Primary Human Limbal Epithelial Cells Research Articles

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.

A decellularized human corneal scaffold for anterior corneal surface reconstruction

Allogenic transplants of the cornea are prone to rejection, especially in repetitive transplantation and in scarred or highly vascularized recipient sites. Patients with these ailments would particularly benefit from the possibility to use non-immunogenic decellularized tissue scaffolds for transplantation, which may be repopulated by host cells in situ or in vitro. So, the aim of this study was to develop a fast and efficient decellularization method for creating a human corneal extracellular matrix scaffold suitable for repopulation with human cells from the corneal limbus. To decellularize human donor corneas, sodium deoxycholate, deoxyribonuclease I, and dextran were assessed to remove cells and nuclei and to control tissue swelling, respectively. We evaluated the decellularization effects on the ultrastructure, optical, mechanical, and biological properties of the human cornea. Scaffold recellularization was studied using primary human limbal epithelial cells, stromal cells, and melanocytes in vitro and a lamellar transplantation approach ex vivo. Our data strongly suggest that this approach allowed the effective removal of cellular and nuclear material in a very short period of time while preserving extracellular matrix proteins, glycosaminoglycans, tissue structure, and optical transmission properties. In vitro recellularization demonstrated good biocompatibility of the decellularized human cornea and ex vivo transplantation revealed complete epithelialization and stromal repopulation from the host tissue. Thus, the generated decellularized human corneal scaffold could be a promising biological material for anterior corneal reconstruction in the treatment of corneal defects.

Unique Expression Pattern and Functional Role of Periostin in Human Limbal Stem Cells

Periostin is a non-structural matricellular protein. Little is known about periostin in human limbal stem cells (LSCs). This study was to explore the unique expression pattern and functional role of periostin in maintaining the properties of human LSCs. Fresh donor corneal tissues were used to make cryosections for evaluation of periostin expression on ex vivo tissues. Primary human limbal epithelial cells (HLECs) were generated from limbal explant culture. In vitro culture models for proliferation and epithelial regeneration were performed to explore functional role of periostin in LSCs. The mRNA expression was determined by reverse transcription and quantitative real-time PCR (RT-qPCR), and the protein production and localization were detected by immunofluorescent staining and Western blot analysis. Periostin protein was found to be exclusively immunolocalized in the basal layer of human limbal epithelium. Periostin localization was well matched with nuclear factor p63, but not with corneal epithelial differentiation marker Keratin 3. Periostin transcripts was also highly expressed in limbal than corneal epithelium. In primary HLECs, periostin expression at mRNA and protein levels was significantly higher in 50% and 70% confluent cultures at exponential growth stage than in 100% confluent cultures at slow growth or quiescent condition. This expression pattern was similar to other stem/progenitor cell markers (p63, integrin β1 and TCF4). Periostin expression at transcripts, protein and immunoreactivity levels increased significantly during epithelial regeneration in wound healing process, especially in 16-24 hours at wound edge, which was accompanied by similar upregulation and activation of p63, integrin β1 and TCF4. Our findings demonstrated that periostin is exclusively produced by limbal basal epithelium and co-localized with p63, where limbal stem cells reside. Periostin promotes HLEC proliferation and regeneration with accompanied activation of stem/progenitor cell markers p63, integrin β1 and TCF4, suggesting its novel role in maintaining the phenotype and functional properties of LSC.

The Effect of Amniotic Membrane De-Epithelialization Method on its Biological Properties and Ability to Promote Limbal Epithelial Cell Culture

We characterized the de-epithelialized human amniotic membrane (HAM), and compared cell attachment and proliferation efficiencies. HAM was de-epithelialized by 20% ethanol (AHAM), 1.2 U/mL Dispase (DHAM), 0.02% EDTA (EHAM), 0.25% trypsin-EDTA (THAM), and 5 M urea (UHAM), respectively, followed by gentle scraping with a #15 blade. Surface topology, extracellular matrix (ECM), and growth factor content were characterized and compared to intact HAM by electron microscopies (EM), atomic force microscopy (AFM), immunohistochemistry, and Western blotting. Primary human limbal epithelial cells (LEC) attachment and proliferation efficiencies were assayed. Statistical significance was calculated by SPSS and Fisher's least significant difference test. EHAM, THAM, and UHAM had intact basal lamina and smooth basement membrane surface shown under transmission and scanning EM, and AFM. Cell remnants stayed on AHAM. Disrupted basement membrane and stroma was found in DHAM. Immunostaining intensity quantification and hierarchical clustering revealed that ECM composition of EHAM and UHAM resembled intact HAM. In contrast, DHAM and THAM had drastic loss of ECM and growth factor content. LEC attachment efficiency at 24 hours after seeding was the highest in EHAM (51% as on conventional culture surface), followed by UHAM and AHAM. However, cell proliferation indices at day 10 of culture were similar among different HAM substrates, suggesting repair of ECM and basement membrane by growing epithelial cells. Urea denudation preserved the basement membrane integrity, ECM, and growth factor composition, and had higher cell attachment and proliferation efficiencies. With its short processing time, urea treatment offers a novel alternative for HAM de-epithelialization.

An Immunoprotective Privilege of Corneal Epithelial Stem Cells Against Th17 Inflammatory Stress by Producing Glial Cell-Derived Neurotrophic Factor

Adult stem cells are well known for their self-renewal and regenerative capacity. The mechanisms protecting these cells from inflammatory damage have not been well elucidated. This study investigated the immunoprotective properties of corneal epithelial stem cells from inflammation by producing glial cell-derived neurotrophic factor (GDNF). Primary human limbal epithelial cells (HLECs) cultured from limbal explants were treated with interleukin (IL)-17A, tumor necrosis factor (TNF)-α, or hyperosmotic media, with or without GDNF or nuclear factor kappa B (NF-κB) inhibitor (NF-κB-I) for 4-48 hours. Inflammatory mediators and Th17-inducing cytokines were determined by real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and immunobead assays. NF-κB activation was detected by p65 phosphorylation, immunostaining and Western blotting. GDNF and its receptor, GDNF family receptor α-1, were exclusively immunolocalized in the basal layer of limbal epithelium, whereas IL-17 receptor was negative in these cells. Exogenous IL-17A stimulated the expression and production of inflammatory cytokines (TNF-α, IL-6, and IL-1β) and chemokine IL-8 by HLECs. Th17-inducing cytokines, transforming growth factor (TGF)-β1, IL-6, IL-23, and IL-1β, were significantly increased at mRNA and protein levels by HLECs exposed to TNF-α or hyperosmotic media. IL-17 activated NF-κB by p65 phosphorylation at serine 536 and nuclear translocation. GDNF or NF-κB-I blocked IL-17-induced NF-κB p65 activation and production of inflammatory mediators. Furthermore, GDNF suppressed the production of Th17-inducing cytokines through inhibiting NF-κB activation. These findings demonstrate that limbal progenitor cell-produced neurotrophic factor GDNF suppresses IL-17-mediated inflammation via NF-κB signaling pathway. This may represent a unique immunoprotective property of limbal stem cells against inflammatory challenges on the ocular surface.

N-Cadherin in the Maintenance of Human Corneal Limbal Epithelial Progenitor Cells In Vitro

To demonstrate the role of N-cadherin (N-cad) in maintaining the progenitor status of primary human limbal epithelial cells in vitro. 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. 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. N-cad plays a pivotal role in the maintenance of the progenitor phenotype in cultured limbal epithelial cells.

Culture Conditions for Primary Human Limbal Epithelial Cells

The cornea at the front of the eye is covered by an epithelium. This epithelium is maintained by stem cells located at the periphery of the cornea, in a region known as the limbus. Because this region harbors the stem cells for the corneal epithelium, the so-called limbal stem cells, its culture provides considerable interest. Limbal epithelial culture is used for two main reasons. The first is to further our understanding of limbal stem-cell biology. The second is for the culture expansion of limbal stem cells for transplantation purposes in patients with limbal stem-cell deficiency. However, considerable variations in the culture methods for limbal epithelium exist. These include culture media, sera used in the culture, use of 3T3 fibroblasts or amniotic membrane or both, the culture of whole pieces of limbal tissue or enzymatically digested tissue, and the use of airlifting.

Correlation of Cell Cycle Kinetics with p63 Expression in Human Limbal Epithelial Cells Expanded on Intact Human Amniotic Membrane

Aim: To analyse the correlation of p63 expression and cell cycle kinetics of human limbal epithelial cells (HLECs) expanded on amniotic membrane (AM) or plastic. Methods: Primary HLECs were cultured either on cryopreserved intact AM or plastic dishes for 2 weeks. Cells were labelled with 5 μM 5-bromo-2′-deoxyuridine (BrdU) for 3 days, followed by an interval of either 7 or 14 days in BrdU-free medium. The expression of p63 and BrdU labelling was detected by immunocytochemistry. Results: More cells on AM (56%) than on plastic (24%) retained their BrdU label after the 7-day interval (p < 0.001). The difference was even more pronounced after 14 days (20 and 2%, p < 0.001). All BrdU-labelled cells were also p63 positive. 2.5-fold more cells on AM (54%) than on plastic (21%) were BrdU positive/p63 positive after 7 days (p < 0.001). It increased to a 9-fold difference after 14 days (p < 0.001). The BrdU label was lost more quickly than the p63 expression during the observation period, indicating that p63 expression was not confined to stem cells but existed also in transient amplifying cells. Conclusions: The combination of p63 expression and BrdU label retention is a better criterion to characterize stemness than either marker alone. AM as a culture substrate preserves stemness better than plastic.

Hypoxia Enhances the Expansion of Human Limbal Epithelial Progenitor Cells In Vitro

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

Plasma polymer coated surfaces for serum-free culture of limbal epithelium for ocular surface disease

The potential use of plasma polymer coatings as substrates for serum-free expansion of limbal epithelial cells was investigated. Preliminary studies using a human corneal epithelial cell line showed that acrylic acid-coated surfaces performed better than allyl amine and allyl alcohol coated surfaces in terms of cell metabolic activity and confluence as assessed using the MTT assay. Subsequently, the proliferation and maturity of primary human limbal epithelial cells in co-culture with growth arrested 3T3 fibroblasts on a range of acrylic acid plasma coated surfaces, octadiene plasma coated surfaces and tissue culture plastic was investigated using MTT and cytokeratin 3 immunostaining. The cells performed better in the presence of serum on all surfaces. However, the acrylic acid coated surfaces successfully sustained a serum-free fibroblast/epithelial cell co-culture. The metabolic activity of the epithelial cells was superior on the acrylic acid coated surfaces than on tissue culture plastic in serum-free conditions and their levels of differentiation were not significantly higher than in the presence of serum. These results suggest that these surfaces can be used successfully for the serum-free expansion of human limbal epithelial cells.

Expression of Delta Np63 in response to phorbol ester in human limbal epithelial cells expanded on intact human amniotic membrane.

To evaluate the effect of phorbol 12-myristate 13-acetate (PMA) on the expression of Delta Np63 in human limbal epithelial cells (HLECs) during ex vivo expansion on amniotic membrane (AM). Primary HLECs were cultured either on AM or plastic surfaces and were treated with 1 micro g/mL PMA for 24 hours. Expression of Delta Np63 and the differentiation-associated gap junctional protein connexin 43 (Cx43) were studied by laser scanning microscopy. The labeling index (LI) of Delta Np63 was higher in HLECs cultured on AM than in HLECs grown on plastic (81.4% +/- 12.2% and 66.6% +/- 16.5%, respectively; P < 0.001). After PMA treatment, Delta Np63 expression in HLECs on plastic dramatically decreased to 20.4% +/- 11.4%. However, HLECs cultured on AM showed only a moderate decrease in Delta Np63 expression (56.4% +/- 10.9%, P < 0.001) after PMA treatment. It was also observed that 72.8% +/- 17.5% of the Delta Np63-positive cells in untreated HLECs cultured on plastic coexpressed Cx43, in contrast to only 21.9% +/- 3.7% of the Delta Np63-positive cells in HLECs cultured on AM (P < 0.001). The latter indicates that growth over AM preserves limbal phenotype, whereas growth over plastic surface induces or allows transition toward corneal peripheral phenotype. Delta Np63 protein is typically detected in human corneal epithelial cells with high proliferative capacity including, limbal epithelial stem cells (SCs) and probably also transient amplifying cells (TACs). AM supports Delta Np63 protein expression in HLECs and maintains a higher resistance against phorbol ester-induced differentiation, indicating that characteristic signs of limbal epithelial progenitor cells may be preserved during ex vivo expansion on AM.