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Purposes: To determine the best protocol in obtaining the higher yield of conditioned culture medium to be used for the bone marrow mesenchymal stem cell differentiation into corneal epithelial cells, five techniques for the primary culture of human corneal epithelial cells were evaluated., Methods: The studied culture techniques of corneal epithelial cells were: explants in culture flasks with and without hydrophilic surface treatment, on amniotic membrane, with enzymatic digestion, and by corneal scraping. The conditioned culture medium collected from these cultures was used to differentiate human bone marrow mesenchymal stem cells into corneal epithelial cells, which were characterized using flow cytometry with pan-cytokeratin and the corneal-specific markers, cytokeratin 3 and cytokeratin 12., Results: The culture technique using flasks with hydrophilic surface treatment resulted in the highest yield of conditioned culture medium. Flasks without surface treatment resulted to a very low success rate. Enzymatic digestion and corneal scraping showed contamination with corneal fibroblasts. The culture on amniotic membranes only allowed the collection of culture medium during the 1st cell confluence. The effectiveness of cell differentiation was confirmed by cytometry analysis using the collected conditioned culture medium, as demonstrated by the expressions of cytokeratin 3 (95.3%), cytokeratin 12 (93.4%), and pan-cytokeratin (95.3%)., Conclusion: The culture of corneal epithelial cell explants in flasks with hydrophilic surface treatment is the best technique for collecting a higher yield of conditioned culture medium to be used to differentiate mesenchymal stem cells.

Mammalian corneal development is a multistep process, including formation of the corneal epithelium (CE), endothelium and stroma during embryogenesis, followed by postnatal stratification of the epithelial layers and continuous renewal of the epithelium to replace the outermost corneal cells. Here, we employed the Cre-loxP system to conditionally deplete Pax6 proteins in two domains of ocular cells, i.e., the ocular surface epithelium (cornea, limbus and conjunctiva) (OSE) or postnatal CE via K14-cre or Aldh3-cre, respectively. Earlier and broader inactivation of Pax6 in the OSE resulted in thickened OSE with CE and limbal cells adopting the conjunctival keratin expression pattern. More restricted depletion of Pax6 in postnatal CE resulted in an abnormal cornea marked by reduced epithelial thickness despite increased epithelial cell proliferation. Immunofluorescence studies revealed loss of intermediate filament Cytokeratin 12 and diffused expression of adherens junction components, together with reduced tight junction protein, Zonula occludens-1. Furthermore, the expression of Cytokeratin 14, a basal cell marker in apical layers, indicates impaired differentiation of CE cells. Collectively, our data demonstrate that Pax6 is essential for maintaining proper differentiation and strong intercellular adhesion in postnatal CE cells, whereas limbal Pax6 is required to prevent the outgrowth of conjunctival cells to the cornea., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Primary canine corneal epithelial cells (CCECs) easily become senescent, and cell proliferation is limited. Therefore, sampling for experimentation requires a large number of animals, which is problematic in terms of animal welfare and fails to maintain the stability of the cells for in vitro analyses., Results: In this study, CCECs were separated and purified by trypsin and dispase II enzymatic analysis. Next, the cells were immortalized by transfection with a lentiviral vector expressing Simian vacuolating virus 40 large T (SV40T). The immortalized canine corneal epithelial cell line (CCEC-SV40T) was established by serial passages and monoclonal selection. The biological characteristics of CCEC-SV40T cells were evaluated based on the cell proliferation rate, cell cycle pattern, serum dependence, karyotype, and cytokeratin 12 immunofluorescence detection. In addition, we infected CCEC-SV40T cells with Staphylococcus pseudintermedius (S. pseudintermedius) and detected the inflammatory response of the cells. After the CCEC-SV40T cells were passaged continuously for 40 generations, the cells grew in a cobblestone pattern, which was similar to CCECs. The SV40T gene and cytokeratin 12 can be detected in each generation. CCEC-SV40T cells were observed to have a stronger proliferation capacity than CCECs. CCEC-SV40T cells maintained the same diploid karyotype and serum-dependent ability as CCECs. After CCEC-SV40T cells were infected with S. pseudintermedius, the mRNA expression levels of NLRP3, Caspase-1 and proinflammatory cytokines, including IL-1β, IL-6, IL-8 and TNF-α, were upregulated, and the protein levels of MyD88, NLRP3 and the phosphorylation of Iκbα and p65 were upregulated., Conclusions: In conclusion, the CCEC-SV40T line was successfully established and can be used for in vitro studies, such as research on corneal diseases or drug screening., (© 2022. The Author(s).)

The cornea is an anterior eye structure specialized for vision. The corneal endothelium and stroma are derived from the periocular mesenchyme (POM), which originates from neural crest cells (NCCs), while the stratified corneal epithelium develops from the surface ectoderm. Activating protein-2β (AP-2β) is highly expressed in the POM and important for anterior segment development. Using a mouse model in which AP-2β is conditionally deleted in the NCCs (AP-2β NCC KO), we investigated resulting corneal epithelial abnormalities. Through PAS and IHC staining, we observed structural and phenotypic changes to the epithelium associated with AP-2β deletion. In addition to failure of the mutant epithelium to stratify, we also observed that Keratin-12, a marker of the differentiated epithelium, was absent, and Keratin-15, a limbal and conjunctival marker, was expanded across the central epithelium. Transcription factors PAX6 and P63 were not observed to be differentially expressed between WT and mutant. However, growth factor BMP4 was suppressed in the mutant epithelium. Given the non-NCC origin of the epithelium, we hypothesize that the abnormalities in the AP-2β NCC KO mouse result from changes to regulatory signaling from the POM-derived stroma. Our findings suggest that stromal pathways such as Wnt/β-Catenin signaling may regulate BMP4 expression, which influences cell fate and stratification.

Transplantation of human cultured limbal epithelial stem/progenitor cells (LESCs) has demonstrated to restore the integrity and functionality of the corneal surface in about 76% of patients with limbal stem cell deficiency. However, there are different protocols for the expansion of LESCs, and many of them use xenogeneic products, being a risk for the patients' health. We compared the culture of limbal explants on the denuded amniotic membrane in the culture medium-supplemental hormone epithelial medium (SHEM)-supplemented with FBS or two differently produced human sera. Cell morphology, cell size, cell growth rate, and the expression level of differentiation and putative stem cell markers were examined. Several bioactive molecules were quantified in the human sera. In a novel approach, we performed a multivariate statistical analysis of data to investigate the culture factors, such as differently expressed molecules of human sera that specifically influence the cell phenotype. Our results showed that limbal cells cultured with human sera grew faster and contained similar amounts of small-sized cells, higher expression of the protein p63α, and lower of cytokeratin K12 than FBS cultures, thus, maintaining the stem/progenitor phenotype of LESCs. Furthermore, the multivariate analysis provided much data to better understand the obtaining of different cell phenotypes as a consequence of the use of different culture methodologies or different culture components.

Background: Limbal stem cells (LSC) sustain the corneal integrity and homeostasis. LSC deficiency (LSCD) leads to loss of corneal transparency and blindness. A clinical approach to treat unilateral LSCD comprises autologous cultured limbal epithelial stem cell transplantation (CLET). CLET uses xenobiotic culture systems with potential zoonotic transmission risks, and regulatory guidelines make necessary to find xenofree alternatives., Methods: We compared two xenofree clinical grade media and two feeder layers. We used CnT07, a defined commercial medium for keratinocytes, and a modified xenofree supplemented hormonal epithelial medium with human serum (XSHEM). Optimal formulation was used to compare two feeder layers: the gold standard 3T3 murine fibroblasts and human processed lipoaspirate cells (PLA). We tested the expressions of ΔNp63α and cytokeratin 3 and 12 by qPCR and immunofluorescence. Morphology, viability, clonogenicity, proliferation, and cell growth assays were carried out. We also evaluated interleukin 6 (IL-6) and stromal-derived factor 1 (SDF-1) by qPCR and ELISA., Results: XSHEM maintained better LSC culture viability and morphology than CnT07. Irradiated PLA feeder cells improved the undifferentiated state of LSC and enhanced their growth and clonogenicity stimulating IL-6 secretion and SDF-1 expression, as well as increased proliferation and cell growth when compared with irradiated 3T3 feeder cells., Conclusions: The combination of XSHEM and PLA feeder cells efficiently sustained LSC xenofree cultures for clinical application. Moreover, PLA feeder layers were able to improve the LSC potential characteristics. Our results would have direct clinical application in CLET for advanced therapy.

Purpose: Subconjunctival injection of antagomir-21 attenuates the progression of corneal neovascularization. We examined the underlying mechanism by investigating the regulation of microRNA (miR)-21 expression and the involvement of miR-21 in the homeostasis of corneal epithelial cells., Methods: Corneal epithelial cells were cultured with TGF-β1 and/or under hypoxia conditions. miR-21 expression was measured by quantitative PCR. The direct targets of miR-21 were validated by the 3'-UTR luciferase reporter assay. Alterations of proangiogenic signaling and the epithelial-mesenchymal transition (EMT) phenotype after miR-21/Sprouty2 (SPRY2) knockdown were examined by Western blotting. The effect of conditioned medium on angiogenesis was assessed using the tube formation assay. Wound healing was evaluated by the migration and scratch assays., Results: TGF-β1 or hypoxia upregulated miR-21, and miR-21 silencing abolished TGF-β1/hypoxia-induced hypoxia inducible factor (HIF)-1α and VEGF expression. miR-21 inhibited SPRY2 by directly targeting its 3'-UTR. Simultaneous silencing of miR-21 and SPRY2 significantly upregulated p-ERK, HIF-1α, and VEGF and promoted angiogenesis. Induction of miR-21 or inhibition of SPRY2 reduced the levels of cytokeratin (CK)-3 and CK-12 and promoted EMT. Transwell and wound healing assays indicated that miR-21 promoted cell migration., Conclusions: TGF-β1 or hypoxia induced miR-21 and inhibited SPRY2, thereby enhancing proangiogenic signaling, suppressing the epithelial phenotype, and promoting wound healing in corneal epithelial cells.

Purpose: To report genetic mutational analysis and in vivo histology of Meesmann corneal dystrophy., Study Design: Prospective, case control study., Methods: Six patients from three independent families with clinically diagnosed Meesmann corneal dystrophy were enrolled in this study. Slit-lamp biomicroscopy with fluorescein vital staining, anterior segment optical coherence tomography (AS-OCT), and in vivo laser confocal microscopy (IVCM) were performed on selected patients. Mutational screening for the keratin genes KRT3 and KRT12 was performed in all six patients and selected unaffected family members., Results: Slit-lamp biomicroscopy revealed numerous intraepithelial microcysts in all affected individuals. AS-OCT revealed hyperreflectivity and high corneal epithelial layer thickness (mean, 64.8μm) in all individuals tested (3/3). By using IVCM, multiple epithelial microcysts and hyperreflective materials (6/6), subepithelial nerve abnormalities (6/6), tiny punctate hyperreflective material (6/6), and needle-like hyperreflective materials (4/6) were observed in the corneal stromal layer. A heterozygous genetic mutation in the KRT12 gene (c.394 C>G, p.L132V) was identified in all six patients. No pathological mutation was observed in the KRT3 gene., Conclusion: We identified a heterozygous genetic mutation (c.394 C>G, p.L132V) in the KRT12 gene in six Japanese patients with inherited Meesmann corneal dystrophy. This is the first study to confirm this genetic mutation in Japanese Meesmann corneal dystrophy patients. This mutation has been independently reported in an American Meesmann corneal dystrophy patient, confirming its pathogenicity. AS-OCT and IVCM proved to be useful tools for observing corneal epithelial layer pathology in this dystrophy. Furthermore, IVCM reveals corneal stromal layer pathological changes not previously reported in this dystrophy.

Purpose: To report a case of limbal stem cell deficiency (LSCD) secondary to diffuse non-necrotizing anterior scleritis (DNNAS)., Method: Interventional case report with clinicopathologic correlation. A 69-year-old white woman with known Crohn disease presented with DNNAS. The acute inflammatory phase was treated with topical and systemic steroids. After DNNAS, she developed secondary LSCD with loss of limbal palisades of Vogt and conjunctivalization of the corneal surface and corneal haze. She underwent superficial keratectomy combined with autologous limbal stem cell grafting from the fellow eye. The keratectomy specimen was sent for pathological examination., Results: There were no intraoperative or post-operative complications. Histopathology and immunohistochemistry showed a cytokeratin 19-positive and cytokeratin 3- and cytokeratin 12 negative epithelium in keeping with a conjunctival phenotype on the corneal surface., Conclusions: LSCD can be a rare complication of DNNAS. After control of ocular surface inflammation, autologous limbal stem cell grafting and amniotic membrane transplantation can be effective in normalizing the ocular surface.

Aniridia is a rare disease of the eye that affects the iris, lens and the cornea. In about 90% of the cases, patients showed a loss of PAX6 function. Patients with aniridia often develop aniridia-related keratopathy (ARK), due to limbal stem cell insufficiency. The aim of this study was to determine the differentiation status of limbal epithelial cells (LECs) in patients with ARK. Epithelial cells were isolated from the limbus region of two patients with aniridia and cultured in KSFM medium supplemented with EGF and BPE. Normal cells were obtained from limbus region of cadaveric control patients. Cells were analyzed with RT-PCR, qPCR and Western blot to evaluate expression of the developmental transcription factor, PAX6, potential stem cell markers, ΔNp63α and ABCG2, and corneal differentiation markers, keratin 12 (K12) and K3. Conjunctival differentiation markers, keratin 13 (K13) and K19 were also investigated. Cells were immunostained to evaluate K3, PAX6, and p63α protein expression. Protein coding sequence of PAX6 from patient LEC-cDNA was cloned and sequenced. RT-PCR showed that K3 and K12 transcripts were absent from patient cells, but present in healthy control preparations. Transcription levels of PAX6, ABCG2, and p63α of aniridia patients show no differences compared to normal control cells. Western blot showed reduced PAX6, protein levels in aniridia-LECs compared to control-LECs. Immunostaining also showed reduced PAX6 and K3 expression in aniridia-LECs compared to control-LECs. One aniridia patient showed a loss of stop codon in half of the cloned transcripts. In the second aniridia patient mRNA degradation through nonsense mediated decay seems to be very likely since we could not identify the mutation c.174C > T (Refseq. NM_000280), or misspliced transcripts in cDNA. We identified decreased PAX6 protein levels in aniridia patients in addition to decreased K12 mRNA levels compared to control cells. This result indicates an altered differentiation of limbal epithelial cells of aniridia patients. Further studies are necessary to evaluate the mechanism of differentiation of limbal epithelial cells in aniridia., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Purpose: This study is to summarize the concurrent keratoconus (KC) and granular corneal dystrophy (GCD) phenotype and identify the underlying genetic cause in a 23-year-old male patient., Methods: A detailed family history and clinical data from the patient and his parents were collected by ophthalmologic examination. The candidate genes were captured and sequenced by targeted next-generation sequencing, and the results were confirmed by Sanger sequencing., Results: The proband was clinically diagnosed as a case of concurrent KC and GCD, which is a very rare presentation. His father and grandmother were diagnosed as GCD in both eyes. There was no character of KC in his father's and grandmother's eyes. A heterozygous TGFBI mutation in exon 4 (c.370G > A) was identified in the proband, which was predicted to generate a missense mutation (p.R124H). The mutation also existed in his father and grandmother. A heterozygous KRT12 mutation in exon 8 (c.1456-1457ins GTA) was identified in the proband, which was predicted to generate an insert mutation and created a premature termination codon. The mutation did not exist in his father and grandmother. The two mutations did not exist in his mother and 200 unrelated normal controls., Conclusions: KC can co-exist with GCD. The missense mutation (c.370G > A) in the TGFBI gene and insert mutation (c.1456-1457ins GAT) in the KRT12 gene were identified in a 23-year-old male patient with concurrent KC and GCD.

The aim of this study was to examine the effect of molecular hydrogen (H 2 ) on the healing of alkali-injured cornea. The effects of the solution of H 2 in phosphate buffered saline (PBS) or PBS alone topically applied on the alkali-injured rabbit cornea with 0.25 M NaOH were investigated using immunohistochemical and biochemical methods. Central corneal thickness taken as an index of corneal hydration was measured with an ultrasonic pachymeter. Results show that irrigation of the damaged eyes with H 2 solution immediately after the injury and then within next five days renewed corneal transparency lost after the injury and reduced corneal hydration increased after the injury to physiological levels within ten days after the injury. In contrast, in injured corneas treated with PBS, the transparency of damaged corneas remained lost and corneal hydration elevated. Later results-on day 20 after the injury-showed that in alkali-injured corneas treated with H 2 solution the expression of proinflammatory cytokines, peroxynitrite, detected by nitrotyrosine residues (NT), and malondialdehyde (MDA) expressions were very low or absent compared to PBS treated injured corneas, where NT and MDA expressions were present. In conclusion, H 2 solution favorably influenced corneal healing after alkali injury via suppression of oxidative stress.

Key issues in corneal epithelium biology are the mechanism for corneal epithelium stem cells to maintain the corneal epithelial homeostasis and wound healing responses, and what are the regulatory molecular pathways involved. There are apparent discrepancies about the locations of the progenitor populations responsible for corneal epithelial self-renewal. We have developed a genetic mouse model to trace the corneal epithelial progenitor lineages during adult corneal epithelial homeostasis and wound healing response. Our data revealed that the early corneal epithelial progenitor cells expressing keratin-12 originated from limbus, and gave rise to the transit amplifying cells that migrated centripetally to differentiate into corneal epithelial cells. Our results support a model that both corneal epithelial homeostasis and wound healing are mainly maintained by the activated limbal stem cells originating form limbus, but not from the corneal basal epithelial layer. In the present study, we further demonstrated the nuclear expression of transcriptional coactivator YAP1 in the limbal and corneal basal epithelial cells and its essential role for maintaining the high proliferative potential of those corneal epithelial progenitor cells in vivo.

Purpose: The aim of this study was to develop nanofibrous polycaprolactone (PCL) substrate for limbal stem cell (LSC) expansion that can serve as a potential alternative substrate to replace human amniotic membrane (AM)., Materials and Methods: The human limbus stem cell was used to evaluate the biocompatibility of substrates (nanofibrous scaffold and, human AM) based on their phenotypic profile, viability, proliferation and attachment ability., Results: Biocompatibility results indicated that the all substrates were highly biocompatible, as LSCs could favorably attach and proliferate on the nanofibrous surface. Microscopic figures showed that the human LSCs were firmly anchored to the substrates and were able to retain a normal corneal stem cell phenotype. Microscopic analyses illustrated that cells infiltrated the nanofibers and successfully formed a three-dimensional corneal epithelium, which was viable for two weeks. Immunocytochemistry (ICC) and real time-PCR results revealed no change in the expression profile of LECs grown on nanofibrous substrate when compared to those grown on human AM., Conclusion: In addition, electrospun nanofibrous PCL substrate provides not only a milieu supporting LSCs expansion, but also serve as a useful alternative carrier for ocular surface tissue engineering and could be used as an alternative substrate to AM.

CRISPR/Cas9-based therapeutics hold the possibility for permanent treatment of genetic disease. The potency and specificity of this system has been used to target dominantly inherited conditions caused by heterozygous missense mutations through inclusion of the mutated base in the short-guide RNA (sgRNA) sequence. This research evaluates a novel approach for targeting heterozygous single-nucleotide polymorphisms (SNPs) using CRISPR/Cas9. We determined that a mutation within KRT12, which causes Meesmann's epithelial corneal dystrophy (MECD), leads to the occurrence of a novel protospacer adjacent motif (PAM). We designed an sgRNA complementary to the sequence adjacent to this SNP-derived PAM and evaluated its potency and allele specificity both in vitro and in vivo. This sgRNA was found to be highly effective at reducing the expression of mutant KRT12 mRNA and protein in vitro. To assess its activity in vivo we injected a combined Cas9/sgRNA expression construct into the corneal stroma of a humanized MECD mouse model. Sequence analysis of corneal genomic DNA revealed non-homologous end-joining repair resulting in frame-shifting deletions within the mutant KRT12 allele. This study is the first to demonstrate in vivo gene editing of a heterozygous disease-causing SNP that results in a novel PAM, further highlighting the potential for CRISPR/Cas9-based therapeutics.

Transplantation of ex vivo cultured limbal epithelial cells is proven effective in restoring limbal stem cell deficiency. The present study aimed to investigate the promoting effect of Y-27632 on limbal epithelial cell proliferation. Limbal explants isolated from human donor eyes were expanded three weeks on culture dishes and outgrowth of epithelial cells was subsequently subcultured for in vitro experiments. In the presence of Y-27632, the ex vivo limbal outgrowth was accelerated, particularly the cells with epithelial cell-like morphology. Y-27632 dose-dependently promoted the proliferation of in vitro cultured human limbal epithelial cells as examined by phase contrast microscopy and luminescent cell-viability assay 30 hours after the treatment. The colony forming efficacy determined 7 days after the treatment was enhanced by Y-27632 also in a dose-dependent manner. The number of p63- or Ki67-positive cells was dose-dependently increased in Y-27632-treated cultures as detected by immunofluorescent staining and western blotanalysis. Cell cycle analysis by flow cytometric method revealed an increase in S-phase proliferating cells. The epithelial woundclosure rate was shown to be faster in experimental group received topical treatment withY-27632 than the sham control using a rat corneal wounding model. These resultsdemonstrate that Y-27632 can promote both the ex vivo and in vitro proliferation oflimbal epithelial cell proliferation. The in vivo enhanced epithelial wound healingfurther implies that the Y-27632 may act as a new strategy for treating limbal stem cell deficiency.

Purpose: To correlate clinical, impression cytologic, and in vivo confocal microscopy findings on the corneal surface after cultured limbal stem cell transplantation., Design: Prospective, interventional, noncomparative, masked case series., Participants: Thirteen patients with limbal stem cell deficiency after unilateral (9 eyes) or bilateral (2 eyes) chemical burn, liquid nitrogen injury (1 eye), or herpes simplex virus infection (1 eye)., Methods: Limbal cells were harvested from healthy or less affected eyes, cultured on 3T3 cells and fibrin glue, and transplanted to the patient's injured eye. Patients underwent clinical examination and impression cytologic examination of the central cornea before and 1 year after intervention. In vivo confocal microscopy scans were obtained in all corneal quadrants after 1 year. The interexamination agreement was established by calculation of the Cohen's κ coefficient., Main Outcome Measures: Results of surgery were assessed considering clinical signs (successful: restoration of transparent, avascular, and stable corneal epithelium without neovascularization in central corneal surface; partially successful: recurrence of superficial neovascularization; failed: recurrent epithelial defects, pannus, and inflammation), phenotype of cells covering the corneal surface (conjunctivalized corneal surface: cytokeratin 12 [cK12]-negative and mucin 1 [MUC1]-positive cells; mixed epithelium: cK12-positive and MUC1-positive cells; corneal epithelium: cK12-positive and MUC1-negative cells), and cell morphologic features (corneal epithelium: multilayered polygonal and flat cells with hyperreflective nuclei; conjunctival epithelium: stratified cuboidal or polygonal cells, hyperreflective cytoplasm, and barely defined borders; epithelial transition: transition of epithelial cells from the cornea to the conjunctiva over the corneal surface)., Results: We found a moderate to substantial degree of concordance between confocal microscopy and clinical evaluation (κ = 0.768) and between confocal microscopy and impression cytologic analysis (κ = 0.629). Confocal microscopy showed that 46.2% of patients exhibited corneal epithelium in the central and peripheral cornea, 30.8% showed an irregular mixed corneal and conjunctival epithelium, and 23.0% showed conjunctival epithelium. Palisades of Vogt were absent in all (100.0%) patients, and the cornea-conjunctiva epithelial transition localized approximately 1 mm internally on the cornea., Conclusions: Confocal microscopy provides objective measures of the corneal epithelium and may significantly improve the evaluation of outcomes after cultured limbal stem cell graft., (Copyright © 2015 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Purpose: To evaluate and compare corneal and conjunctival biomarkers for immunocytochemical diagnosis of limbal stem cell deficiency (LSCD)., Methods: In accordance with the current literature, we selected K12 as the corneal biomarker and K7/K13/K19/MUC5AC as the conjunctival ones. The specificity and accuracy for each biomarker were assessed and compared on 10 healthy subjects and tissues of deceased donors. Twelve eyes of 9 patients clinically suspected of LSCD were enrolled. Epithelial cells (ECs) from the central cornea were collected using impression cytology (IC) and assessed for each biomarker. The presence of conjunctival cells in the central cornea was diagnostic proof of LSCD, whereas the detection of corneal residual cells would quantify the degree of LSCD., Results: K12 and K7/K13/MUC5AC are, respectively, highly specific of corneal and conjunctival differentiation, whereas K19 is not. Normal corneal ECs are not desquamative enough to be suitable for IC. Among 12 eyes with suspected LSCD, 84% (10 of 12) of IC samples were suitable for analysis. K3/K7/K19 immunostaining was positive in 100%, MUC5AC in 40%, and K12 was never observed., Conclusions: Clinical examination can lead to misdiagnosis of LSCD. Immunocytochemical detection of K7/K13 on corneal ECs collected by IC is reproducible, noninvasive, and highly effective in this indication, but without any quantification of the degree of the disease. This time-consuming technique requires skilled technicians and laboratory facilities, reserving it for planned limbal reconstruction.

Purpose: The purpose of this study was to develop a novel primary epithelial cell toxicity assay using porcine corneal explant and evaluate the assay using benzalkonium chloride (BAK)., Methods: Circular corneal explants were trephined from the peripheral cornea of porcine eyes using 2-mm biopsy punches, and placed on 6-well culture dishes with a culture medium. After incubation for 12 hours, 50 μL of BAK at 0.00001%, 0.0001%, 0.001%, or 0.01% was applied to each well for 2 minutes. After washing, explants were cultured for another 24 hours, then epithelial outgrowth was photographed and measured. Corneal immunohistochemical characteristics were evaluated by cytokeratin (CK) 3, CK12, and ZO-1. Cell toxicity was evaluated by WST-8 assay, Ki-67 staining, and TUNEL assay., Results: Epithelial cells migrated outward concentrically from the corneal explant as time elapsed and were positive for CK3, CK12, and ZO-1. The outgrowth rate decreased significantly with 0.0001%, 0.001%, and 0.01% BAK compared with the phosphate-buffered saline (PBS) control (P < 0.01), and the decrease was BAK concentration dependent. Numbers of viable cells and Ki-67-positive cells also decreased significantly with 0.01% BAK compared with the PBS control (P < 0.05). TUNEL-positive cells were present in the epithelial outgrowth. Moreover, TUNEL-positive cell density tended to increase with 0.01% BAK compared with the PBS control (P = 0.14) and 0.00001% BAK (P = 0.081)., Conclusions: Our novel toxicity assay using porcine corneal explant is simple and reflects the effect of single and short-duration instillation of eye drops. The method is useful for evaluation of corneal toxicity at low concentrations of BAK.

The ocular surface consists of the cornea, conjunctiva, and the limbus that is located in the transitional zone between the cornea and conjunctiva. The corneal epithelial cells are generated through the mitosis of corneal epithelial stem cells in the limbus. This study investigated a rabbit corneal deficiency model prepared by the surgical removal of the corneal and limbal epithelia, which express cytokeratin 12 (K12). After the surgery, K13-expressing conjunctival epithelium migrated onto the corneal surface and completely covered the surface, leading to neovascularization and corneal opacification. However, at 24 and 48 weeks after the surgery, K12-expressing cornea-like cells reappeared on the model ocular surface. These cells formed an island surrounded by invaded conjunctiva and were isolated from the limbus. Interestingly, in the 24-week model surface, α1(IV) and α2(IV) collagen chains, which are normally found in the basement membrane of the native limbus and conjunctiva, and not in the cornea, were continuously deposited throughout the entire basement membrane, including the basement membrane under cornea-like cells. By contrast, in the 48-week model surface, α1(IV) and α2(IV) collagen chains were absent from the basement membrane beneath the central part of cornea-like cells and were localized below the invaded conjunctiva and the transitional zone between cornea-like cells and the invaded conjunctiva, which had similar distribution to the native ocular basement membrane. Moreover, K12, K14, p63, vimentin, and α1(IV) and α2(IV) collagen chains, which are colocalized in the native limbus, were all present at the transitional zone of the 48-week model surface. Therefore, a limbus-like structure appeared to be reconstructed on the surface of the 48-week model as a stem cell niche. This study should aid in the understanding of human corneal deficiency, the correlation between the epithelial cell phenotype and the composition of the basement membrane, and the epithelial stem cell niche.

Purpose: The aim of this study is to further assess our previously reported keratin 12 (K12)-Leu132Pro specific siRNA in silencing the mutant allele in Meesmann's Epithelial Corneal Dystrophy (MECD) in experimental systems more akin to the in vivo situation through simultaneous expression of both wild-type and mutant alleles., Methods: Using KRT12 exogenous expression constructs transfected into cells, mutant allele specific knockdown was quantified using pyrosequencing and infrared Western blot analysis, while the silencing mechanism was assessed by a modified rapid amplification of cDNA ends (5'RACE) method. Corneal limbal biopsies taken from patients suffering from MECD were used to establish cultures of MECD corneal limbal epithelial stem cells and the ability of the siRNA to silence the endogenous mutant KRT12 allele was assessed by a combination of pyrosequencing, qPCR, ELISA, and quantitative-fluorescent immunohistochemistry (Q-FIHC)., Results: The siRNA displayed a potent and specific knockdown of K12-Leu132Pro at both the mRNA and protein levels with exogenous expression constructs. Analysis by the 5'RACE method confirmed siRNA-mediated cleavage. In the MECD cells, an allele-specific knockdown of 63% of the endogenous mutant allele was observed without effect on wild-type allele expression., Conclusions: Combined with an effective delivery vehicle this siRNA approach represents a viable treatment option for prevention of the MECD pathology observed in K12-Leu132Pro heterozygous individuals., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

The current standard method to culture human limbal stem/progenitor cells (LSCs) in vitro is to culture limbal epithelial cells directly on a layer of murine 3T3 feeder cells (standard method). The direct contact between human cells and murine feeder cells poses the potential risk of incomplete removal of feeder cells after culture and cross-contamination in clinical applications. We present here a novel three-dimensional (3D) sandwich method in which LSCs and feeder cells were separately cultured on opposite sides of a porous membrane. Limbal epithelial cells in the form of single-cell suspensions, cell clusters, and tissue explants were subjected to standard culture or to a 3D sandwich culture method. The 3D sandwich method consistently yielded LSCs derived from cell clusters and tissue explants. The expanded LSCs exhibited a small, compact, cuboidal stem-cell morphology and stem cell phenotypes comparable to those of LSCs derived from the standard culture method. Limbal epithelial cell clusters cultured with the sandwich method had a significantly higher proliferation rate than did those cultured with the standard method. The 3D sandwich method did not favor the propagation of single LSCs. In summary, the 3D sandwich method permits complete separation between cultured cells and feeder cells, while providing an even and maximal proximity between them. This alternative method permits culturing of LSCs without the risk of feeder cell contamination.

Purpose: To identify an allele-specific short interfering RNA (siRNA), against the common KRT12 mutation Arg135Thr in Meesmann epithelial corneal dystrophy (MECD) as a personalized approach to treatment., Methods: siRNAs against the K12 Arg135Thr mutation were evaluated using a dual luciferase reporter gene assay and the most potent and specific siRNAs were further screened by Western blot. Off-target effects on related keratins were assessed and immunological stimulation of TLR3 was evaluated by RT-PCR. A modified 5' rapid amplification of cDNA ends method was used to confirm siRNA-mediated mutant knockdown. Allele discrimination was confirmed by quantitative infrared immunoblotting., Results: The lead siRNA, with an IC(50) of thirty picomolar, showed no keratin off-target effects or activation of TLR3 in the concentration ranges tested. We confirmed siRNA-mediated knockdown by the presence of K12 mRNA fragments cleaved at the predicted site. A dual tag infrared immunoblot showed knockdown to be allele-specific, with 70% to 80% silencing of the mutant protein., Conclusions: A potent allele-specific siRNA against the K12 Arg135Thr mutation was identified. In combination with efficient eyedrop formulation delivery, this would represent a personalized medicine approach, aimed at preventing the pathology associated with MECD and other ocular surface pathologies with dominant-negative or gain-of-function pathomechanisms.

Purpose: Previous studies have shown that the stem cells of corneal epithelia are located at the limbal basal layer. Limbal stem cells are believed to be the source of corneal epithelial cell proliferation and differentiation. This study tested the replacement hypothesis, which suggests that corneal stem cell origin epithelia may be replaced by limbal stem cell origin epithelia after 2 weeks of age in mice., Methods: The cytokeratin 12 expression pattern in the cornea was examined using K12 IRES-Cre and Cre reporter mice., Results: Before 2 weeks of age, K12 expression in corneal epithelia showed a mosaic pattern. After 2 weeks of age, centripetal K12 IRES-Cre expression gradually elongated from the limbal area. Around 12 weeks of age, the mosaic expression pattern disappeared from the center of the cornea. Temporal and spatial observations of K12 IRES-Cre expression patterns suggested that the mosaic pattern cells proliferated and amassed at the same position from day 15.5 of the embryonic stage at the latest., Conclusions: Therefore, these cells were considered corneal stem cell origin epithelia. In contrast, centripetal pattern cell populations were considered limbal stem cell origin epithelia because they originated from the limbal area and moved to the center of the cornea. These observations suggest that corneal stem cell origin epithelia are replaced by limbal stem cell origin epithelia after 2 weeks of age in mice.

Purpose: The present study was conducted to evaluate the therapeutic effects of topically applied bone marrow (BM) cells and CD117-positive haematopoietic stem (CD117(+)) cells on alkali-induced corneal ulcers., Methods: Bone marrow cells and CD117(+) cells were isolated from syngenic mice and labelled with an intracellular cell tracer. Defined corneal wounds were produced in 89 eyes of syngenic mice and allowed to partially heal in vivo for 6 hr. The alkali-burned eyes were enucleated 6 hr postinjury and randomly divided into three groups. Control group (33 eyes) was incubated with medium only. The treatment groups received either BM cells (30 eyes) or CD117(+) cells (26 eyes) suspended in medium. Re-epithelialization process of corneal defects was qualitatively and quantitatively assessed and statistically analysed. The corneas were examined by histological and immunohistochemical methods., Results: We found that the re-epithelialization of corneal wounds in both treatment groups was significantly accelerated as compared to the control group. During the follow-up period (85 hr), the corneal transparency was comparable in all groups. Morphological investigations of corneas from control and treatment group showed no evident differences in the phenotype of the regenerated epithelium. Additionally, corneas in the treatment groups were devoid of donor-derived BM cells and CD117(+) cells, respectively., Conclusions: This study provides evidence that topical application of BM cells or CD117(+) cells can be used to reconstruct corneal surfaces. Because neither BM cells nor CD117(+) cells were integrated into the corneal epithelium, we suggest that soluble factors could be responsible for the positive effect of BM cells and CD117(+) cells on corneal wound healing., (© 2012 The Authors. Acta Ophthalmologica © 2012 Acta Ophthalmologica Scandinavica Foundation.)

This study was designed to demonstrate the effects of pluripotin on the proliferation, senescence and colony formation efficiency of rabbit limbal epithelial cells (RLECs) in vitro. Rabbit primary limbal epithelial cells were harvested and cultured in the presence of pluripotin. The cell proliferation was measured using the 3-(4,5)-dimethylthiahiazo(-z-y1)-3 5-di-phenytetrazoliumromide (MTT) assay and was also observed by confocal microscopy with Ki67 staining, whereas cell senescence was detected by senescence-associated β-galactosidase (SA-β-gal) staining. The colony morphology, colony-forming efficiency and colony size were observed and compared. The characteristics of the proliferating cells were examined by immunofluorescent staining using antibodies against deltaNP63, ABCG2 and Keratin 3/12. The results showed that pluripotin significantly promoted the proliferation of RLECs and increased the dividing cells with positive Ki67 staining at the concentrations lower than 400 nM. The colony-forming efficiency increased from 13.5% in the control cells to 26.4% in the 200 or 400 nM pluripotin-treated cells. The number of colonies of moderate size (600-900 μm) increased significantly in the presence of pluripotin (above 60.0% at 200 nM or 400 nM) compared with the untreated normal cells (18.6%), whereas the number of small-sized colonies (<600 μm) decreased from 79.5% for the control cells to lower than 35.0% at 200 nM or 400 nM pluripotin. Moreover, the cells treated with pluripotin stained negative with SA-β-gal, while the untreated cells showed visible positive staining. Immunofluorescent staining suggested that the pluripotin treatment resulted in higher positive staining for the limbal stem cell markers (deltaNP63 and ABCG2) and down-regulated of differentiated corneal epithelial cell marker (Keratin 3/12). This study confirmed that the small molecular compound pluripotin promoted the proliferation of rabbit limbal epithelial cells by improving the expansion of limbal stem/progenitor cells in vitro., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In patients with limbal stem cell deficiency (LSCD), transplantation of ex vivo expanded human limbal epithelial cells (HLECs) can restore the structural and functional integrity of the corneal surface. However, the protocol for cultivation and transplantation of HLECs differ significantly, and in most protocols growth additives such as cholera toxins, exogenous growth factors, hormones and fetal calf serum are used. In the present article, we compare for the first time human limbal epithelial cells (HLECs) cultivated on human amniotic membrane (HAM) in a complex medium (COM) including fetal bovine serum to a medium with human serum as single growth supplement (HSM), and report on our first examinations of HLECs expanded in autologous HSM and used for transplant procedures in patients with LSCD. Expanded HLECs were examined by genome-wide microarray, RT-PCR, Western blotting, and for cell viability, morphology, expression of immunohistochemical markers and colony forming efficiency. Cultivation of HLECs in HSM produced a multilayered epithelium where cells with markers associated with LESCs were detected in the basal layers. There were few transcriptional differences and comparable cell viability between cells cultivated in HSM and COM. The p63 gene associated with LESCs were expressed 3.5 fold more in HSM compared to COM, and Western blotting confirmed a stronger p63α band in HSM cultures. The cornea-specific keratin CK12 was equally found in both culture conditions, while there were significantly more CK3 positive cells in HSM. Cells in epithelial sheets on HAM remaining after transplant surgery of patients with LSCD expressed central epithelial characteristics, and dissociated cells cultured at low density on growth-arrested fibroblasts produced clones containing 21 ± 12% cells positive for p63α (n = 3). In conclusion, a culture medium without growth additives derived from animals or from animal cell cultures and with human serum as single growth supplement may serve as an equivalent replacement for the commonly used complex medium for ex vivo expansion of HLECs on HAM., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Aims: To investigate the optimum conditions for the differentiation of a mouse embryonic stem cell line towards corneal epithelial cell fate., Materials & Methods: The effect of conditioned media from both metabolically active (to produce lineage A) and growth-arrested limbal fibroblasts (lineage G) were compared with basal media (lineage N) in terms of morphology and marker expression, assessed by immunocytochemistry and reverse transcription PCR. Cultures were transplanted into a porcine ex vivo model to investigate their ability for wound healing and cornea repair., Results: Lineage N exhibited cobblestone morphology and expressed CK12 and p63α, while OCT4 and SSEA1 were downregulated. Post-transplantation, these cells were able to multilayer and heal after wounding while maintaining marker expression., Conclusion: Lineages with corneal epithelial-like characteristics, which are derived from embryonic stem cells, have potential for use in the study of corneal wound healing and therapy.

Purpose: The limbus of the cornea is said to be the niche for limbal stem cells (LSCs) and the primary source of corneal epithelial maintenance. Previously, we aimed to have shown that central human epithelial cells are capable of corneal regeneration after wounding. In this study, we aimed to investigate whether central epithelial cells in human corneas have LSC properties., Methods: Human corneal epithelial cells were separated from the central cornea and the limbus. Isolated cells were collected for sphere-forming assay, and spheres formed subsequently were analyzed using immunohistochemistry. Fluorescence-activated cell sorting (FACS) was also used to analyze epithelial cells from central cornea, limbal rim, older donors, younger donors, and dissociated spheres. These analyses were based on cell size and Hoechst 33342 dye efflux ability, and side populations and non-side populations were isolated for colony growth measurement and sphere-forming assay., Results: Human central and limbal epithelial cells were capable of forming spheres, in a 1:2 ratio, that were positive for p63 immunolabeling. In FACS, central and limbal epithelial cells showed no significant difference in cell size and dye efflux ability. There were almost 10 times more large cells with good dye efflux ability from younger donors than from older donors, and the gated side population showed more than 4 times faster rate of colony growth than the non-side population. Dissociated sphere cells, however, did not follow a similar pattern to tissue-derived cells using FACS analysis. In these, there were more than twice as many large cells than small cells with good dye efflux ability., Conclusions: Both limbal and central epithelial cells are capable of forming spheres in cultures that have stem cell properties. Central and limbal epithelial cells cannot be differentiated using FACS, but younger donor tissues give rise to greater numbers of large cells with high dye efflux. Therefore, results indicate that human central corneal epithelium contains cells with stem/progenitor properties, and these stem properties decline with age.

Purpose: To assess whether Pax6 functions directly in the cornea, a corneal-preferred promoter was used to overexpress Pax6 specifically in the cornea., Methods: Transgenic mice harboring a construct containing mouse Pax6 coding sequences fused downstream of the aldehyde dehydrogenase 3a1 (Aldh3a1) promoter were generated (Pax6 Tg). Pax6 expression was analyzed by Western blot and immunohistochemistry. Eye sections were stained with hematoxylin and eosin, Schiff reagent, and fluorescein, to assess morphologic changes, the presence of goblet cells, and barrier integrity, respectively. Gene expression changes in mildly affected Pax6 Tg corneas were compared to age-matched, wild-type (WT) corneas by microarray analysis and quantitative PCR. Promoter regulation of several differentially expressed genes was examined by monitoring luciferase activity of reporter constructs after cotransfection with Pax6 in COS7 cells., Results: Corneal overexpression of Pax6 produces an abnormal cornea with altered epithelial cell morphology, neovascularization, immune cell invasion, and a compromised barrier; the lens appeared normal. Major changes in expression of genes involved in immune function, vascularization, and epithelial differentiation occurred in corneas from Pax6 Tg versus WT mice. The keratin (K) profile was dramatically altered in the Pax6 Tg corneas, as were several components of the Wnt signaling pathway. In severely affected Pax6 Tg corneas, K12 was reduced, and Pax6 was redistributed into the cytoplasm. Promoters from the chitinase 3-like 3, Wnt inhibitory factor 1, and fms-related tyrosine kinase 1/soluble VEGF receptor genes were upregulated five-, seven-, and threefold, respectively, by Pax6 in transfected COS7 cells., Conclusions: Pax6 functions directly to maintain normal, corneal epithelial cells.

Purpose: Limbal fibroblasts are known to regulate the maintenance and differentiation of the corneal epithelium including the limbal epithelial stem cells. This study examined the effect of limbal fibroblast conditioned media in a mouse model of limbal stem cell deficiency., Methods: Limbal stem cell deficiency was created in C57/Bl6 mice by performing a limbus to limbus epithelial debridement. The mice were treated topically for 3 weeks with conditioned media derived from human limbal fibroblasts. The control mice were treated with skin fibroblast conditioned media or Dulbecco's serum-free medium., Results: The mice treated with limbal fibroblast conditioned media demonstrated substantial growth of corneal type epithelial cells on the corneal surface with less conjunctival goblet cells. By contrast, the control treated corneas were found to be covered primarily by conjunctival type epithelium., Conclusions: Cell culture media conditioned by limbal fibroblasts appear to contain factor(s) that are therapeutically beneficial in a model of limbal stem cell deficiency. The current results further support the notion that the essential limbal stem cell niche is provided by limbal fibroblasts and suggest a new, non-invasive option in the treatment of limbal stem cell deficiency.

Purpose: We investigated the immunohistochemical characteristics of corneal specimens in congenital aniridia and pemphigoid using various corneal markers to determine the status of the corneal epithelium., Methods: Conjunctivalization was clinically suspected in all corneas. Ten aniridia and seven pemphigoid paraffin-embedded corneal specimens were stained with periodic Schiff reagent (PAS) and antibodies against CK3/12, CK12, CK19, breast cancer resistance protein 1 (BCRP) and p63., Results: Aniridia: six cases contained goblet cells, four were negative. Both groups had cases with (three of six; one of four) and without CK19 positivity and cases with (two of six; three of four) and without p63 positivity. All aniridia cases except two in the goblet cell group were CK3/12- and CK12-positive and BCRP-negative. Pemphigoid: only one of the seven cases contained goblet cells. This case stained positively for CK19, 3/12, 12 and p63 and negatively for BCRP. The other six cases were positive for CK3/12, five of which were positive for CK12; only one case was CK19-positive. Three cases were p63-positive and two BCRP-positive. The CK12 staining was heterogenous in most cases and was often found in the superficial layer., Conclusion: Three different stages of epithelial characteristics were found in congenital aniridia and pemphigoid: (i) CK19-negative and inhomogenous CK12-positive cases indicating epithelium mainly from (partly) CK12-deficient limbal stem cells; (ii) CK19- and/or goblet cell-positive and CK12-positive cases with their epithelia originating from CK12-deficient limbal stem cells and from incursing conjunctival cells; and (iii) CK19-positive and CK12-negative cases consisting of conjunctival cells alone., (© 2009 The Authors. Journal compilation © 2009 Acta Ophthalmol.)

Purpose: To determine the long-term epithelial lineage of origin of surgically removed grafts after allogeneic cultivated corneal limbal epithelial transplantation (CLET)., Design: Interventional case reports., Participants: We studied 2 eyes from 2 patients with total corneal stem cell destruction; 1 eye was from a patient with Stevens-Johnson syndrome and 1 eye had sustained chemical injury., Methods: Allogeneic cultivated corneal limbal epithelial sheets on human amniotic membrane (AM) were transplanted onto the ocular surface. Regrafting (1 eye, 42 months later) or penetrating keratoplasty (1 eye, 75 months later) were performed after the initial transplantation procedure for further visual rehabilitation., Main Outcome Measures: The excised grafts were subjected to clinical evaluation and to light- and transmission electron microscopy (TEM) examination and to immunohistochemical analysis., Results: In clinically conjunctival grafts, TEM and immunohistochemical analysis disclosed only small areas where the original cultivated corneal epithelial cells persisted. Neighboring conjunctival epithelial cells had apparently invaded a large portion of the corneal surface (keratin 3/12(-), Muc5ac(+)). In clinically corneal grafts, transplanted allogeneic cultivated corneal epithelial cells clearly survived for a long period of time (keratin 3/12(+), Muc5ac(-)); there was no infiltration by inflammatory cells, nor was there dissolution of the AM substrate., Conclusions: We theorize that the process of graft opacification after allogeneic CLET is responsible for the loss of transplanted cultivated corneal epithelial cells and that this is followed by conjunctival cell invasion onto the corneal surface. The results of this study confirmed that in the clinically evaluated corneal graft, transplanted cultivated corneal epithelial cells indeed survived for a long period of time on the corneal surface and maintained ocular surface integrity, even though the transplanted cells were allogeneic., (Copyright © 2010 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Corneal development and structure were studied in the Iberian mole Talpa occidentalis, which has permanently closed eyelids, and the European mole Talpa europaea, in which the eyes are open. The vertebrate cornea typically maintains a three-layered structure - a stratified epithelium with protective and sensory function, an avascular, hypocellular, collagenous stroma, and an endothelium with both barrier and transport functions that regulates corneal hydration, hence maintaining transparency. Compared to mouse, both mole species had significant corneal specializations, but the Iberian mole had the most divergent phenotype, with no endothelium and a flattened monolayer epithelium. Nevertheless, normal epithelial cell junctions were observed and corneal transparency was maintained. Corneas of European moles have a dysmorphic phenotype that recapitulates the human disorder keratoconus for which no mouse model exists. Mole corneas are vascularized - a situation only previously observed in the manatee Trichechus- and have non-radial patterns of corneal innervation indicative of failure of corneal epithelial cell migration. The transcription factor Pax6 is required for corneal epithelial differentiation in mice, but was found to be dispensable in moles, which had mosaic patterns of PAX6 localization uniquely restricted, in European moles, to the apical epithelial cells. The apparently stalled or abnormal differentiation of corneas in adult moles is supported by their superficial similarity to the corneas of embryonic or neonatal mice, and their abnormal expression of cytokeratin-12 and cytokeratin-5. European moles seem to have maintained some barrier/protective function in their corneas. However, Iberian moles show a more significant corneal regression likely related to the permanent eyelid fusion. In this mole species, adaptation to the arid, harder, Southern European soils could have favoured the transfer of these functions to the permanently sealed eyelids., (© 2010 The Authors. Journal of Anatomy © 2010 Anatomical Society of Great Britain and Ireland.)

Purpose: To determine the epithelial phenotype in patients with a limbal stem cell deficiency (LSCD) after ocular surface reconstruction with autologous cultured stem cells. To correlate the epithelial phenotype with the clinical outcome., Methods: Six eyes affected by LSCD, verified and graded by impression cytology, were treated with an autologous fibrin-cultured limbal stem cell graft. The clinical outcome was defined as a "success" or a "failure," depending on ocular surface stability. To improve their visual function, 4 patients underwent lamellar or penetrating keratoplasty after the stem cell graft. The phenotype of the regenerated corneal epithelium was determined by immunofluorescence of the corneal button to detect CK12, CK3, CK19, and Muc1 as corneal and conjunctival markers., Results: After a mean follow-up of 24 months, 5 cases were defined as successes; 1 case presented an epithelial defect 4 months after grafting and was defined as a failure. Immunofluorescence performed on 4 patients after lamellar and penetrating keratoplasty confirmed the presence of epithelial corneal markers (CK12 and CK3) in 2 of the success cases and the presence of conjunctival markers (CK19 and Muc1) in the 1 failure case. In one of the success cases, both corneal and conjunctival markers were detected on the corneal button. All success cases showed maintenance of marker accounting for high proliferative potential (DeltaNp63alpha) after transplantation., Conclusions: Autologous cultures of limbal stem cells can regenerate a functional corneal epithelium in patients affected by unilateral LSCD. We showed a correlation between the clinical outcome and the molecular marker expression.

Purpose: To report a novel missense mutation of the cornea specific keratin 12 (KRT12) gene in two generations of a German family diagnosed with Meesmann;s corneal dystrophy., Methods: Ophthalmologic examination of the proband and sequencing of keratin 3 (KRT3) and KRT12 of the proband and three other family members were performed. Restriction enzyme analysis was used to confirm the detected mutation in affected individuals of the family., Results: Slit-lamp biomicroscopy of the proband revealed multiple intraepithelial microcysts comparable to a Meesmann dystrophy phenotype. A novel heterozygous A-->G transversion at the first nucleotide position of codon 129 (ATG>GTG, M129V) in exon 1 of KRT12 was detected in the proband, her two affected sons but not in her unaffected husband or 50 control individuals., Conclusions: We have identified a novel missense mutation within the highly conserved helix-initiation motif of KRT12 causing Meesmann;s corneal dystrophy in a German family.

Purpose: We previously reported the successful transplantation of corneal epithelium-like cells derived from mouse embryonic stem (ES) cells onto injured mouse cornea. Here, we tested whether nonhuman primate ES cells have ability to differentiate into corneal epithelial cells and whether monkey ES cell-derived corneal epithelium-like cells were applicable for the experimental transplantation to damaged cornea., Methods: Monkey ES cells were cultivated on type IV collagen-coated dishes for various days to induce differentiation into corneal epithelium-like cells. The differentiation was evaluated by reverse transcription-polymerase chain reaction and immunostaining. The corneal epithelium-like cells were transplanted to the injured mouse cornea. Reconstitution of the corneal epithelium was evaluated by immunostaining., Results: The cells cultured on type IV collagen showed cobblestone-like appearance resembling epithelial cells. They expressed messenger RNA of pax6, p63, E-cadherin, CD44, proliferating cell nuclear antigen, keratin 3, and keratin 12. Protein expressions of pax6, keratin 3/12, p63, proliferating cell nuclear antigen, E-cadherin, and CD44 were confirmed by immunostaining. When the corneal epithelium-like cells were transplanted, they adhered to the corneal stroma, leading to formation of multiple cell layers. The grafted cells were stained with anti-human nuclear protein antibody, which cross-reacted with nuclei of monkey cells but not with those of mouse cells. They retained the expressions of keratin 3/12, E-cadherin, and CD44., Conclusions: We induced corneal epithelium-like cells from monkey ES cells with moderate efficiency. The cells were successfully transplanted onto the injured mouse cornea. This is the first demonstration that nonhuman primate ES cells were induced to differentiate into corneal epithelium-like cells, which were applicable for transplantation to an animal model of corneal injury.

Purpose: To determine the feasibility of cultivated conjunctiva as a viable epithelial sheet for transplantation and corneal resurfacing in eyes with limbal stem cell deficiency (LSCD)., Methods: Human corneal epithelial (HCE) and human conjunctival epithelial (HCjE) cells were cultivated on human amniotic membrane (AM) to confluence and then air lifted to allow further stratification and differentiation. Denuded AM and cultivated HCE and cultivated HCjE cells were then transplanted into 18 eyes of rabbits with induced LSCD. The cultivated and engrafted epithelia were examined by transmission electron microscopy (TEM) and immunohistochemistry. Two weeks after transplantation, the eyes were examined by slit lamp biomicroscopy and scored on epithelial integrity, corneal haze, and corneal neovascularization., Results: Both cultivated and engrafted HCjE sheets demonstrated confluent epithelial sheets with five to six layers of well-stratified epithelium. TEM examination of engrafted HCjE revealed numerous microvilli, desmosomes, and hemidesmosomes, identical with in vivo corneal epithelium. Immunohistochemical analysis of both HCjE and HCE cells showed the presence of CK3, CK4, and CK12, with absence of Muc5AC. Clinical outcomes for eyes receiving HCjE transplants and HCE transplants were comparable, with most having transparent, smooth corneas, free of epithelial defects., Conclusions: The study showed that microscopically, HCjE cells have features similar to HCE cells, with clinically equivalent outcomes. The ex vivo cultivation of conjunctiva to form transplantable epithelial sheets for corneal replacement is a promising new treatment modality in patients with LSCD.

Purpose: To determine the growth potential of p63-positive cell clusters maintained in human limbal epithelial sheets., Methods: Intact human limbal epithelial sheets were isolated from corneoscleral rims and cultured with or without being rendered into single cells by trypsinization on either plastic or 3T3 fibroblast feeder layers. Clonal growth on 3T3 fibroblast feeder layers was compared between monolayers in sheets or single cells and between areas with or without laser-microdissected, p63-enriched cell clusters. Immunostaining, immunoblot analysis, and reverse transcription-polymerase chain reaction of such differentiation markers as keratin (K)-3 and -12 and such progenitor markers as p63, ABCG2, and MDR-1 were also compared., Results: Clusters of small p63-positive cells were enriched in limbal palisades of dispase-isolated epithelial sheets immediately or after brief cultivation on plastic in SHEM. Clonal growth of p63-rich cell clusters was higher than areas without clusters (P < 0.001). Clonal growth of epithelial monolayers derived from sheets was also higher than that derived from single cells (P < 0.01). Although expression of ABCG2 and MDR-1 transcripts was similar, cells from epithelial sheets expressed higher protein levels of p63 and lower protein levels of K3 and -12 than single cells, whether they were cultured on plastic or as 3T3 fibroblast feeder layers., Conclusions: Limbal palisades contain clusters of p63-rich progenitor cells. Maintenance of such cluster architecture during ex vivo expansion yields higher growth potential than being dispersed into single cells.

Purpose: To demonstrate the role of N-cadherin (N-cad) in maintaining the progenitor status of primary human limbal epithelial cells in vitro., Methods: Immunohistochemistry and immunoelectron microscopy against N-cad was performed in human limbal tissue. The expression of N-cad, cytokeratin (K) 12, and K14 was also observed in primary cultured limbal epithelial cell colonies in 3T3 feeder cells, which also express N-cad. Laser microdissection of individual colonies was performed to separate central cells from peripheral cells to compare secondary colony formation. Finally, an artificial small interfering RNA (siRNA) expression vector was used to downregulate N-cad in 3T3 cells (N-cad(low) 3T3) to observe changes in colony formation and cultivated epithelial sheet phenotype., Results: N-cad was expressed in clusters of basal limbal epithelial cells. Limbal epithelial cell colonies cocultured with N-cad(+) 3T3 feeder cells showed N-cad expression along the edge of each colony. When individual colonies were divided into peripheral and central sections by laser microdissection, peripheral cells had a significantly higher secondary colony-forming efficiency than did central cells. Furthermore, colonies using N-cad(low) 3T3 cells were significantly smaller than mock-transfected cells. Then a duplex-feeder model with two layers of either 3T3 or N-cad(low) 3T3 cells was used to produce stratified epithelial sheets. Only N-cad(+) 3T3 cells produced epithelial sheets with basal K15/ suprabasal K12 expression observed in limbal tissue., Conclusions: N-cad plays a pivotal role in the maintenance of the progenitor phenotype in cultured limbal epithelial cells.

Several types of adult stem cells are capable of transdifferentiaton into other types of tissues. The hair follicle bulge area is an abundant and easily accessible source of pluripotent adult stem cells. We demonstrate that the bulge KSCs have the potential for transdifferentiation into corneal epithelial-like cells. Bulge KSCs isolated by collagen type IV adhesiveness possessed the highest colony formation efficiency (CFE), and expressed specific markers (CD34 and alpha6-integrin). The isolated cells transdifferentiate into corneal epithelial-like cells in conditioned medium containing corneal limbus soluble factors, including their specific marker, keratin12. The transdifferentiation depends on upregulation of pax6 and downregulation of beta-catenin and Lef-1. Furthermore, overexpression of pax6 in bulge KSCs induced their expression of k12. The expressions of beta-catenin and Lef-1 were not suppressed in the pax6-transfected bulge KSCs, but which were downregulated pax6-transfected cells cultured in the conditioned medium. Bulge KSCs may have potential therapeutic application as cell source for the construction of bioengineered corneas.

Background: This study was undertaken to establish the characterization of cultured oral mucosal epithelium and introducing them as an alternative source for reconstruction of ocular surface disease., Methods: Human oral epithelial cells were cultured on simple media (DMEM/HF12) as control and co-cultured on mitomycin C-treated 3T3 feeder layer, on the amniotic membrane (AM) without nitrocellulose and the mitotically inactivated 3T3 fibroblast, and on the sandwich layer of AM fastened on the nitrocellulose as insert and 3T3 fibroblast. After 3 weeks, the characteristics of the cells were assessed morphologically and also ultrastructurally using scanning electron microscopy and transmission electron microscopy and immuno-cytochemically., Results: The epithelial cells were cultured on AM spread on nitrocellulose insert and 3T3 feeder layer showed better growth than other groups and all groups of study were shown similar characteristics. The cultured oral epithelial shared the characteristics with corneal epithelium., Conclusion: Thus the oral epithelial could be an alternative source for transplantation.

To compare the stem niche in different culture conditions of limbal epithelial cells, the suspended human limbal epithelial cells (HLECs) were seeded on the 3T3-pretreated plates and the other suspended cells were plated on amniotic membranes (AMs) which were either cryo-preserved or freeze-dried. All were cultured for 10 to 12 days. Reverse transcription-polymerase chain reaction (RT-PCR) for ATP-binding cassette, subfamily G, member 2 (ABCG2), p63, cytokeratin 12, and connexin 43 were performed in cultivated HLECs and their expression levels were compared. The mRNA expression of all markers examined showed no statistically significant differences between the cells on cryo-preserved and on freeze-dried AM. The expression of p63 and cytokeratin 12 in cultivated cells on AMs were significantly lower than those in 3T3-cocultured cells on RT-PCR and immunofluorescent staining. Cultivated HLECs on AMs showed reduced proliferation and differentiation while maintaining stem-property regardless of the preservative method of AM.

Purpose: To investigate the effect of human serum, an ocular therapy, on human corneal fibroblast (HCF) wound-healing activities., Methods: The water soluble tetrazolium reagent-1, chemotactic chambers, fibroblast-populated type-I collagen gels, zymography, and Western blotting were used to assess HCF proliferation, migration, contraction, and matrix metalloproteinase (MMP) activity and levels, respectively. Fibroblasts were obtained from human donor corneas. Human serum, fibroblast culture medium (FCM; Dulbeccos Minimal Essential Medium/10% newborn calf serum) with and without calf serum supplementation, and 0.3% hypromellose were compared., Results: Proliferation and migration were maximal in 1% human serum. Relaxed gel contraction was maximal for fibroblasts cultured in 10%, 50%, and 100% serum and FCM. Whereas in stressed gels, maximal contraction was induced when fibroblasts were cultured in 50% and 100% serum. In low serum concentrations, greater MMP-2 activity was detected than MMP-1 and MMP-9., Conclusions: Low concentrations of human serum stimulated HCF migration, proliferation, and MMP activity. High concentrations produced greater matrix contraction. This may have implications for the therapeutic use of autologous serum.

Purpose: To establish a culture technique for human corneal epithelial equivalents that do not require fetal bovine serum (FBS), feeder cells, or bovine pituitary extracts and compare this system with conventional culture medium with FBS and mouse 3T3 fibroblasts., Methods: Human corneal limbal tissue from donor corneas was dissociated on denuded amniotic membranes and then cultured for 3 weeks in feeder-cell- and serum-free medium containing epidermal growth factor and B-27. Then, the cell sheet was evaluated by light microscopy, immunohistochemistry, and electron microscopy. The epithelial proliferative capacity was compared between serum- and feeder-cell-free medium and conventional medium. The cultured cell sheets were transplanted onto the denuded rabbit ocular surface to cover the resected area., Results: A stratified cell sheet expressing cytokeratin-3 and -12 was grown in serum- and feeder-cell-free medium without unknown growth factors. The epithelial proliferative capacity in feeder-cell- and serum-free medium determined by WST-1 and colony-forming efficiency was significantly higher than that in conventional medium. Scanning and transmission electron microscopy showed well-formed stratified epithelium with clear cell boundaries, microvilli, and hemidesmosomal/desmosomal junctions. The transplanted cell sheets remained transparent without epithelial defects during the follow-up period., Conclusions: This method using serum- and feeder-cell-free medium not containing unknown growth factors allows the highly proliferative culture of human corneal epithelium. It avoids exposure of the corneal epithelial equivalent to FBS and animal feeder cells, thus minimizing the risk of contamination by pathogens that could transmit diseases to recipients.

Purpose: To examine whether promiscuous Cre/LoxP recombination happens during gametogenesis in double transgenic mice carrying LoxP modified alleles and Cre transgene driven by tissue-specific promoter outside the gonads of adult mice., Methods: Cre driver mice were crossbred with reporter mouse lines (e.g., ZEG and Rosa26R) to obtain Cre/ZEG and Cre/Rosa26R double transgenic mice. The frequency of promiscuous LoxP/Cre recombination was determined by the expression of second reporter genes in the offspring of double transgenic mice., Results: The frequency of promiscuous LoxP/Cre recombination varied in different lines of Cre driver mice and in the sex of the same driver mice with higher penetrance in male than in female double transgenic mice. Polymerase chain reaction (PCR) and recombination analysis demonstrate that the recombination of floxed allele occurs during the transition from spermatogonia (diploid) to primary spermatocyte (tetraploid) in the testis. Thereby, target-floxed allele(s) may be ubiquitously ablated in experimental animals intended for tissue-specific gene deletion., Conclusions: Gametogenesis-associated recombination should always be examined in tissue-specific gene ablation studies.

Purpose: To describe a case of bilateral corneal epithelial dysplasia in which each lesion was characterized by both laser confocal biomicroscopy and cytokeratin immunofluorescence., Methods: A 52-year-old Japanese woman with bilateral corneal epithelial dysplasia was treated by corneal epithelial debridement. We observed the affected area with laser confocal biomicroscopy before and after treatment and examined the immunofluorescence of cytokeratins to examine the characteristics of the abnormal epithelial cells., Results: Laser confocal biomicroscopy revealed the atypical epithelial cells in all layers of the corneal epithelium as well as the reconstituted normal structure of the corneal epithelium after epithelial debridement. Immunofluorescence of cytokeratin 12 (K12) and K4 revealed the presence of four types of cells (those positive for one, both or neither of these cytokeratins) in each lesion., Conclusion: Cells expressing both K12 and K4 probably represented dysplastic cells that had invaded the cornea via the limbus and adopted characteristics of corneal epithelial cells. Cells lacking both K12 and K4 were probably either undifferentiated cells or epithelial cells in which cytokeratin expression had not been initiated.

Pax6 is the universal master control gene for eye morphogenesis. Other than retina and lens, Pax6 also expressed in the ocular surface epithelium from early gestation until the postnatal stage, in which little is known about the function of Pax6. In this study, corneal pannus tissues from patients with ocular surface diseases such as Stevens-Johnson syndrome (SJS), chemical burn, aniridia and recurrent pterygium were investigated. Our results showed that normal ocular surface epithelial cells expressed Pax6. However, corneal pannus epithelial cells from the above patients showed a decline or absence of Pax6 expression, accompanied by a decline or absence of K12 keratin but an increase of K10 keratin and filaggrin expression. Pannus basal epithelial cells maintained nuclear p63 expression and showed activated proliferation, evidenced by positive Ki67 and K16 keratin staining. On 3T3 fibroblast feeder layers, Pax6 immunostaining was negative in clones generated from epithelial cells harvested from corneal pannus from SJS or aniridia, but positive in those from the normal limbal epithelium; whereas western blots showed that some epithelial clones expanded from pannus retained Pax6 expression. Transient transfection of an adenoviral vector carrying EGFP-Pax6 transgenes into these Pax6(-) clones increased both Pax6 and K12 keratin expression. These results indicate that Pax6 helps to maintain the normal corneal epithelial phenotype postnatally, and that down-regulation of Pax6 is associated with abnormal epidermal differentiation in severe ocular surface diseases. Reintroduction of activation of the Pax6 gene might be useful in treating squamous metaplasia of the ocular surface epithelium.

Purpose: AP-1 is a transcription factor that plays a pivotal role in regulating cellular homeostasis and which may modulate the differentiation of corneal epithelial cells. We examined the role of c-Fos in the differentiation of corneal epithelial cells by using c-Fos-deficient (c-fos (-/-)) mice., Methods: Ten adult c-fos (-/-) mice and ten control (c-fos (+/-) or c-fos (+/+)) mice were used. The expression patterns of the mRNA and protein of keratin 12 (K12) were determined to examine the differentiation of cornea-type epithelium. To evaluate the intraepithelial differentiation from basal cells to superficial cells, the ultrastructure of the corneal epithelium was studied. We focused on the formation of desmosomes in the superficial, suprabasal, and basal cell layers, and also on the hemidesmosomes. The number of desmosomes in each epithelial layer was statistically analyzed by using an unpaired t test. The expressions of keratin 14 (K14), desmoglein, E-cadherin, occludin, connexin 43, filaggrin, loricrin, and involucrin were examined to analyze epithelial differentiation., Results: The mRNA and protein of K12 were expressed in the corneal epithelium of c-fos (-/-) and control mice. Ultrastructural observations showed that the number of desmosomes between the basal cells of the corneal epithelia was similar in c-fos (-/-) and control mice. However, there were fewer desmosomes between suprabasal cells and between superficial cells in c-fos (-/-) mice than in control mice. The number of hemidesmosomes in the corneal epithelial cells in c-Fos-null mice was similar to that in control mice. The expressions of the other epithelial cell differentiation markers were not affected by the absence of c-Fos. Ultrastructural observations showed a disarrangement of the corneal epithelium in the c-Fos-null mice., Conclusions: The absence of c-Fos disturbs the formation of desmosomes in the superficial layers of the corneal epithelium, suggesting a perturbation of intraepithelial differentiation from the basal epithelial cells to the suprabasal and superficial epithelial cells.

Purpose: Corneal epithelial stem cells are deficient in cases of limbal disorders, leading to conjunctival epithelial ingrowth, vascularization, and eventually visual disturbance. We introduced the eye development-associated transcription factor pax6 to embryonic stem (ES) cells and tested whether pax6-transfected cells resembling purified corneal epithelial cells were applicable as a cell source for corneal transplantation., Methods: pax6 cDNA with green fluorescence protein was electrotransfected to ES cells and the cells were cultured with G418 for 14 days. They were characterized by reverse transcription-polymerase chain reaction and immunohistochemistry. The cells were transplanted onto experimentally damaged mouse corneas. Histologic reconstitution of the corneal epithelium was assessed., Results: pax6-transfected cells formed a monolayer of epithelium-like cells in vitro. They expressed cytokeratin12, a specific keratin of corneal epithelial cells, E-cadherin, and CD44, which are important adhesion molecules of corneal epithelial cells on the cell membrane. They accumulated to make a colony that gave a staining pattern of reticular configuration for cytokeratin 12, E-cadherin, and CD44. When the cells were transplanted onto damaged cornea, they have been kept alive on the cornea., Conclusions: The purified corneal epithelium-like cells derived from ES cells transfected with pax6 gene adapted to the injured cornea and were kept alive on it. These results suggested application of ES cell-derived corneal epithelial cells for treating corneal injuries.