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H. Ueno, M. S. Kurokawa, Y. Kumagai, M. Kayama, R. Homma, C. Masuda, E. Takada, K. Tubota, S. Ueno, N. Suzuki; Characterization of Corneal Epithelium Like Cells Induced by Pax6 Gene Transfection of Mouse Embryonic Stem Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):480.
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We previously reported that highly purified corneal epithelium like cells were induced by pax6 gene transfection of mouse ES cells, and that they adhered onto damaged cornea when transplanted. Herein, we characterized the pax6 transfected cells, focusing whether the cells shared the characteristics with the corneal limbal stem cells.
The eye development associated transcription factor, pax6 gene with a C-terminal tag encoding green fluorescence protein (GFP) was electrotransfected to ES cells and the transfected cells were cultivated with G418 for 14 days. These cells were transplanted onto a mouse cornea damaged by n-heptanol treatment. Twenty-four hours after the transplantation, reconstitution of the corneal epithelial layers was assessed and their expressions of keratin 12, E-cadherin, CD44, p63 and PCNA were evaluated.
The pax6 transfected cells formed a mono layer with cobble stone like appearance and expressed keratin12. They expressed putative corneal limbal stem cell marker, p63, and PCNA. When the pax6 transfected cells were transplanted onto the damaged cornea, they formed bi-layers of epithelial cells expressing GFP 12 hours after the transplantation. Transplantation of the pax6 transfected cells reduced the extent of stromal edema and inflammatory cell infiltration in the corneal stroma. The pax6 transfected cells expressed keratin12, E-cadherin and CD44 on the grafted site. Positive staining of p63 and PCNA was noted in the basal layer of the limbal and corneal epithelium in normal mice. We recognized the expressions of p63 and PCNA of the transplanted cells on the grafted site.
The corneal epithelium like cells derived from ES cells by pax6 transfection have kept alive on the injured cornea and had proliferative ability. The cells shared the characteristics with the corneal limbal stem cells. The ES derived corneal epithelium like cells may become applicable for clinical transplantation.
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