Mechanistic and Kinetic Insights into Cellular Uptake of Biomimetic Dinitrosyl Iron Complexes and Intracellular Delivery of NO for Activation of Cytoprotective HO-1.

Dinitrosyl iron unit (DNIU), [Fe(NO)2], is a natural metallocofactor for biological storage, delivery, and metabolism of nitric oxide (NO). In the attempt to gain a biomimetic insight into the natural DNIU under biological system, in this study, synthetic dinitrosyl iron complexes (DNICs) [(NO)2Fe(µ-SCH2CH2COOH)2Fe(NO)2] (DNIC-COOH) and [(NO)2Fe(µ-SCH2CH2COOCH3)2Fe(NO)2] (DNIC-COOMe) were employed to investigate the structure-reactivity relationship of mechanism and kinetics for cellular uptake of DNICs, intracellular delivery of NO, and activation of cytoprotective heme oxygenase (HO)-1. After rapid cellular uptake of dinuclear DNIC-COOMe through a thiol-mediated pathway (tmax = 0.5 h), intracellular assembly of mononuclear DNIC [(NO)2Fe(SR)(SCys)]n-/[(NO)2Fe(SR)(SCys-protein)]n- occurred, followed by O2-induced release of free NO (tmax = 1-2 h) or direct transfer of NO to soluble guanylate cyclase, which triggered the downstream HO-1. In contrast, steady kinetics for cellular uptake of DNIC-COOH via endocytosis (tmax = 2-8 h) and for intracellular release of NO (tmax = 4-6 h) reflected on the elevated activation of cytoprotective HO-1 (∼50-150-fold change at t = 3-10 h) and on the improved survival of DNIC-COOH-primed mesenchymal stem cell (MSC)/human corneal endothelial cell (HCEC) under stressed conditions. Consequently, this study unravels the bridging thiolate ligands in dinuclear DNIC-COOH/DNIC-COOMe as a switch to control the mechanism, kinetics, and efficacy for cellular uptake of DNICs, intracellular delivery of NO, and activation of cytoprotective HO-1, which poses an implication on enhanced survival of postengrafted MSC for advancing the MSC-based regenerative medicine.

Differences in change of post-operative antioxidant levels between laser-assisted lenticule extraction and femtosecond laser in situ keratomileusis.

To evaluate the change of total antioxidant capacity (TAC) and ascorbic acid (AA) between femtosecond laser in situ keratomileusis (FS-LASIK) and laser-assisted lenticule extraction (LALEX). A prospective non-randomized study was conducted, and 33 and 75 eyes that had undergone FS-LASIK or LALEX surgeries were enrolled, respectively. The tear films near corneal incisions were collected, and the concentrations of TAC and AA were determined. The generalized linear mixed model was adopted to calculate the adjusted odds ratio (aOR) with 95% confidence interval (CI) of TAC and AA between the two groups. The AA reduction was significant 1 month after the LALEX and FS-LASIK procedures (both p < 0.05), and the decrement in AA level was significantly larger in the FS-LASIK group compared to the LALEX group (p = 0.0002). In the subgroup analysis, the LALEX group demonstrated a lower decrement in TAC level in the individuals with dry eye disease (DED) than the FS-LASIK group (p = 0.0424), and the LALEX group demonstrated a significantly lower AA decrement in the participants with high myopia (p = 0.0165) and DED (p = 0.0043). The LALEX surgery causes lesser AA decrement compared to FS-LASIK surgery especially for the patients with DED.

Transcription Factor ATF3 Participates in DeltaNp63-Mediated Proliferation of Corneal Epithelial Cells.

Understanding the regulatory mechanisms underlying corneal epithelial cell (CEC) proliferation in vitro may provide the means to boost CEC production in cell therapy for ocular disorders. The transcription factor ΔNp63 plays a crucial role in the proliferation of CECs, but the underlying mechanisms is yet to be elucidated. TP63 and ΔNp63 are encoded by the TP63 gene via alternative promoters. We previously reported that both ΔNp63 and activating transcription factor (ATF3) are substantially expressed in cultured CECs, but the regulatory relationship between ΔNp63 and ATF3 is unknown. In the present study, we found that ΔNp63 increased ATF3 expression and ATF3 promoter activity in cultured CECs. The deletion of the p63 binding core site reduced ATF3 promoter activity. CECs overexpressing ATF3 exhibited significantly greater proliferation than control CECs. ATF3 knockdown suppressed the ΔNp63-induced increase in cell proliferation. Overexpression of ATF3 in CECs significantly elevated protein and mRNA levels of cyclin D. The protein levels of keratin 3/14, integrin ß1, and involucrin did not differ between ATF3-overexpressing CECs, ATF3-downregulated CECs, and control cells. In conclusion, our results suggest that ΔNp63 increases CEC proliferation via the ΔNp63/ATF3/CDK pathway.

Association of serum levels of inflammatory cytokines with retinopathy of prematurity in preterm infants.

Introduction: Retinopathy of prematurity (ROP) is a retinal vascular developmental disease associated with risks factors such as supplementary oxygen use or low birth weight/early gestational age. Multiple studies have reported associations between ROP and systemic inflammation. In this study, we investigated serum cytokines associated with ROP development and severity and assessed their applicability as potential biomarkers of ROP. Methods: This prospective study was conducted at an institutional referral center between 2019 and 2021. To measure the serum levels of 40 inflammatory cytokines in eligible premature patients, we collected their serum samples during the enrollment of patients or the intravitreal injection of anti-vascular endothelial growth factor (VEGF) agents and after 2 and 4 weeks. Results: Fifty patients were enrolled. In patients with type 1 ROP who received anti-VEGF agents (n = 22), the levels of serum intercellular adhesion molecule-1 decreased significantly (p < 0.05) at 4 weeks compared with the baseline level, whereas those of serum granulocyte-macrophage colony-stimulating factor increased significantly (p < 0.05). In patients with ROP who did not require any treatment (n = 14), no significant change was noted in the level of any of the 40 inflammatory cytokines. In control infants without ROP (n = 14), the serum levels of tumor necrosis factor-α, interleukin (IL)-15, and IL-12p40 increased significantly (p < 0.05) at 4 weeks. The changes in the levels of serum inflammatory cytokines did not vary significantly among the aforementioned three groups. A generalized estimating equation indicated that zone 1 ROP, stage 3 ROP, older postmenstrual age, respiratory distress syndrome, necrotizing enterocolitis, and sepsis were associated with the changes in serum cytokine levels. Conclusions: Although significant changes (compared with baseline) were observed in the serum levels of certain inflammatory cytokines in patients with type 1 ROP and infants without ROP, no significant difference in cytokine level fluctuations were noted among the three groups. Changes in serum inflammatory cytokine levels may not predict ROP development or severity. Additional comprehensive studies are warranted to establish their definitive role and significance in ROP, emphasizing the need for continued research in this area.

Successful Phakic Intraocular Lens Implantation with the Usage of Topical Ascorbic Acid in Patient with Reduced Corneal Endothelial Cell Density.

We aimed to describe the use of topical ascorbic acid (AA) in a patient with reduced endothelial cells density (ECD) who was scheduled for phakic intraocular lens (pIOL) implantation. A 28-year-old woman presenting with dry eye and reduced ECD would like to have her high myopia (spherical equivalence >−15.0 D) corrected. The procedure of laser refractive surgery or even pIOL was not indicated for the reduced ECD of 1865/mm2 in the right eye and 2188/mm2 in the left eye, as well as level 3 dry eye. Fortunately, the ECD increased to 3144/mm2 in the right eye and 2538/mm2 in the left eye after topical AA was prescribed for one year preoperatively and one month postoperatively, with concomitant improvement of dry eye to level 1. Finally, bilateral pIOL implantation was performed smoothly and no sign of corneal decompensation was found postoperatively. Three months postoperatively, the ECD showed a satisfactory level of 2983/mm2 in the right eye and 3003/mm2 in the left eye. In conclusion, topical AA instillation might increase and maintain the density of central human corneal endothelial cells (HCECs) even after pIOL implantation.

Corneal Endothelial Changes Following Early Capsulotomy Using Neodymium:Yttrium-Aluminum-Garnet Laser.

We aimed to survey whether the timing of neodymium:yttrium-aluminum-garnet (Nd:YAG) laser capsulotomy would alter the corneal endothelial morphology and density. A retrospective cohort study was conducted, and 48 patients with unilateral posterior capsular opacity (PCO) and Nd:YAG laser capsulotomy performance were enrolled. The participants were divided into the early Nd:YAG group (timing ≤ 12 months, n = 20) and late Nd:YAG group (timing > 12 months, n= 28) depending on elapsed months between phacoemulsification and Nd:YAG laser capsulotomy. Endothelial cell density (ECD), coefficient of variant (CV), hexagonality (HEX), and central corneal thickness (CCT) between the two groups were collected. A generalized estimate equation was conducted to evaluate the corneal endothelial parameters between the two groups with an adjusted odds ratio (aOR) and 95% confidence interval (CI). The CDVA was improved after treatment in both groups (both p < 0.001). Chronically, ECD in the early group was significantly decreased one week after treatment (2221.50 ± 327.73/mm2 vs. 2441.55 ± 321.80/mm2, p < 0.001), which recovered to 2369.95 ± 76.37/mm2 four weeks after the treatment but was still lower than the preoperative status (p < 0.001). In addition, the HEX percentage showed a significant reduction at four weeks after treatment (p = 0.028). The ECD in the early group was significantly lower than that in the late group (aOR: 0.167, 95% CI: 0.079-0.356, p = 0.003) in both week 1 (p < 0.001) and week 4 (p = 0.004) after laser treatment. In conclusion, the early application of Nd:YAG laser capsulotomy within one year after cataract surgery may be the reason for postoperative ECD decrement without known etiology.

Long-term survival of cultivated oral mucosal epithelial cells in human cornea: generating cell sheets using an animal product-free culture protocol.

Previously, we reported a collagenase-based, animal product-free protocol for cultivated oral mucosal epithelial cell sheets for transplantation (COMET). Here, we reported the long-term outcomes of first 2 clinical cases. A 27-year-old man suffered from thermal burn, which resulted in symblepharon of lower fornix OD. COMET was performed, and the cornea remained clear with few peripheral NV and no more symblepharon 34 months postoperatively. Another 42-year-old man suffered from severe alkaline burn OD. He underwent COMET, followed by corneal transplantation half a year later. A biopsy taken two years after COMET showed stratified epithelium positive for keratin 4, 13, and 3 in the suprabasal layer. Staining for p63 and p75NTR was both positive in the basal layer. The graft remained clear up to post-OP 4 years. Our study confirmed the long-term survival of the transplanted OMECs, suggesting that collagenase-based spheroidal suspension culture is a promising technique for COMET.Trial registration ClinicalTrials.gov, ClinicalTrials.gov ID: NCT03943797 Registered 9 May 2019-Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT03943797 .

MicroRNA-126 inhibits pathological retinal neovascularization via suppressing vascular endothelial growth factor expression in a rat model of retinopathy of prematurity.

Vascular endothelial growth factor (VEGF) is the principal growth factor responsible for the retinal neovascularization in the pathogenesis of retinopathy of prematurity (ROP). Current therapies for ROP include laser ablation and intravitreal anti-VEGF injection. However, these treatments either destroy the peripheral retina or associate with problems of persistent peripheral avascular retina or later recurrence of ROP. In the present study we investigated a new therapeutic approach by exploring the potential role of a specific microRNA, miR-126, in regulating VEGFA expression and retinal neovascularization in a rat oxygen-induced retinopathy (OIR) model. We demonstrated that miR-126 mimic and plasmid effectively suppresses VEGFA mRNA expression in both human and rat retinal pigment epithelium cell lines, quantified with qRT-PCR. Animal experiments on rat OIR model revealed that intravitreal injection of miR-126 plasmid efficiently downregulated VEGFA expression in the intraocular fluid and retinal tissues measured by ELISA, and significantly suppressed retinal neovascularization, which was confirmed by calculating sizes of neovascularization areas on fluorescence microscopic images of flat mounted retina stained with Alexa Fluor 594-conjugated isolectin B4 to visualize blood vessels. Together, these results showed that intravitreal injection of miR-126 plasmid could inhibit retinal neovascularization by down-regulating VEGFA expression, suggesting a potential therapeutic effect for ROP.

Accelerated corneal endothelial cell loss in two patients with granulomatosis with polyangiitis following phacoemulsification.

BACKGROUND: Generally, the loss rate of human endothelial cells (HCEC) in routine cataract surgery is 8.5%. When the corneal endothelial cells density (ECD) drops, the HCEC may decompensate to keep cornea dehydration which leads to corneal edema. Granulomatosis with polyangiitis (GPA) is an uncommon autoimmune disease involving multiple organs including eyes such as conjunctivitis, scleritis, uveitis, and corneal ulcer. In this study, we report two cases of GPA whose corneal ECD decreased significantly after phacoemulsification cataract surgery. CASE PRESENTATION: In the first case of 69-year-old male with GPA, the ECD dropped 39.6% (OD) four months after phacoemulsification and 38.1% (OS) six months postoperatively respectively. At the final follow-up, the residual ECD was only 55% in the right eye in the 49th month, and 56% remained in the left eye in the 39th month. In the second case of 54-year old female, left ECD dropped 63.9% at the 4th month after surgery and 69.6% ECD remained at the 15th month postoperatively while similar ECD of right eye before and after left eye surgery. CONCLUSION: Extensive preoperative ophthalmic evaluation and meticulous postoperative inflammation control should be applied to prevent severe loss of HCEC in GPA patients.

Perioperative topical ascorbic acid for the prevention of phacoemulsification-related corneal endothelial damage: Two case reports and review of literature.

BACKGROUND: The current case report describes successful phacoemulsification with the aid of perioperative topical ascorbic acid (AA) in two patients with corneal endothelial disorders to prevent postoperative corneal endothelial decompensation. CASE SUMMARY: Two eyes of two patients underwent phacoemulsification with pre-existing corneal endothelial disorders including Fuchs corneal endothelial dystrophy (Patient 1) and endotheliitis (Patient 2). Topical AA was applied to both patients at least one month before and after with a frequency of four times per day. After the surgery, both eyes improved best-corrected visual acuity (BCVA) and there was limited human corneal endothelial cell loss without signs of corneal endothelial decompensation, such as deteriorated BCVA or persistent corneal edema during the follow-up of at least two years. CONCLUSION: Perioperative administration of topical AA may be an alternative therapy to the triple procedure in patients expecting to undergo cataract surgery.

Preservation of epithelial progenitor cells from collagenase-digested oral mucosa during ex vivo cultivation.

To avoid xenogeneic infection, we report a novel protocol for producing animal-derived component-free oral mucosal epithelial cells (OMECs) sheet for transplantation, in which collagenase was used to replace dispase II/trypsin-EDTA for digesting oral mucosal tissue, and human platelet-derived PLTMax to replace fetal bovine serum. The resulting epithelial aggregates were expanded on de-epithelialized amniotic membranes without 3T3 feeder cells, and serum-free EpiLife was used to reduce contamination by submucosal mesenchymal cells. The OMEC sheets thus generated showed similar positive keratin 3/76-positive and keratin 8-negative staining patterns compared with those generated by the original protocol. Colony formation efficiency assay, BrdU label retention assay, and p63 and p75NTR immunostaining results indicated that higher proliferative potentials and more progenitor cells were preserved by the modified protocol. TaqMan array analysis revealed that the transcription of integrin-linked kinase (ILK) was up-regulated along with an increase in ß-catenin signaling and its downstream cell cycle modulators, cyclin D1 and p27KIP1. Furthermore, ILK silencing led to the inhibition of nuclear ß-catenin accumulation, suppressed p63 expression, and reduced the expression of cyclin D1 and p27KIP1; these observations suggest that ILK/ß-catenin pathway may be involved in cell proliferation regulation during the ex vivo expansion of OMECs for transplantation purposes.

Preservation of human limbal epithelial progenitor cells on carbodiimide cross-linked amniotic membrane via integrin-linked kinase-mediated Wnt activation.

The Wnt pathway is a major signaling pathway that regulates corneal epithelial stem cells. However, little is known about how the ultrastructure of the limbal epithelial basement membrane (EBM) affects Wnt activity. Due to its enhanced matrix stability, the cross-linked amniotic membrane (AM) has gained increasing interest in the field of regenerative medicine. For the first time, we used EDC/NHS cross-linked denuded AM (CLDAM) as a simulated EBM substrate to investigate this mechanism. Human limbal epithelial (HLE) cells were cultured on dishes (HLE/dish), denuded AM (HLE/DAM) or CLDAM (HLE/CLDAM). Compared with HLE/dish or HLE/DAM cultures, HLE/CLDAM cultures showed greater BrdU retention and colony formation efficiency and expressed higher levels of p63, ABCG2, integrin ß1, and integrin-linked kinase (ILK). Nuclear ß-catenin and TCF-4 levels were higher in HLE/CLDAM cultures compared with HLE cells cultured on collagen IV, laminin, Matrigel, or DAM. Silencing of ILK in HLE/CLDAM cultures resulted in decreased levels of nuclear ß-catenin, TCF-4 and deltaNp63α, whereas cytokeratin 12 expression increased. Over-expression of ILK in HLE/dish cultures had the opposite effects. Accordingly, we proposed that the CLDAM matrix, with its higher rigidity and rougher ultrastructure, better preserved HLE progenitor cells in vitro, possibly by activating integrin ß1/ILK, which indirectly activated Wnt/ß-catenin and subsequently deltaNp63α. Crosstalk between the integrin ß1/ILK and Wnt/ß-catenin pathways appears to play a crucial role in limbal progenitor cell survival on EBM. STATEMENT OF SIGNIFICANCE: We demonstrated the superior capability of carbodiimide cross-linked denuded amniotic membrane (CLDAM) than natural DAM to preserve limbo-corneal epithelial progenitor cells in vitro, then we used CLDAM as a simulated epithelial basement membrane (EBM) to study how EBM maintains limbal epithelial stem cells (LESCs). We found that integrin-linked kinase (ILK) is an important mediator that transfers survival signals detected by integrin ß1 to the Wnt/ß-catenin pathway, which in turn up-regulates deltaNp63α, a master gene that regulates LESC function. The rougher surface of the limbal EBM suggests that the surface complexity of the LESC niche may be important in regulating LESC function, which is triggered by the recognition of topographic cues by integrin ß1, followed by activation of the ILK/Wnt/ß-catenin/p63 cascade.

Transcriptional regulators of the ΔNp63: their role in limbal epithelial cell proliferation.

The surface cells of corneal epithelium are regularly shed off and replaced by new cells that are derived from limbal epithelial stem cells (LESC). LESC are believed to reside in the basal layer of the limbal epithelium and are characterized with high expression levels of ΔNp63, a transcription factor (TF) which is believed to play roles in the regulation of LESC proliferation. In this study, we examined the transcriptional regulation of ΔNp63 in limbal epithelial cell. We employed DNA pull down assay followed by LC/MS analysis and cDNA microarray analysis to identify the TFs that were capable of binding to ΔNp63 promoter or were expressed at higher levels in limbus over cornea. The TFs thus selected were further examined for their in vivo ΔNp63 promoter binding by chromatin immunoprecipitation assay. We identified six putative TFs (PAX6, EGR1, CEBPB, JUN, ATF3, and ARID5B) through the aforementioned approaches. Among them, PAX6 and EGR1 were shown to promote the transcription of ΔNp63 and led to increased cell proliferation. In contrast, CEBPB and ATF3 appeared to exert little or no effect on ΔNp63 expression, however, their silencing suppressed cell proliferation. Although JUN exhibited low promoter-binding specificity, however, it affected ΔNp63 expression and limbal epithelial cell proliferation in ways similar to that of PAX6 and EGR1. Intriguingly, ARID5B was highly expressed in the limbal epithelial cell, however, its silencing by siRNA did not obviously affect the expression of ΔNp63, nor did it reduce cell proliferation of the limbal epithelial cell.

STAT3 regulates the proliferation and differentiation of rabbit limbal epithelial cells via a ΔNp63-dependent mechanism.

PURPOSE: To explore the roles of STAT3 in the regulation of ΔNp63-dependent proliferation and differentiation of rabbit limbal keratinocytes. METHODS: siRNAs were designed to specifically suppress the expression of STAT3 and ΔNp63, and their effects on limbal epithelial cell proliferation and differentiation were examined. Ectopically expressed ΔNp63 was used to compensate for the decreased endogenous ΔNp63. Immunoblot was used to examine the expressions of STAT3, ΔNp63, K3, integrin ß1, and involucrin. RESULTS: Limbal tissue expresses higher level of phosphorylated and nuclear translocated STAT3 compared with that of the cornea. Knockdown of STAT3 expression reduces the expression of ΔNp63, inhibits the expansion of limbal epithelial outgrowth, suppresses the expression of integrin ß1, and promotes the expression of involucrin. CONCLUSIONS: STAT3 enhances the proliferation of limbal keratinocytes through a ΔNp63-dependent mechanism. Suppression of this pathway inhibits cell proliferation with a concomitant increase of cell differentiation.

Regulation of limbal keratinocyte proliferation and differentiation by TAp63 and DeltaNp63 transcription factors.

PURPOSE: To examine the effects of TAp63 and DeltaNp63 on the proliferation and differentiation of rabbit limbal keratinocytes cultured on human amniotic membrane. METHOD: Real-time Q-RT-PCR was used to quantify the relative abundance of TAp63 and DeltaNp63 transcripts in limbal, peripheral corneal, and central corneal epithelia. Antisense oligonucleotides were designed specifically to suppress the expression of TAp63 or DeltaNp63 in limbal keratinocytes, and their effects on cell proliferation and differentiation were examined. Immunofluorescence was used to examine the expressions of p63 and keratin-3 and -14. RESULTS: The expressions of TAp63 and DeltaNp63 transcripts appeared to be site specific. TAp63 was expressed at the highest level in limbus, decreased by approximately 10-fold in peripheral cornea and was undetectable in the central cornea. DeltaNp63 was also expressed at the highest level in limbus, decreased by approximately 35% in peripheral cornea, and was undetectable in the central cornea. Suppression of TAp63 expression inhibited limbal keratinocyte proliferation but promoted differentiation. Suppression of DeltaNp63 expression also inhibited cell proliferation but had no obvious effect on cell differentiation. CONCLUSIONS: TAp63 and DeltaNp63 affect the proliferation of limbal keratinocytes by a different mechanism. The inhibition by TAp63 antisense oligos appeared to be secondary to the promotion of cell differentiation. In contrast, the inhibition by DeltaNp63 antisense oligos appeared to be independent of cell differentiation.

Ischemic preconditioning ameliorates microcirculatory disturbance through downregulation of TNF-alpha production in a rat cremaster muscle model.

Ischemia-reperfusion (I/R) injury is a complex process involving the generation and release of inflammatory cytokines, and the accumulation and infiltration of neutrophils and macrophages, which disturbs the microcirculatory hemodynamics. Nonetheless, ischemic preconditioning (IPC) is known to produce immediate tolerance to subsequent prolonged I/R insults, although its underlying mechanism largely remains unknown. Our study investigated the role of the IkappaB-alpha-NF-kappaB-TNF-alpha (tumor necrosis factor-alpha) pathway in IPC's ability to ameliorate I/R-induced microcirculatory disturbances in rat cremaster muscle flaps. Male Sprague-Dawley rats were randomized (n = 8 per group) into 3 groups: a sham-operated control group, an I/R group (4 h of pudic epigastric artery ischemia followed by 2 h of reperfusion), and an IPC+I/R group (3 cycles of 10 min of ischemia followed by 10 min reperfusion before I/R). Intravital microscopy was used to observe leukocyte/endothelial cell interactions and quantify functional capillaries in cremaster muscles. I/R markedly increased the number of rolling, adhering, and migrating leukocytes. It was also observed that I/R significantly increased TNF-alpha expression in these injured tissues. On the other hand, IPC prevented I/R-induced increases in leukocyte rolling, adhesion, and transmigration. Moreover, TNF-alpha protein production and its mRNA expression were downregulated in the IPC group. Finally, I/R-induced IkappaB-alpha phosphorylation and NF-kappaB (p65) nuclear translocation were both suppressed by IPC. These results indicated that IPC attenuated NF-kappaB activation and subsequently reduced TNF-alpha expression, which resulted in the amelioration of microcirculatory disturbances in I/R-injured cremaster muscles.

Propagation and phenotypic preservation of rabbit limbal epithelial cells on amniotic membrane.

PURPOSE: To describe the phenotypic characteristics of a limbal epithelial cell sheet outgrowth from a limbal explant cultured on amniotic membrane. METHOD: Immunofluorescent staining and confocal microscopy were used to examine the expressions of p63, Ki-67, keratins 3 and 14, connexin 43, and the integrin alpha6/beta4 and alpha3/beta1 subunits in corneal and limbal tissues in a limbal explant and epithelial outgrowth cultured for 2 weeks on amniotic membrane. RESULTS: The expression patterns of p63, Ki-67, keratins, integrins, and connexin 43 in a limbal explant with an epithelial outgrowth cultured for 2 weeks on amniotic membrane resembled those in freshly prepared limbus. Moreover, the distribution of integrin subunits in positive cells of the limbal explant and its epithelial outgrowth was similar to that of the corneal epithelial cells during wound repair. CONCLUSIONS: The epithelial cell sheet grown from a limbal explant on amniotic membrane exhibited a phenotype similar to that of the limbus, suggesting that amniotic membrane is a substrate capable of supporting the propagation and preservation of p63-positive limbal epithelial cells.