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R. Homma, S. Ueno, N. Suzuki; Experimental Corneal Transplantation by Using Mouse Embryonic Stem (ES) Cell Derived Epithelial Cell Sheets . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3148.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Mouse embryonic stem (ES) cells are pluripotent and can differentiate into any cell type. Allogeneic ES cells can in theory be applicable as universal donor cells to any recipient because cornea is an immune privileged organ. The aim of this study was to establish 1) culture method for induction of epithelium cell differentiation of ES cells in vitro and 2) subsequent transplantation protocol of the epithelial cells to the damaged mouse cornea. Methods: ES cells were cultivated on collagen coated dishes for several days to induce epithelial differentiation. The differentiation was evaluated by using RT-PCR, western blotting, flow cytometry, and immunochemical staining. Corneal injury was developed by n-heptanol treatment of corneal epithelium. The epithelial cells derived from ES cells harboring Y chromosome were transplanted onto a female mouse cornea. Reconstitution of corneal epithelium was evaluated by RT-PCR and immunochemical staining for up to 12 hours after transplantation. Results: The ES cells have differentiated exclusively, if not all, into epithelial cells and formed a sheet of the epithelial cells. The cells expressed mRNA and proteins of cytokeratins and Pax6 transcription factor. They were negative for ICAM-1, and contained a small number of CD44+ cells. E-cadherin expression was upregulated upon epithelial cell differentiation. They became H2Kb+ concomitant with differentiation. When the epithelial sheet was transplanted onto corneal stroma, they overlaid the corneal lesion for at least 12 hours. Sry gene on the Y choromosome (ES cell origin) was detected on the recipient cornea (female) by PCR, suggesting that ES cell derived epithelial cells have kept alive on the cornea. Conclusions: We have successfully induced epithelial cell differentiation in vitro of mouse ES cells that can be applicable for corneal injury. ES cells may become an unlimited donor source of corneal epithelial cells for corneal transplantation.
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