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Y. Kumagai, M. S.Kurokawa, H. Ueno, M. Kayama, K. Tsubota, N. Nakatsuji, Y. Kondo, S. Ueno, N. Suzuki; Induction of Corneal Epithelium-Like Cells From Non Human Primate Embryonic Stem Cells and Their Experimental Transplantation to Damaged Cornea. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5733.
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We previously reported the successful transplantation of corneal epithelium-like cells derived from mouse embryonic stem (ES) cells onto damaged mouse cornea. Here we tested whether cynomolgus monkey (CM) ES cells have ability to differentiate into corneal epithelial cells and whether the CMES derived corneal epithelium-like cells were applicable for the experimental transplantation to damaged cornea of mice.
CMES cells were cultivated on typeIV collagen-coated dishes for various days to induce differentiation into corneal epithelium-like cells. The differentiation was evaluated by RT-PCR and immunohistocmistry. Mouse corneas were scraped and corneal epithelial cells were peeled off to make the damaged cornea. The corneal epithelium-like cells were transplanted to the injured cornea, and the recipient mice were sacrificed at the various time points. Reconstitution of the corneal epithelium was evaluated by immunostaining.
The cells cultured on typeIV collagen showed cobble stone like appearance. They expressed mRNA of pax6, p63, e-cadherin, CD44, PCNA, keratin3 and keratin12 in vitro. Confocal analysis confirmed the protein expressions of Pax6, p63, E-cadherin, CD44, and Keratin3/12, suggesting differentiation to corneal epithelium-like cells of CMES cells and a part of the cells shared protein expression pattern of limbal stem cells. When the corneal epithelium-like cells of CMES origin were transplanted onto injured cornea, they adhered to the corneal stroma, leading to the formation of monolayer to bilayer of corneal epithelium-like cells. The overlaid cells were stained with anti-human nuclei antibody which crossreacted with nuclei of CM cells but not those of mouse cells. They retained the expressions of E-cadherin and Keratin3/12.
We have succeeded in the induction of corneal epithelium-like cells from CMES cells, some of which expressed limbal stem cell markers. The cells were successfully transplanted onto the injured mouse cornea. This is the first demonstration that corneal epithelium-like cells were differentiated from nonhuman primate ES cells which were applicable for transplantation to an animal model of corneal injury.
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