論文

© Copyright 2018, Mary Ann Liebert, Inc. Corneal blindness is the third leading cause of blindness in the world, and one of the main etiologies is dysfunction of the corneal endothelium. Current treatment of corneal endothelial disease is allogenic corneal transplantation, which is limited by the global shortage of donor corneas and immunological rejection. The corneal endothelium consists of a monolayer of cells derived from the neural crest and mesoderm. Its main function is to prevent corneal edema by tight junctions formed by zonular occludens-1 (ZO-1) and Na, K-ATPase pump function. The human umbilical cord (UC) is a rich source of mesenchymal stem cells (MSCs). UC-MSCs that have multi-lineage potential may be an accessible allogenic source. After inducing differentiation with medium containing glycogen synthase kinase (GSK) 3-β inhibitor, UC-MSCs formed polygonal corneal endothelial-like cells that functioned as tissue-engineered corneal endothelium (UTECE). Expressions of major corneal endothelial markers were confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and quantitative RT-PCR (qRT-PCR). Western blotting confirmed the expression of Na,K-ATPase and PITX2, the functional and developmental markers of corneal endothelial cells. Immunohistochemistry revealed the localization of Na,K-ATPase and ZO-1 in cell-cell junctions, suggesting the presence of tight junctions. In vitro functional analysis revealed that UTECE had significantly high pump function compared with UC-MSCs. Moreover, UTECE transplanted into a rabbit model of bullous keratopathy successfully maintained corneal thickness and transparency. Our findings suggest that UTECE may be used as a source of allogenic cells for the treatment of corneal endothelial disease.

Cultures of epithelial cells are limited by the proliferative capacity of primary cells and cell senescence. Herein we show that primary human epithelial cell sheets cultured without dermal equivalents maintained homeostasis in vitro for at least 1 year. Transparency of these sheets enabled live observation of pigmented melanocytes and Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) labeled epithelial cells during wound healing. Cell turn over and KRT15 expression pattern stabilized within 3 months, when KRT15 bright clusters often associated with niche-like melanocytes became apparent. EdU labels were retained in a subset of epithelial cells and melanocytes after 6 months chasing, suggesting their slow cell cycling property. FUCCI-labeling demonstrated robust cell migration and proliferation following wounding. Transparency and long-term (1 year) homeostasis of this model will be a powerful tool for the study of wound healing and cell linage tracing.

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

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

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