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Recent advances in ocular surface reconstruction for patients with severe ocular surface diseases have significantly improved the prognosis of patients with vision-impairing corneal abnormalities. The history of cultivated epithelial sheet transplantation is short, and debate on the current approaches for these procedures is continuing. Limbal stem cell transplantation, including conjunctivolimbal autograft and keratolimbal allograft, has brought opportunities for vision improvement. In addition, the use of cultivated limbal epithelial transplantation from both allogeneic and autologous sources has provided further options for immediate postoperative epithelialization of the corneal surface. Finally, cultivated oral mucosal epithelial transplantation, which allows autologous transplantation for patients with bilateral limbal stem cell deficiency, has provided the best overall midterm and long-term results. Its biggest advantages are the absence of rejection reactions and the reduction of postoperative complications associated with steroid therapy. However, a solitary surgical approach is not sufficient for obtaining a good clinical outcome. To maximize the possibility of success using these procedures, it is important to preoperatively enhance aggressive treatment of the ocular surface, especially with factors that facilitate moisture retention. In this review article, we also discuss our clinical experience in relation to these surgical procedures.

Corneal epithelial stem cells are located in the limbus, the junction between the cornea and the conjunctiva. A limbal epithelium model in vitro would be useful for the study of epithelial stem cells, as well as improving the quality of cultivated epithelial sheets for the treatment of limbal stem cell deficiency. In this study, we succeeded in constructing a limbal epithelium-like structure that could be maintained for at least 5 months in vitro. We modified conventional medium by replacing epidermal growth factor with keratinocyte growth factor (KGF) and adding Y-27632, a rho kinase inhibitor. Using this medium, epithelial cells freshly isolated from human limbus were cocultured with human mesenchymal stem cell-derived feeder cells. Cells formed a stratified layer without air exposure, and both basal and suprabasal layers maintained their unique morphologies for up to 5 months. Basal layers expressed the progenitor marker p63 uniformly and K15 heterogeneously. Expressions of PAX6, K3, and K12 indicated that cell sheets underwent normal differentiation in the corneal epithelium lineage. Although medium was changed daily after day 7, cell debris was observed every day, suggesting that cell sheets underwent turnover. Furthermore, secondary colonies were observed from cells dissociated from 1-month and 3-month cultured sheets. In conclusion, human limbal epithelial cell sheet cultures with KGF and Y-27632 maintained stratification, high expression of both stem/progenitor markers and differentiation markers, and colony-forming cells long-term. This protocol may be useful as an in vitro limbal epithelial model for basic studies.

Epithelial-mesenchymal transition (EMT), via activation of Wnt signaling, is prevailing in embryogenesis, but postnatally it only occurs in pathological processes, such as in tissue fibrosis and tumor metastasis. Our prior studies led us to speculate that EMT might be involved in the loss of limbal epithelial stem cells in explant cultures. To examine this hypothesis, we successfully grew murine corneal/limbal epithelial progenitors by prolonging the culture time and by seeding at a low density in a serum-free medium. Single cell-derived clonal growth was accompanied by a gradient of Wnt signaling activity, from the center to the periphery, marked by a centrifugal loss of E-cadherin and β-catenin from intercellular junctions, coupled with nuclear translocation of β-catenin and LEF-1. Large-colony-forming efficiency at central location of colony was higher than peripheral location. Importantly, there was also progressive centrifugal differentiation, with positive K14 keratin expression and the loss of p63 and PCNA nuclear staining, and irreversible EMT, evidenced by cytoplasmic expression of α-SMA and nuclear localization of S100A4; and by nuclear translocation of Smad4. Furthermore, cytoplasmic expression of α-SMA was promoted by high-density cultures and their conditioned media, which contained cell density-dependent levels of TGF-β1, TGF-β2, GM-CSF, and IL-1α. Exogenous TGF-β1 induced α-SMA positive cells in a low-density culture, while TGF-β1 neutralizing antibody partially inhibited α-SMA expression in a high-density culture. Collectively, these results indicate that irreversible EMT emerges in the periphery of clonal expansion where differentiation and senescence of murine corneal/limbal epithelial progenitors occurs as a result of Smad-mediated TGF-β-signaling., (Copyright © 2012 Wiley Periodicals, Inc.)

Purpose: Ultraviolet (UV) acts as low-dose ionizing radiation. Acute UVB exposure causes photokeratitis and induces apoptosis in corneal cells. Astaxanthin (AST) is a carotenoid, present in seafood, that has potential clinical applications due to its high antioxidant activity. In the present study, we examined whether topical administration of AST has preventive and therapeutic effects on UV-photokeratitis in mice., Methods: C57BL/6 mice were administered with AST diluted in polyethylene glycol (PEG) in instillation form (15 μl) to the right eye. Left eyes were given vehicle alone as controls. Immediately after the instillation, the mice, under anesthesia, were irradiated with UVB at a dose of 400 mJ/cm². Eyeballs were collected 24 h after irradiation and stained with H&E and TUNEL. In an in vitro study, mouse corneal epithelial (TKE2) cells were cultured with AST before UV exposure to quantify the UV-derived cytotoxicity., Results: UVB exposure induced cell death and thinning of the corneal epithelium. However, the epithelium was morphologically well preserved after irradiation in AST-treated corneas. Irradiated corneal epithelium was significantly thicker in eyes treated with AST eye drops, compared to those treated with vehicles (p<0.01), in a doses dependent manner. Significantly fewer apoptotic cells were observed in AST-treated eyes than controls after irradiation (p<0.01). AST also reduced oxidative stress in irradiated corneas. The in vitro study showed less cytotoxicity of TKE2 cells in AST-treated cultures after UVB-irradiation (p<0.01). The cytoprotective effect increased with the dose of AST., Conclusions: Topical AST administration may be a candidate treatment to limit the damages by UV irradiation with wide clinical applications.

Previous observations in a rat model of a non-Sjögren's syndrome (non-SS) type of dry eye seen in users of visual display terminals (VDT) indicated that secretory vesicle (SV) accumulation in the lacrimal gland epithelia contributes to the condition. Here, to examine this possibility in humans, we compared the lacrimal gland histology and percent SV area in the cytoplasm of acinar epithelial cells using light microscopy and transmission electron microscopy, in patients with VDT work-related non-SS dry-eye (VDT group), SS-induced dry-eye, and autopsied normal controls. In addition, the VAMP8 (vesicle-associated membrane protein 8, an exocrine-pathway molecule) and Rab3D (mature vesicle marker) were histochemically examined in lacrimal gland tissue sections. The lacrimal gland acini were larger in the VDT group than in the SS group, and the percent SV area was significantly higher in the VDT group than in the normal controls (P = 0.021) or SS group (P = 0.004). Immunostaining revealed abnormal distributions of VAMP8 in the VDT and SS groups. Rab3D was more strongly expressed in the cytoplasm of acinar epithelial cells in the VDT group than in that of normal controls. The duration of VDT use was significantly longer in the VDT group than in the other groups. These findings suggest that excessive SV accumulation in the acinar epithelia may contribute to the reduced tear secretion in VDT users.

The ocular surface is strongly affected by oxidative stress, and anti-oxidative systems are maintained in corneal epithelial cells and tear fluid. Dry eye is recognized as an oxidative stress-induced disease. Selenium compound eye drops are expected to be a candidate for the treatment of dry eye. We estimated the efficacy of several selenium compounds in the treatment of dry eye using a dry eye rat model. All of the studied selenium compounds were uptaken into corneal epithelial cells in vitro. However, when the selenium compounds were administered as eye drops in the dry eye rat model, most of the selenium compounds did not show effectiveness except for Se-lactoferrin. Se-lactoferrin is a lactoferrin that we prepared that binds selenium instead of iron. Se-lactoferrin eye drops suppressed the up-regulated expression of heme oxygenase-1, cyclooxygenase-2, matrix metallopeptidase-9, and interleukin-6 and also suppressed 8-OHdG production in the cornea induced by surgical removal of the lacrimal glands. Compared with Se-lactoferrin, apolactoferrin eye drops weakly improved dry eye in high dose. The effect of Se-lactoferrin eye drops on dry eye is possibly due to the effect of selenium and also the effect of apolactoferrin. Se-lactoferrin is a candidate for the treatment of dry eye via regulation of oxidative stress in the corneal epithelium.

Purpose: To date, mouse lacrimal gland epithelial cells have been cultured successfully but only in cases involving newborn mouse lacrimal glands. In this work, we attempted to cultivate and characterize adult mouse lacrimal gland epithelial cells., Methods: Lacrimal glands were removed from newborn mice (C57B/6) and isolated lacrimal gland epithelial cells were seeded onto tissue culture treated or low adherent culture dishes in Cnt-07 culture medium with or without cholera toxin. Cultivated cells were characterized by immunostaining with pan-cytokeratin, α-smooth muscle actin, and lactoferrin antibodies. Lacrimal gland cells from 7-week-old green fluorescent protein (GFP) and non-GFP (C57B/6) mice were mixed and seeded onto uncoated dishes to assess sphere-forming efficiency. Cells were also seeded onto 3T3 cell feeder layers to assess colony forming efficiency., Results: Lacrimal gland epithelial cells were selectively cultured with cholera toxin, and cell type was verified by pan-cytokeratin and α-smooth muscle actin immunostaining. Sphere formation from single cells of adult mice was observed using specific medium and low adherent culture dishes. These cells could also undergo colony formation on 3T3 feeder cells., Conclusions: Adult mouse lacrimal gland epithelial cells were successfully cultivated in cholera toxin-containing medium, and were observed to form spheres from single cells.

Purpose: To investigate the effect of desiccation on secretion of inflammatory cytokines in corneal epithelial cells and in the rat desiccation model., Methods: A human corneal epithelial cell line (CEPI) was grown in keratinocyte growth medium 2 (KGM2) to approximately 80% confluence. The medium was aspirated and dishes were left for 0 to 30 min with the cover left open to dry the cells (short-term desiccation). After desiccation, KGM2 was added to the dishes and collected from the dishes 15 min later to measure the concentrations of cytokines in the medium by sandwich enzyme immunoassay (ELISA). Viability of the cells was estimated with alamer blue. To study the effect of long-term desiccation, cultivated cells on transwells were used. After dessiccation for up to 8 h, the viability of the cells and levels of cytokines in the culture medium were examined. The expression of cytokines in the cornea of the dry eye model rat was measured by real-time PCR., Results: Short-term dessication of CEPI cells significantly increased the interleukin (IL)-6 level and slightly increased the tumor necrosis factor (TNF)-α level. Anti-IL-6 antibody partially suppressed cell death caused by desiccation. Upon long-term desiccation, IL-6 and IL-8 levels were increased. 
In the dry eye model rats, the IL-6 mRNA level in the cornea significantly increased, whereas TNF-α mRNA level slightly increased., Conclusions: Desiccation induced IL-6 expression in corneal epithelial cells, suggesting that IL-6 participates in desiccation-induced cell death.

Chronic graft-versus-host disease (cGVHD) of the ocular surface and lacrimal gland is a vision-threatening condition that occurs after allogeneic bone marrow transplantation. In this study, we used immunohistochemistry and electron microscopy to investigate whether epithelial mesenchymal transition (EMT) contributed to the pathogenesis of ocular cGVHD. We detected down-regulation of E-cadherin and translocation of beta-catenin from the intercellular junction to the cytoplasm and nucleus of cGVHD conjunctival basal epithelia and lacrimal gland myoepithelia. Notable findings included expression of Snail, an inducer of EMT, in the nucleus of ocular cGVHD epithelia. The fibrosis markers heat shock protein 47, alpha-smooth muscle actin, and fibroblast specific protein-1 were overexpressed in ocular cGVHD epithelia. In addition, p63, a marker of conjunctival basal epithelia, was observed in the nuclei of subconjunctival cells beneath disrupted basal lamina. Disrupted basal lamina and the presence of altered collagen bundles were observed in the cytoplasm and beneath cGVHD epithelia. In contrast, these observations were rarely observed in the normal conjunctiva and in Sjögren's syndrome lacrimal gland epithelia. These findings together indicate that ocular cGVHD epithelia gain the mesenchymal phenotype and the capacity to migrate into the subepithelial stroma. Our findings suggest that EMT may be partially responsible for the conjunctival and lacrimal gland fibrosis found in patients with cGVHD.

Purpose: Nucleostemin, a nuclear protein involved in the regulation of cell cycle and proliferation, is a candidate marker for various stem cells. We examined the expression of nucleostemin as a marker of differentiation and senescence in corneal epithelial progenitor cells., Methods: Nucleostemin expression in normal mouse corneal epithelium was examined by RT-PCR, as well as in cultured mouse corneal epithelial cells by immunohistochemistry. Co-expression with the progenitor markers p63 and Bmi-1, cytokeratin 14 and 19, the proliferating marker Ki67, differentiation markers cytokeratin 12 and involucrin, and with the senescent marker SA-beta-gal were examined by immunohistochemistry. Correlation of cellular size and nucleostemin expression was also examined., Results: Nucleostemin expression was detected in mouse corneal epithelium by RT-PCR. Immunohistochemistry revealed that nucleostemin was expressed predominantly in basal and suprabasal cells of the whole cornea. The expression of nucleostemin was not associated with the expression of Ki67, K14, and K19, but with the expression of Bmi-1 and particularly with p63. Nucleostemin was not co-expressed with SA-beta-gal in same cell., Conclusions: Nucleostemin can be used as a progenitor marker analogous to p63.

Purpose: To report the efficacy of human bone marrow-derived mesenchymal stem cells as a source of feeder cells for the cultivation of transplantable corneal epithelial cell sheets., Methods: Human mesenchymal stem cells (marrow adherent stem cells; MASCs) were cultured in alpha-modified Eagle's medium with 10% serum and were treated with mitomycin C. Expression of cytokines in MASCs was confirmed by reverse transcription-polymerase chain reaction. Human limbal epithelial cells were cocultured with MASCs or 3T3 feeder cells to compare colony-forming efficiency (CFE). Limbal epithelial cells were cultured on MASCs or 3T3 feeder cells at the air-liquid interface to allow stratification, and stratified epithelial sheets were analyzed by immunohistochemistry against cytokeratin 3 (K3), K15, p63alpha, and ABCG2. Rabbit limbal epithelial cell sheets were cultivated with MASC feeder cells and transplanted to the ocular surface of the limbal-deficient rabbits. Epithelial grafts were observed by slit lamp microscopy for 4 weeks and then evaluated by histology and immunohistochemistry against K3 and K4., Results: MASC feeder cells expressed keratinocyte growth factor, hepatocyte growth factor, and N-cadherin. The CFE of human limbal epithelial cells was similar in MASC and 3T3 feeder groups. Stratified cell sheets were successfully cultivated with MASC feeder cells expressing K3, K15, p63alpha, and ABCG2. Transplanted epithelial sheets regenerated the corneal phenotype in limbal-deficient rabbits., Conclusions: MASC-derived feeder cells are suitable for the engineering of epithelial sheets, avoiding the use of potentially hazardous xenologic feeder cells.

The limbal region of the adult cornea contains stem cells which are ultimately responsible for regeneration of the corneal epithelium during wound repair. However, primarily-isolated murine corneal/limbal epithelial cells rapidly senesce on plastic in a serum-free low [Ca(2+)] medium, suggesting only transit amplifying cells are promoted. We developed a novel expansion method by seeding at a low cell density (<500 cells/cm(2)) and prolonging each culture time beyond the lifespan of transit amplifying cells (4 weeks). Expanded cells were uniformly small, negative to K12 keratin, but positive for p63 nuclear staining, and could be subcultured beyond 100 passages. After limiting dilution, one clone (TKE2) was selected that exhibited single cell clonal expansion with a doubling time of 34.2 hrs, and had normal karyotyping, but no anchorage-independent growth. A single cell could be continually expanded to a confluent monolayer on denuded amniotic membrane and became stratified by exposing to the air-medium interface. The resultant stratified epithelium expressed K14 keratin, involucrin, connexin 43 and p63, but not K12 keratin or Pax 6. However, expression of K12 could be up-regulated by increasing extracellular calcium concentration and addition of foetal bovine serum (FBS) at P12, but less so at P85. Therefore, this murine lim-bal/corneal epithelium-derived progenitor cell line still retained the plasticity for adopting corneal lineage differentiation, could be useful for investigating limbal niche cues that may promote corneal epithelial fate decision.

Corneal epithelial cell sheets co-cultivated with feeder cells are used to reconstruct the ocular surface in stem cell-depleted eyes. The present study was conducted to investigate the optimal method of using feeder cells in the interest of preserving progenitor cells in cultivated sheets. We compared the phenotype and secondary colony forming efficiency (CFE) of cell sheets that were engineered using 3T3 feeder cells as a separate layer or as a contact layer. We also devised a novel "duplex feeder" system that consists of two separate layers of feeder cells. After cells reached confluence, cells were cultured at the air-liquid interface to allow full stratification. Stratified sheets were then analyzed using immunohistochemistry and secondary colony formation. Contact feeder cultures and duplex feeder cultures yielded epithelial sheets with small, cuboid basal cells with strong expression of keratin (K)3, K12, and K 15. Furthermore, only duplex feeder layers reproduced the basal K 15, suprabasal K12 limbal phenotype where epithelial stem cells reside. A similar effect was observed when cornea stroma-derived progenitor cells were used as feeder cells. Duplex feeder sheets also produced significantly more secondary colonies than cells dissociated from single layer sheets, suggesting that the duplex feeder system produces transplantable sheets with a higher yield of progenitor cells.

Purpose: To demonstrate the effects of hypoxia on proliferation and differentiation of human limbal epithelial cells in vitro., Methods: Primary human limbal epithelial cells were harvested from the rim of donor corneas. Colony-forming efficiency (CFE) and cell proliferation were observed in standard (20% O(2)) or hypoxic (2% O(2)) culture conditions. Cell cycle, forward scatter (FSC) and side scatter (SCC) of cells were analyzed by flow cytometry. Proliferating cells were also observed by pulse labeling (2 hours) with BrdU and Ki67 staining. Apoptosis was detected by TUNEL assay. Isolated colonies were examined by immunohistochemistry against K15, p63, involucrin, and K3. Involucrin expression was also analyzed by Western blot analysis., Results: Both CFE and proliferation of limbal epithelial cells was significantly enhanced in hypoxia. Flow cytometry revealed a higher fraction of hypoxic cells in the G(0)/G(1)-phase and fewer cells in the S-phase, compared with normoxia. However, there was no difference in the uptake of BrdU during a 2-hour pulse, suggesting that hypoxic colonies contained rapidly cycling cells. Apoptotic cells were sparse in both groups, and hypoxic cells showed lower FSC compared with normoxic cells. Although there was no difference in the staining pattern of K15, p63, and Ki67, cells cultivated in normoxia expressed higher levels of the differentiation markers involucrin and K3. Significantly higher involucrin expression was also observed by Western blot., Conclusions: Hypoxic culture (2%) enhances proliferation while inhibiting differentiation of limbal epithelial cells in vitro.

Purpose: To analyze factors influencing clinical outcomes in cultivated limbal epithelial transplantation (CLET)., Design: Retrospective, observational case series., Methods: Twenty-seven eyes of 27 patients that had CLET for severe chronic cicatricial ocular surface disorders were studied. Two different cultivation methods were used to prepare epithelial sheets. Method 1 used the explant technique and neither feeder cells nor air-lifting were used. In Method 2, cell suspension technique and 3T3 feeder cells were used, and air-lifting was applied after cultivated cells became confluent. Clinical outcomes including corneal surface epithelialization and incidence of postoperative complications were studied. The relationship between the clinical outcome and type of cultivation method, original diseases, tear function, or preoperative ocular surface status was also studied., Results: Both cultivation methods produced transplantable epithelial sheets with corneal phenotype. With a mean follow-up period of 127 weeks, corneal epithelialization was achieved in 16 eyes (59.3%). Eyes that had CLET with Method 1 suffered more severe postoperative complications such as infection, ulceration, and perforation (P = .053). Eyes with Stevens-Johnson syndrome (SJS) had poor final corneal epithelialization compared with other diseases (P = .034). CLET was more successful when performed to conjunctivalized corneas compared with eyes with persistent epithelial defects or with dermalized corneas., Conclusions: CLET offers new treatment modalities to chronic cicatricial ocular surface disorders with moderate success rates. The treatment is feasible for eyes with non-immune-mediated disorders with stable ocular surface conditions. Epithelial sheets with better structural integrity seem to be superior to obtain early postoperative epithelialization and to avoid serious postoperative complications.

Purpose: To investigate whether beta-catenin activation and epithelial-mesenchymal transition (EMT) is involved in the pathogenesis of pterygium., Methods: beta-Catenin and E-cadherin expression were examined in surgically excised tissue and eye bank corneas with intact pterygium. Snail and Slug, the transcriptional repressors of E-cadherin, and matrix metalloproteinase (MMP)-7, a down-stream gene regulated by beta-catenin were also investigated. Epithelial cells undergoing EMT-like changes were identified by double immunostaining for alpha-smooth muscle actin (SMA)/vimentin and cytokeratin 14. Transmission electron microscopy was used to examine the ultrastructure of the pterygial head., Results: Histopathology showed aberrant fibrotic proliferation beneath the pterygium epithelium, with epithelial processes extending into the stroma. Transmission electron microscopy revealed the dissociation of epithelial cells, which were surrounded by activated fibroblast-like cells. Characteristic downregulation of E-cadherin and intranuclear accumulation of beta-catenin and lymphoid-enhancer-factor-1 in pterygial epithelium were also observed by immunohistochemistry. Of note, epithelial cells extending into the stroma were positive for both alpha-SMA/vimentin and cytokeratin 14. Snail and Slug were immunopositive in the nuclei of pterygial epithelial cells, but not in normal corneal epithelial cells., Conclusions: EMT of basal epithelial cells may play a key role in the pathogenesis of pterygium.

Purpose: To elucidate the expression pattern of K15, K19, K14, and K12 in human and mouse ocular surface epithelium as putative markers of epithelial phenotype., Methods: Immunohistochemical staining with specific antibodies for K15, K19, K14, and K12 was performed in human donor cornea tissue and normal ICR mouse corneas, with emphasis on localization of immunopositive cells. Immunohistochemistry was performed in a limbus-deficient mouse model as well as in clinical samples of pannus surgically removed from a thermal burn and a patient with Saltzmann's dystrophy. Staining patterns were classified as limited to the most basal layer (K(bas)), basal and suprabasal layers (K(bas-sup)), predominantly in suprabasal layers (K(sup)) and negative staining (K(-))., Results: In human conjunctival epithelium, strong expression of K15 was observed in basal cells, whereas K19 was expressed in both basal and suprabasal layers (K15(bas)/K19(bas-sup)/K12(-)). Limbal epithelial cells were K15(bas-sup)/K19(bas-sup)/K12(sup), whereas epithelial cells in the central cornea were K15(-)/K19(bas-sup)/K12(bas-sup). In contrast, the mouse ocular surface demonstrated a different expression pattern of K15 and K19 than did the human tissue in the conjunctiva (K15(bas-sup)/K19(bas)/K12(-)) and the limbus (K15(bas-sup)/K19(bas)/K12(sup)). Neither K15 nor K19 was expressed in the central mouse cornea (K15(-)/K19(-)/K12(bas-sup)). Similar cytokeratin expression was observed in conjunctivalized corneas in mice and in surgically removed pannus tissue., Conclusions: Although the expression of K15 and K19 differ in humans and mice, specific staining patterns can be used to characterize the epithelial phenotype in normal and diseased ocular surface.

Purpose: Ex vivo expansion of limbal epithelial progenitor cells on amniotic membrane (AM) without 3T3 fibroblasts is a new surgical approach to treat limbal stem cell deficiency. Such expansion requires NGF-TrkA-mediated signaling, and this study was conducted to delineate the downstream signaling pathways., Methods: The human corneolimbal ring was cut into explants and cultured on intact human AM. At day 0 or 10, low-molecular-weight inhibitors were added, whereas the control group received dimethyl sulfoxide (DMSO). The epithelial outgrowth rate was monitored for 17 days, and the epithelial cells were collected for Western blot analysis., Results: In the control, most expansion of human limbal epithelial cells started from the limbus from days 5 to 7 and reached approximately 80% confluence at day 17. Compared with the control, the outgrowth was completely inhibited by 50 microM LY294002 or 50 microM SR13668 and was significantly suppressed by 10 microM U0126, but was not affected by 10 microM of either SB203580 or JNK inhibitor 1. The inhibition of outgrowth by LY294002, SR13668, and U0126 was reversible. Western blot analysis showed that phosphorylation of Akt and FKHRL1was abolished by LY294002 and SR13668, but downregulated by U0126, which also abolished phosphorylation of p44/42 mitogen-activated protein kinase (MAPK). The phosphorylation of p38 and JNK MAPK were downregulated or abolished during ex vivo expansion., Conclusions: Ex vivo expansion of human limbal epithelial progenitor cells on intact AM is mediated by the survival signaling pathway mediated by PI3K-Akt-FKHRL1 and by the mitogenic MAPK pathway mediated by p44/42 at the expense of p38 and JNK MAPK.

Purpose: The adult rabbit limbal basal epithelium contains corneal epithelial stem cells, which have been characterized by a negative expression of keratin-3 (K3) and a lower expression of connexin 43 (Cx43). This study was conducted to determine whether the limbal stroma dictates the plasticity of limbal and corneal epithelial differentiation., Methods: Viable epithelial sheets of the central cornea and the pigmented limbus were isolated from Dutch belted rabbits by incubation of 50 mg/mL of dispase II in supplemental hormonal epithelial medium (SHEM) for 18 hours at 4 degrees C. The cleavage plane was studied by immunostaining with antibodies against K3, Cx43, integrin beta1, and collagen IV. Viability of single cells derived from these sheets was assessed by a live-dead assay. Such limbal (L) and corneal (K) epithelial sheets were recombined with either limbal (Ls) or corneal (Ks) stroma, and cultured in SHEM for 10 days before lifting to the air-fluid interface for 1 week. The resultant epithelial phenotype was determined by histology and immunostaining to K3 and Cx43, and apoptosis was investigated by Hoechst and TUNEL nuclear staining., Results: Viability of isolated limbal and corneal epithelial sheets was determined to be 91.1% +/- 2.9%. The basal epithelium of isolated limbal epithelial sheets was positive for integrin beta1, negative for K3, but weakly positive for Cx43, and still retained patches of collagen IV. All recombinants showed stratified epithelia, with intraepithelial cysts with desquamated debris noted only in K/Ks, and epithelial outgrowth onto the insert filter from L/Ls. As expected, expression of K3 was negative in the basal layer of L/Ls, but positive in that of K/Ks. Expression of K3 was sporadically positive in the basal layer of L/Ks but largely negative in that of K/Ls. Expression of Cx43 was uniformly expressed in the basal layer of the K/Ks, but weak in that of L/Ls, K/Ls, and L/Ks. A higher apoptosis index was only noted in intraepithelial cysts of K/Ks., Conclusions: These results strongly indicate that the limbal stroma modulates epithelial differentiation, proliferation and apoptosis in the direction favoring stemness, whereas the corneal stroma promotes differentiation. Further investigation into elements constituting such a niche should help unveil the secrecy whereby stemness is controlled.

Objective: To develop a reproducible method of isolating an intact viable human limbal epithelial sheet., Methods: Human pigmented limbus was incubated at 4 degrees C for 18 hours in supplemental hormonal epithelial medium (SHEM) containing 50 mg/mL dispase II and 100 mM sorbitol. A loose limbal epithelial sheet was separated by a spatula. The remaining stroma was digested and subcultured. The viability of isolated cells was assessed. Isolated epithelial sheets and remaining stroma were subjected to immunostaining. Sheets 1.5 mm in length were cultured in SHEM on plastic until confluence, and cell extracts were subjected to Western blot analysis., Results: Intact limbal epithelial sheets were consistently isolated. Pigmented palisades of Vogt revealed large superficial squamous cells and small basal cuboidal cells. No epithelial cells grew from the remaining stroma. Mean viability was 80.7% +/- 9.1%. The basal epithelium was negative to keratin 3 and connexin 43, but was scatter positive for p63. The epithelial sheet showed negative staining for laminin 5 and collagen VII, but interrupted linear basal staining for collagen IV. The remaining stroma showed negative staining for laminin 5, positive linear staining for collagen IV in the basement membrane, and diffuse staining for collagen VII in the superior stroma subjacent to the basement membrane. Western blot analysis revealed that cells originating from the limbal sheets expressed keratin 3 and p63., Conclusions: An intact limbal epithelial sheet can be consistently and reproducibly isolated and contains stem cell characteristics in the basal epithelium by degrading laminin 5 and part of collagen IV, and disassembling collagen VII.