels, and clear stroma, i.e were considered successful. Of the other two eyes receiving HAM-LC transplantation, one was infected, and the transplanted HAM on the other eye peeled off 4 days after surgery. Figure 4B shows completed epithelialization 40 days after HAM-LC transplantation, displaying an avascular surface and negative fluorescein staining. Healing occurred approximately ten days after surgery.

In comparison, 5 of 6 eyes receiving amniotic membrane transplantation (AMT) alone, i.e. HAM treated similarly and cultured in the same medium, omitting seeded cells (Table 1, row B, HAM-Transplantation) did not show recovery, as indicated by non-uniform fluorescein staining during the whole follow-up period. Figure 4D shows neovascularization and epithelial defects with fluorescein staining 40 days post operation of AMT. One cornea that received AMT was completely recovered with a normal ocular surface, perhaps due to regrowth of endogenous limbal stem cells.

As a positive control, 2 eyes received autologous limbal transplantation using limbal tissue obtained from the contralateral corneal limbus (Table 1, row C). These eyes showed a smooth surface and non-neovascularization (not shown). As a negative control, 2 eyes receiving keratectomy only without transplantation exhibited delayed healing, with diffuse vascularization and severe stromal opacity (Figure 4F and Table 1, row D).

Using the Fisher Exact Test [24], we found that there were significant differences between HAM transplantation with and without cells (Table 1, rows A and B), HAM with cells and no transplantation (Table 1, rows A and D), and between autologous limbal transplantation and no transplantation (Table 1, rows C and D) at the p<0.05 level, but there was no significant difference between rows B and D (p>0.05).

Epithelial cells of reconstructed rabbit corneas were of human origin

Forty days after reconstruction, the rabbit corneal epithelia were evaluated by immunofluorescent staining. Anti-human nuclear, anti-p63, anti-K3, and anti-connexin43 antibodies were used to determine the origin and type of cells. Almost all the epithelial cells in corneas with transplants of human limbal cells cultured on HAM were positive for anti-human nuclear antibody (Figure 5A), while the non-transplanted corneas were negative (Figure 5C). Anti-p63 antibody staining was positive both in non-wounded and transplanted rabbit corneas, but the distribution of p63 positive cells was different in that p63 positive cells were distributed throughout the transplanted corneas (Figure 5B), but in rabbit corneas without transplantation p63 positive cells were located at the basal layer exclusively (Figure 5D). K3 was present in both xenotransplanted and non- transplanted rabbit corneas (Figure 5A-D). Some cells were positive for p63 and negative for connexin43, while some cells were positive for both p63 and connexin43. Reaction of anti-p63 and anti-K3 antibodies with both human and rabbit proteins was confirmed by western blot analysis (data not shown).

Discussion

Expression of p63 was previously suggested as a marker for limbal stem cells [20], but its specificity has not been confirmed up to now. Our results show that there is a significant difference of p63 expression between cultured cells derived from limbal cells and corneal epithelial cells. RT-PCR shows that cultured cells from LCs are p63 positive while corneal epithelial cells negative (Figure 2). By immunofluorescent staining, we found that many of the cells cultured on plastic were positive for both p63 and connexin43 (Figure 3G). In vivo, gap junction protein connexin43 (Cx43) expression has been shown to be absent in the limbal basal epithelium but present in the corneal basal epithelium [25]. Thus expression of Cx43 serves as a negative marker for LSCs. Co-expression of p63 and Cx43 suggests that p63 is not limited to stem cells. On the other hand, the inverse response of p63 compared to K12, K3, and Cx43 suggests that p63 may be a marker of undifferentiated precursor cells (stem cells or transient amplifying cells) in limbal cultures. Thus we speculate that p63 may not be specific for LSC, but is a marker for both limbal stem cells (LSC) and transient amplifying cells (TAC). Our results suggest that LSCs are p63 positive and connexin43 negative, whereas TAC are positive for both p63 and connexin43.

When cells from the limbal region were passaged, the cultures were positive for both p63, K3, and K12. This suggests that these cultured cells were heterogeneous, containing both precursor cells and differentiated epithelial cells. When passaged, some stem cells kept their stem cell-like character while some differentiated into transient amplified cells and terminal differentiated epithelial cells. It remains an open question as to how stem cells in culture might be induced to proliferate without differentiation.

We found that the proportion of p63-positive cells cultured on HAM is much higher than that of cells cultured on plastic, indicating that HAM limits differentiation in culture, maintaining a greater proportion of functional precursor cells. Cells cultured on HAM can retain their stem cell-like character better than those cultured on plastic. These conclusions are in agreement with several recent studies that have addressed the same question using different experimental approaches [13,15,22,26-30].

The basement membrane and its underlying extracellular matrix of HAM contain collagen alpha 2(IV), type VII, and laminins-1 and -5 [7]. Therefore, HAM may provide an appropriate microenvironment in which LSC can proliferate. Grueterich, et al. [15] proposed that removal of epithelial cells from the amniotic membrane to expose the basement membrane may promote TAC differentiation, but that culture of limbal cells on intact amniotic membrane from which the (previously frozen) epithelial layer has not been removed may preserve and expand the number of progenitor cells. The results of our study differ somewhat, perhaps due to differences in HAM preparation and cell culture. We believe that human amniotic membrane may provide the means to maintain stem cell proliferation while preventing differentiation.

After transplantation of human limbal cells cultured on HAM, injured rabbit corneas with LSCD are intact, recovered by epithelial regeneration, and show neovascular regression (Figure 4B). Immunofluorescent staining of the rabbit corneal sections verified that transplanted human cells remain present (Figure 5A,B). We also detected both p63-positive progenitor cells and K3-positive differentiated epithelial cells on the corneal slides. However, we observed that the distribution of the p63 positive cells was not restricted to the limbal basal cell area where normally LSC are segregated (Figure 5B,D). The reconstruction of a fully functional epithelium is strong evidence that limbal stem cells survive and proliferate on the HAM in vitro. Our conclusions are in agreement with a number of recent studies in which limbal cells cultured on HAM have been successfully transplanted to LSCD corneas [6,12,14,30-33]. Unlike these previous studies, the human-rabbit xenotransplantation format of the present study allowed us to determine the origin of cells in the reconstituted epithelium, demonstrating that a monolayer of cultured limbal cells was as effective as that of stratified epithelial cells in repopulation of the restored epithelial layer [32,34]. This study also appears to be the first to track expression of p63 in the reconstructed epithelium, demonstrating a large population of p63 positive cells from the human donor remaining in the healed epithelium. These results suggest that during repopulation progenitor cells are not limited to the basal layer but are distributed throughout the epithelial layer. Even after 40 days of healing and reformation of the normal stratified arrangement of the epithelial layer, such p63 positive human cells were present throughout the layer.

This report provides new experimental information comparing fetal and adult limbal cells. We found no significant difference in cell morphology, phenotype, and proliferation in cultured fetal and adult limbal cells, indicating that a naturally-aborted fetus could be useful to benefit both research and clinical work.

Based on the results from our research, we conclude that rabbit corneas with LSCD can be successfully reconstructed by transplantation of adult cultured human limbal cells. This technique provides a new perspective with important clinical implications for ocular surface reconstruction using hetero- or xeno- stem cell transplantation.

Acknowledgements

The authors thank Aili Lu, Li Shen, Huiqun Wu, Zuguo Liu, Wenyuan Guo, Shuling Wang, Yinan Liu, Haiyan Yu, Huiyang Zeng, and Juan Du for their help in doing all the work for this paper. The authors also thank Martha L. Funderburgh for reading and critiquing this manuscript. This work is supported by grants from the Chinese National 973 Project (2002CB510100), 863 Project (2001AA216171) to L Li and NIH grants (EY09836 and EY013806) to JL Funderburgh. JL Funderburgh is a Jules and Doris Stein Research to Prevent Blindness Professor.

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