Human Limbal Epithelial Cells Research Articles

The Role of SLIT3-ROBO4 Signaling in Endoplasmic Reticulum Stress-Induced Delayed Corneal Epithelial and Nerve Regeneration.

In the present study, we aim to elucidate the underlying molecular mechanism of endoplasmic reticulum (ER) stress induced delayed corneal epithelial wound healing and nerve regeneration. Human limbal epithelial cells (HLECs) were treated with thapsigargin to induce excessive ER stress and then RNA sequencing was performed. Immunofluorescence, qPCR, Western blot, and ELISA were used to detect the expression changes of SLIT3 and its receptors ROBO1-4. The role of recombinant SLIT3 protein in corneal epithelial proliferation and migration were assessed by CCK8 and cell scratch assay, respectively. Thapsigargin, exogenous SLIT3 protein, SLIT3-specific siRNA, and ROBO4-specific siRNA was injected subconjunctivally to evaluate the effects of different intervention on corneal epithelial and nerve regeneration. In addition, Ki67 staining was performed to evaluate the proliferation ability of epithelial cells. Thapsigargin suppressed normal corneal epithelial and nerve regeneration significantly. RNA sequencing genes related to development and regeneration revealed that thapsigargin induced ER stress significantly upregulated the expression of SLIT3 and ROBO4 in corneal epithelial cells. Exogenous SLIT3 inhibited normal corneal epithelial injury repair and nerve regeneration, and significantly suppressed the proliferation and migration ability of cultured mouse corneal epithelial cells. SLIT3 siRNA inhibited ROBO4 expression and promoted epithelial wound healing under thapsigargin treatment. ROBO4 siRNA significantly attenuated the delayed corneal epithelial injury repair and nerve regeneration induced by SLIT3 treatment or thapsigargin treatment. ER stress inhibits corneal epithelial injury repair and nerve regeneration may be related with the upregulation of SLIT3-ROBO4 pathway.

Anti-inflammatory effect of curcuminoids and their analogs in hyperosmotic human corneal limbus epithelial cells

BackgroundTo assess the efficacy of curcuminoids (curcumin, demethoxycurcumin, bisdemethoxycurcumin [BDC]) and their analogs (tetrahydrocurcumin [THC], tetrahydrodemethoxycurcumin [THDC], tetrahydrobisdemethoxycurcumin) in reducing inflammatory cytokines and their toxicity to primary human corneal limbal epithelial cells, these cells were cultured and exposed to these compounds.MethodsThe PrestoBlue assay assessed cell viability after treatment. Anti-inflammatory effects on hyperosmotic cells were determined using real-time polymerase chain reaction and significance was gauged using one-way analysis of variance and Tukey’s tests, considering p-values < 0.05 as significant.ResultsCurcuminoids and their analogs, at 1, 10, and 100 µM, exhibited no effect on cell viability compared to controls. However, cyclosporin A 1:500 significantly reduced cell viability more than most curcuminoid treatments, except 100 µM curcumin and BDC. All tested curcuminoids and analogs at these concentrations significantly decreased mRNA expression levels of tumor necrosis factor-α, interleukin (IL)-1β, IL-6, IL-17 A, matrix metallopeptidase-9, and intercellular adhesion molecule-1 after 90 mM NaCl stimulation compared to untreated cells. Furthermore, proinflammatory cytokine levels from hyperosmotic cells treated with 1, 10, and 100 µM curcumin, 100 µM BDC, 100 µM THC, 1 and 100 µM THDC mirrored those treated with cyclosporin A 1:500.ConclusionThe anti-inflammatory efficiency of 1 and 10 µM curcumin, 100 µM THC, 1 and 100 µM THDC was comparable to that of cyclosporin A 1:500 while maintaining cell viability.

Conditional reprogrammed human limbal epithelial cell model for anti-SARS-CoV-2 drug screening

To minimize the global pandemic COVID-19 spread, understanding the possible transmission routes of SARS-CoV-2 and discovery of novel antiviral drugs are necessary. We describe here that the virus can infect ocular surface limbal epithelial, but not other regions. Limbal supports wild type and mutant SARS-CoV-2 entry and replication depending on ACE2, TMPRSS2 and possibly other receptors, resulting in slight CPE and arising IL-6 secretion, which symbolizes conjunctivitis in clinical symptoms. With this limbal model, we have screened two natural product libraries and discovered several unreported drugs. Our data reveal important commonalities between COVID-19 and ocular infection with SARS-CoV-2, and establish an ideal cell model for drug screening and mechanism research.

Surface coatings of contact lenses for cell sheet transplantation

AbstractThe field of corneal cell sheet engineering has evolved rapidly in the last decade; however, challenges related to transplantation into target tissue still exist, necessitating a new approach to integrate cell sheet technology onto a contact lens surface. In this study, a novel surface‐modified contact lens was developed for corneal regenerative medicine to ensure the easy and safe integration of cell sheets into the target tissue. The lens surface was coated with a thermo‐responsive polymer using the initiated chemical vapor deposition technique, facilitating the separation of tissue‐integrated cell sheets from the coated lens surface by simply lowering the temperature. BEAS‐2B and L929 cells, along with human limbal epithelial cells, were cultured on a crosslinked poly(2‐(dimethylamino)ethyl methacrylate)‐coated lens, providing temperature sensitivity for 14 days. At the end of the incubation period, the cultured cells were harvested as intact monolayer cell sheets through a simple temperature reduction, without enzymatic treatment or additional surgical procedures. Rapid and complete delamination of the cells planted and incubated on the coated lens surface was achieved at room temperature. Therapeutic contact lenses modified with cell sheet technology allowed targeted delivery of cells to the affected area of the cornea.

NRG1 Regulates Proliferation, Migration and Differentiation of Human Limbal Epithelial Stem Cells.

Limbal epithelial stem/progenitor cells (LESCs) proliferate, migrate and differentiate into mature corneal epithelium cells (CECs) that cover the ocular surface. LESCs play a crucial role in the maintenance and regeneration of the corneal epithelium, and their dysfunction can lead to various corneal diseases. Neuregulin 1 (NRG1) is a member of the epidermal growth factor family that regulates the growth and differentiation of epithelial tissues. Here, we depicted the dynamic transcriptomic profiles during human CEC differentiation, identifying six gene co-expression modules that were specific to different differentiation stages. We found that the expression of NRG1 was high in human LESCs and decreased dramatically upon differentiation. Knockdown of NRG1 significantly inhibited LESC proliferation and upregulated the expression of the terminal differentiation marker genes KRT3, KRT12 and CLU. In addition, the scratch wound closure assay showed that knockdown of NRG1 attenuated wound closure of LESCs over 24 h. Together, we dissected the transcriptional regulatory dynamics during CEC differentiation and identified NRG1 as a key regulator that promoted LESC proliferation and migration and maintained the undifferentiated state.

Culturing Limbal Epithelial Cells of Long-term Stored Corneal Donors (Organ Culture) In Vitro - A Stepwise Linear Regression Algorithm.

To assess various potential factors on human limbal epithelial cell (LEC) outgrowth in vitro using corneal donor tissue following long-term storage (organ culture) and a stepwise linear regression algorithm. Of 215 donors, 304 corneoscleral rings were used for our experiments. For digestion of the limbal tissue and isolation of the limbal epithelial cells, the tissue pieces were incubated with 4.0 mg/mL collagenase A at 37 °C with 95% relative humidity and a 5% CO2 atmosphere overnight. Thereafter, limbal epithelial cells were separated from limbal keratocytes using a 20-µm CellTricks filter. The separated human LECs were cultured in keratinocyte serum-free medium medium, 1% penicillin/streptomycin (P/S), 0.02% epidermal growth factor (EGF), and 0.3% bovine pituitary extract (BPE). The potential effect of donor age (covariate), postmortem time (covariate), medium time (covariate), size of the used corneoscleral ring (360°, 270°180°, 120°, 90°, less than 90°) (covariate), endothelial cell density (ECD) (covariate), gender (factor), number of culture medium changes during organ culture (factor), and origin of the donor (donating institution and storing institution, factor) on the limbal epithelial cell outgrowth was analyzed with a stepwise linear regression algorithm. The rate of successful human LEC outgrowth was 37.5%. From the stepwise linear regression algorithm, we found out that the relevant influencing parameters on the LEC growth were intercept (p < 0.001), donor age (p = 0.002), number of culture medium changes during organ culture (p < 0.001), total medium time (p = 0.181), and size of the used corneoscleral ring (p = 0.007), as well as medium time × size of the corneoscleral ring (p = 0.007). The success of LEC outgrowth increases with lower donor age, lower number of organ culture medium changes during storage, shorter medium time in organ culture, and smaller corneoscleral ring size. Our stepwise linear regression algorithm may help us in optimizing LEC cultures in vitro.

Reversal of dual epigenetic repression of non-canonical Wnt-5a normalises diabetic corneal epithelial wound healing and stem cells

Aims/hypothesisDiabetes is associated with epigenetic modifications including DNA methylation and miRNA changes. Diabetic complications in the cornea can cause persistent epithelial defects and impaired wound healing due to limbal epithelial stem cell (LESC) dysfunction. In this study, we aimed to uncover epigenetic alterations in diabetic vs non-diabetic human limbal epithelial cells (LEC) enriched in LESC and identify new diabetic markers that can be targeted for therapy to normalise corneal epithelial wound healing and stem cell expression.MethodsHuman LEC were isolated, or organ-cultured corneas were obtained, from autopsy eyes from non-diabetic (59.87±20.89 years) and diabetic (71.93±9.29 years) donors. The groups were not statistically different in age. DNA was extracted from LEC for methylation analysis using Illumina Infinium 850K MethylationEPIC BeadChip and protein was extracted for Wnt phospho array analysis. Wound healing was studied using a scratch assay in LEC or 1-heptanol wounds in organ-cultured corneas. Organ-cultured corneas and LEC were transfected with WNT5A siRNA, miR-203a mimic or miR-203a inhibitor or were treated with recombinant Wnt-5a (200 ng/ml), DNA methylation inhibitor zebularine (1–20 µmol/l) or biodegradable nanobioconjugates (NBCs) based on polymalic acid scaffold containing antisense oligonucleotide (AON) to miR-203a or a control scrambled AON (15–20 µmol/l).ResultsThere was significant differential DNA methylation between diabetic and non-diabetic LEC. WNT5A promoter was hypermethylated in diabetic LEC accompanied with markedly decreased Wnt-5a protein. Treatment of diabetic LEC and organ-cultured corneas with exogenous Wnt-5a accelerated wound healing by 1.4-fold (p<0.05) and 37% (p<0.05), respectively, and increased LESC and diabetic marker expression. Wnt-5a treatment in diabetic LEC increased the phosphorylation of members of the Ca2+-dependent non-canonical pathway (phospholipase Cγ1 and protein kinase Cβ; by 1.15-fold [p<0.05] and 1.36-fold [p<0.05], respectively). In diabetic LEC, zebularine treatment increased the levels of Wnt-5a by 1.37-fold (p<0.01)and stimulated wound healing in a dose-dependent manner with a 1.6-fold (p<0.01) increase by 24 h. Moreover, zebularine also improved wound healing by 30% (p<0.01) in diabetic organ-cultured corneas and increased LESC and diabetic marker expression. Transfection of these cells with WNT5A siRNA abrogated wound healing stimulation by zebularine, suggesting that its effect was primarily due to inhibition of WNT5A hypermethylation. Treatment of diabetic LEC and organ-cultured corneas with NBC enhanced wound healing by 1.4-fold (p<0.01) and 23.3% (p<0.05), respectively, with increased expression of LESC and diabetic markers.Conclusions/interpretationWe provide the first account of epigenetic changes in diabetic corneas including dual inhibition of WNT5A by DNA methylation and miRNA action. Overall, Wnt-5a is a new corneal epithelial wound healing stimulator that can be targeted to improve wound healing and stem cells in the diabetic cornea.Data availabilityThe DNA methylation dataset is available from the public GEO repository under accession no. GSE229328 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE229328).Graphical

ABCB5+ Limbal Epithelial Stem Cells Inhibit Developmental but Promote Inflammatory (Lymph) Angiogenesis While Preventing Corneal Inflammation.

The limbus, the vascularized junction between the cornea and conjunctiva, is thought to function as a barrier against corneal neovascularization. However, the exact mechanisms regulating this remain unknown. In this study, the limbal epithelial stem cell (LESC) marker ABCB5 was used to investigate the role of LESCs in corneal neovascularization. In an ABCB5KO model, a mild but significant increase of limbal lymphatic and blood vascular network complexity was observed in developing mice (4 weeks) but not in adult mice. Conversely, when using a cornea suture model, the WT animals exhibited a mild but significant increase in the number of lymphatic vessel sprouts compared to the ABCB5KO, suggesting a contextual anti-lymphangiogenic effect of ABCB5 on the limbal vasculature during development, but a pro-lymphangiogenic effect under inflammatory challenge in adulthood. In addition, conditioned media from ABCB5-positive cultured human limbal epithelial cells (ABCB5+) stimulated human blood and lymphatic endothelial cell proliferation and migration. Finally, a proteomic analysis demonstrated ABCB5+ cells have a pro(lymph)angiogenic as well as an anti-inflammatory profile. These data suggest a novel dual, context-dependent role of ABCB5+ LESCs, inhibiting developmental but promoting inflammatory (lymph)angiogenesis in adulthood and exerting anti-inflammatory effects. These findings are of high clinical relevance in relation to LESC therapy against blindness.

Wnt/β-Catenin Signaling Activation Induces Differentiation in Human Limbal Epithelial Stem Cells Cultured Ex Vivo.

Human limbal epithelial stem cells (hLESCs) continuously replenish lost or damaged human corneal epithelial cells. The percentage of stem/progenitor cells in autologous ex vivo expanded tissue is essential for the long-term success of transplantation in patients with limbal epithelial stem cell deficiency. However, the molecular processes governing the stemness and differentiation state of hLESCs remain uncertain. Therefore, we sought to explore the impact of canonical Wnt/β-catenin signaling activation on hLESCs by treating ex vivo expanded hLESC cultures with GSK-3 inhibitor LY2090314. Real-time qRT-PCR and microarray data reveal the downregulation of stemness (TP63), progenitor (SOX9), quiescence (CEBPD), and proliferation (MKI67, PCNA) genes and the upregulation of genes for differentiation (CX43, KRT3) in treated- compared to non-treated samples. The pathway activation was shown by AXIN2 upregulation and enhanced levels of accumulated β-catenin. Immunocytochemistry and Western blot confirmed the findings for most of the above-mentioned markers. The Wnt/β-catenin signaling profile demonstrated an upregulation of WNT1, WNT3, WNT5A, WNT6, and WNT11 gene expression and a downregulation for WNT7A and DKK1 in the treated samples. No significant differences were found for WNT2, WNT16B, WIF1, and DKK2 gene expression. Overall, our results demonstrate that activation of Wnt/β-catenin signaling in ex vivo expanded hLESCs governs the cells towards differentiation and reduces proliferation and stem cell maintenance capability.

Assessment of Rose Bengal Photodynamic Therapy on Viability and Proliferation of Human Keratolimbal Epithelial and Stromal Cells In Vitro.

To investigate the effect of Rose Bengal photodynamic therapy (RB-PDT) on viability and proliferation of human limbal epithelial stem cells (T-LSCs), human corneal epithelial cells (HCE-T), human limbal fibroblasts (LFCs), and human normal and keratoconus fibroblasts (HCFs and KC-HCFs) in vitro. T-LSCs and HCE-T cell lines were used in this research. LFCs were isolated from healthy donor corneal limbi (n = 5), HCFs from healthy human donor corneas (n = 5), and KC-HCFs from penetrating keratoplasties of keratoconus patients (n = 5). After cell culture, RB-PDT was performed using 0.001% RB concentration and 565 nm wavelength illumination with 0.14 to 0.7 J/cm2 fluence. The XTT and the BrdU assays were used to assess cell viability and proliferation 24 h after RB-PDT. RB or illumination alone did not change cell viability or proliferation in any of the cell types (p ≥ 0.1). However, following RB-PDT, viability decreased significantly from 0.17 J/cm2 fluence in HCFs (p < 0.001) and KC-HCFs (p < 0.0001), and from 0.35 J/cm2 fluence in T-LSCs (p < 0.001), HCE-T (p < 0.05), and LFCs ((p < 0.0001). Cell proliferation decreased significantly from 0.14 J/cm2 fluence in T-LSCs (p < 0.0001), HCE-T (p < 0.05), and KC-HCFs (p < 0.001) and from 0.17 J/cm2 fluence in HCFs (p < 0.05). Regarding LFCs proliferation, no values could be determined by the BrdU assay. Though RB-PDT seems to be a safe and effective treatment method in vivo, its dose-dependent phototoxicity on corneal epithelial and stromal cells has to be respected. The data and experimental parameters applied in this study may provide a reliable reference for future investigations.

WNT16b promotes the proliferation and self-renewal of human limbal epithelial stem/progenitor cells via activating the calcium/calcineurin A/NFATC2 pathway.

Our previous finding revealed that WNT16b promoted the proliferation of human limbal epithelial stem cells (hLESCs) through a β-catenin independent pathway. Here, we aimed to explore its underlying molecular mechanism and evaluate its potential in the treatment of limbal stem cell deficiency (LSCD). Based on the findings of mRNA-sequencing, the expression of key molecules in WNT/calcineurin A/NFATC2 signalling pathway was investigated in WNT16b-co-incubated hLESCs and control hLESCs. An epithelial wound healing model was established on Wnt16b-KO mice to confirm the regulatory effect of WNT16b in vivo. The therapeutic potential of WNT16b-co-incubated hLESCs was also evaluated in mice with LSCD. Our findings showed that WNT16b bound with Frizzled7, promoted the release of Ca2+ and activated calcineurin A and NFATC2. With the translocation of NFATC2 into cell nucleus and the activation of HDAC3, WDR5 and GCN5L2, the expression of H3K4me3, H3K14ac and H3K27ac in the promoter regions of FoxM1 and c-MYC increased, which led to hLESC proliferation. The effect of the WNT16b/calcium/calcineurin A/NFATC2 pathway on LESC homeostasis maintenance and corneal epithelial repair was confirmed in Wnt16b-KO mice. Moreover, WNT16b-coincubated hLESCs could reconstruct a stable ocular surface and inhibit corneal neovascularization in mice with LSCD. In conclusion, WNT16b enhances the proliferation and maintains the stemness of hLESCs by activating the non-canonical calcium/calcineurin A/NFATC2 pathway in vitro and in vivo, and accelerates corneal epithelial wound healing.

ETS1-HMGA2 Axis Promotes Human Limbal Epithelial Stem Cell Proliferation.

This study aimed to investigate the role and molecular mechanism of ETS1 in the proliferation and differentiation of human limbal epithelial stem cells (LESCs). RNA-seq and quantitative real-time PCR were used to determine gene expression changes when ETS1 and HMGA2 was knocked down using short-hairpin RNAs or overexpressed by lentivirus. Immunofluorescence and flow cytometry experiments were performed to assess the roles of ETS1 and HMGA2 in LESC proliferation. ETS1-bound cis-regulatory elements and target genes in LESCs were identified using chromatin immunoprecipitation sequencing. The epigenetic features of ETS1-binding sites were assessed by the published histone modification and chromatin accessibility profiles. ETS1 was robustly expressed in LESCs but dramatically reduced on differentiation into corneal epithelial cells (CECs). ETS1 knockdown in LESCs inhibited cellular proliferation and activated CEC markers (KRT3, KRT12, CLU, and ALDH3A1). When ETS1 was overexpressed during CEC differentiation, LESC-associated genes were upregulated while CEC-associated genes were downregulated. The genome-wide binding profile of ETS1 was identified in LESCs. ETS1 occupied H3K4me3-marked promoters and H3K27ac/H3K4me1-marked enhancers. ETS1-binding sites were also enriched for chromatin accessibility signal. HMGA2 showed a consistent expression pattern with ETS1. ETS1 activates HMAG2 by binding to its promoter. Knockdown and overexpression experiments suggested that HMGA2 can promote LESC proliferation and inhibits its differentiation. ETS1 promotes LESC proliferation and inhibits its differentiation via activating HMGA2.

A novel technique of amniotic membrane suturing for ex vivo cultivated limbal epithelial stem cell expansion increases the number of progenitor cells

AbstractPurpose: Human limbal epithelial stem cells (HLESCs) reside in limbal epithelial crypts and continuously compensate for the loss of cells in the corneal epithelium. The lack or malfunction of HLESCs leads to a severe ocular surface disease called limbal epithelial stem cell deficiency (LSCD). Cultivated limbal epithelial stem cell transplantation (CLET) of expanded autologous limbal tissue on a human amniotic membrane (HAM) carrier is one of the most common methods for monocular LSCD. We hereby present a novel suturing technique of HAM prior to limbal biopsy expansion and cultivation that mimics natural limbal crypts.Methods: HLESCs derived from limbal biopsies were cultured ex vivo in complex media (COM) on flat or crypt‐like sutured HAMs for 3 weeks. The samples were then processed for immunohistochemistry (IHC) and analysed for progenitor markers: p63α and SOX9; markers for quiescence: CEBPD; proliferation: Ki‐67; differentiation: CX43 and CK3/12; transmembrane markers: E‐cadherin and N‐cadherin. The positivity of markers was counted and compared in cells cultivated on flat vs. crypt‐like sutured HAMs.Results: HLESCs cultured on folded, crypt‐like sutured HAMs had significantly more positivity for p63α, SOX9 (progenitor markers) and Ki‐67 (proliferation marker) compared to those cultured on flat HAMs. Fewer cells were positive for CK3/12 (differentiation marker) in cultures on folded compared to flat HAMs. The number of cells positive for CEBPD (quiescence marker), CX43 and E‐cadherin (differentiation markers) were equal in both groups (flat vs. folded HAMs).Conclusions: A novel technique of HAM suturing that forms crypt‐like undulations mimicking limbal epithelial crypts increases the number of early progenitor cells and could hence increase the quality of the ex vivo expanded limbal epithelial tissue and success for transplantation.

The progress in techniques for culturing human limbal epithelial stem cells.

In vitro culture of human limbal epithelial stem cells (hLESCs) is crucial to cell therapy in the treatment of limbal stem cell deficiency, a potentially vision-threatening disease that is characterized by persistent corneal epithelial defects and corneal epithelium conjunctivalization. Traditionally, hLESCs are cultivated based on either limbal tissue explants or single-cell suspensions in culture media containing xenogenous components, such as fetal bovine serum and murine 3T3 feeder cells. Plastic culture dishes and human amniotic membranes are classical growth substrates used in conventional hLESC culture systems. The past few decades have witnessed considerable progress and innovations in hLESC culture techniques to ensure a higher level of biosafety and lower immunogenicity for further cell treatment, including complete removal of xenogenous components from culture media, the application of human-derived feeder cells, and the development of novel scaffolds. Three-dimensional artificial niches and three-dimensional culture techniques have also been established to simulate the real microenvironment of limbal crypts for better cell outgrowth and proliferation. All these progresses ensure that in vitro cultured hLESCs are more adaptable to translational stem cell therapy for limbal stem cell deficiency.

Influence of the Postmortem/Storage Time of Human Corneas on the Properties of Cultured Limbal Epithelial Cells.

Besides being a powerful model to study the mechanisms of corneal wound healing, tissue-engineered human corneas (hTECs) are sparking interest as suitable substitutes for grafting purposes. To ensure the histological and physiological integrity of hTECs, the primary cultures generated from human cornea (identified as human limbal epithelial cells (hLECs) that are used to produce them must be of the highest possible quality. The goal of the present study consisted in evaluating the impact of the postmortem/storage time (PM/ST) on their properties in culture. hLECs were isolated from the entire cornea comprising the limbus and central cornea. When grown as monolayers, short PM/ST hLECs displayed increased daily doublings and generated more colonies per seeded cells than long PM/ST hLECs. Moreover, hLECs with a short PM/ST exhibited a markedly faster wound closure kinetic both in scratch wound assays and hTECs. Collectively, these results suggest that short PM/ST hLECs have a greater number of highly proliferative stem cells, exhibit a faster and more efficient wound healing response in vitro, and produce hTECs of a higher quality, making them the best candidates to produce biomaterial substitutes for clinical studies.

Integrated Stress Response Regulation of Corneal Epithelial Cell Motility and Cytokine Production.

PurposeTo investigate the effect of an active integrated stress response (ISR) on human corneal epithelial cell motility and cytokine production.MethodsISR agonists tunicamycin (TUN) and SAL003 (SAL) were used to stimulate the ISR in immortalized corneal epithelial cell lines, primary human limbal epithelial stem cells, and ex vivo human corneas. Reporter lines for ISR-associated transcription factors activating transcription factor 4 (ATF4) and XBP1 activity were generated to visualize pathway activity in response to kinase-specific agonists. Scratch assays and multiplex magnetic bead arrays were used to investigate the effects of an active ISR on scratch wounds and cytokine production. A C/EBP homologous protein (CHOP) knockout cell line was generated to investigate the effects of ISR ablation. Finally, an ISR antagonist was assayed for its ability to rescue negative phenotypic changes associated with an active ISR.ResultsISR stimulation, mediated through CHOP, inhibited cell motility in both immortalized and primary human limbal epithelial cells. Scratch wounding of ex vivo corneas elicited an increase in the ISR mediators phosphorylated-eIF2α and ATF4. ISR stimulation also increased the production of vascular endothelial growth factor (VEGF) and proinflammatory cytokines. ISR ablation, through CHOP knockout or inhibition with integrated stress response inhibitor (ISRIB) rescued epithelia migration ability and reduced VEGF secretion.ConclusionsWe demonstrate that the ISR has dramatic effects on the ability of corneal epithelial cells to respond to wounding models and increases the production of proinflammatory and angiogenic factors. Inhibition of the ISR may provide a new therapeutic option for corneal diseases in which the ISR is implicated.

P-Cadherin Is Expressed by Epithelial Progenitor Cells and Melanocytes in the Human Corneal Limbus.

Interactions between limbal epithelial progenitor cells (LEPC) and surrounding niche cells, which include limbal mesenchymal stromal cells (LMSC) and melanocytes (LM), are essential for the maintenance of the limbal stem cell niche required for a transparent corneal surface. P-cadherin (P-cad) is a critical stem cell niche adhesion molecule at various epithelial stem cell niches; however, conflicting observations were reported on the presence of P-cad in the limbal region. To explore this issue, we assessed the location and phenotype of P-cad+ cells by confocal microscopy of human corneoscleral tissue. In subsequent fluorescence-activated cell sorting (FACS) experiments, we used antibodies against P-cad along with CD90 and CD117 for the enrichment of LEPC, LMSC and LM, respectively. The sorted cells were characterized by immunophenotyping and the repopulation of decellularized limbal scaffolds was evaluated. Our findings demonstrate that P-cad is expressed by epithelial progenitor cells as well as melanocytes in the human limbal epithelial stem cell niche. The modified flow sorting addressing P-cad as well as CD90 and CD117 yielded enriched LEPC (CD90−CD117−P-cad+) and pure populations of LMSC (CD90+CD117−P-cad−) and LM (CD90−CD117+P-cad+). The enriched LEPC showed the expression of epithelial progenitor markers and better colony-forming ability than their P-cad− counterparts. The cultured LEPC and LM exhibited P-cad expression at intercellular junctions and successfully repopulated decellularized limbal scaffolds. These data suggest that P-cad is a critical cell–cell adhesion molecule, connecting LEPC and LM, which may play an important role in the long-term maintenance of LEPC at the limbal stem cell niche; moreover, these findings led to further improvement of cell enrichment protocols to enhance the yield of LEPC.

Human Oral Mucosal Fibroblasts from Limbal Stem Cell Deficient Patients as an Autologous Feeder Layer for Epithelial Cell Culture

Purpose To investigate if human oral mucosal fibroblasts (HOMF) from patients with limbal stem cell deficiency (LSCD) can be used as an autologous feeder layer to support the culture of epithelial cells for potential clinical use. Methods HOMF were isolated from oral mucosal biopsies obtained from the following groups of patients with LSCD: aniridia, mucous membrane pemphigoid (MMP), Stevens–Johnson syndrome (SJS), and ectodermal dysplasia (ED). The ability of these cells to support the culture of human limbal epithelial cells (HLE) was compared to that of HOMF from non-LSCD donors and 3T3s commonly used to culture epithelial cells for use in the clinic to treat LSCD. Results HOMF were successfully obtained by explant culture for 3/3 aniridia patients, 3/3 MMP patients, 1/3 SJS patients, and 1/1 ED patients. All HOMF cultured from these LSCD groups supported the expansion of HLE with epithelial culture times and total colony forming efficiency (CFE) comparable to those achieved on HOMF isolated from donors without LSCD. PCR showed that all HLE cultured on LSCD donor HOMF expressed p63α, CK15, PAX6, CK12, and MUC16 as did HLE cultured on the control non-LSCD donor HOMF and 3T3s. Western blotting detected CK15 and MUC16 protein expression in all groups. Conclusions HOMF from patients with LSCD can be successfully used to support the expansion of epithelial cells. These cells may therefore be useful as autologous feeder fibroblasts for the expansion of epithelial cells for use in the clinic to treat LSCD.

Glycogene Expression Profile of Human Limbal Epithelial Cells with Distinct Clonogenic Potential.

Glycans function as valuable markers of stem cells but also regulate the ability of these cells to self-renew and differentiate. Approximately 2% of the human genome encodes for proteins that are involved in the biosynthesis and recognition of glycans. In the present study, we evaluated the expression of a small subset of glycogenes in human limbal epithelial cells with distinct clonogenic potential. Individual clones were classified as abortive or clonogenic, based on the fraction of the terminal colonies produced; clones leading exclusively to terminal colonies were referred to as abortive while those with half or fewer terminal colonies were referred to as clonogenic. An analysis of glycogene expression in clonogenic cultures revealed a high content of transcripts regulating the galactose and mannose metabolic pathways. Abortive clones were characterized by increased levels of GCNT4 and FUCA2, genes that are responsible for the branching of mucin-type O-glycans and the hydrolysis of fucose residues on N-glycans, respectively. The expansion of primary cultures of human limbal epithelial cells for 10 days resulted in stratification and a concomitant increase in MUC16, GCNT4 and FUCA2 expression. These data indicate that the clonogenic potential of human limbal epithelial cells is associated with specific glycosylation pathways. Mucin-type O-glycan branching and increased fucose metabolism are linked to limbal epithelial cell differentiation.

Wnt/β-Catenin Signaling Activation Induces Differentiation in Human Limbal Epithelial Stem Cells Cultured Ex Vivo

Wnt/β-Catenin Signaling Activation Induces Differentiation in Human Limbal Epithelial Stem Cells Cultured Ex Vivo