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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; Development of Corneal Epithelial Cell Sheets Derived From Mouse Embryonic Stem (ES) Cells Transfected With Pax6 Gene. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5721.
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© ARVO (1962-2015); The Authors (2016-present)
We previously reported successful transplantation of corneal epithelium like cells derived from mouse ES cells transfected with pax6 gene. Herein, we developed a cell sheet with the corneal epithelium like cells derived from pax6 transfected ES cells for transplantation onto damaged cornea. Furthermore, we examined whether the pax6 transfected cell sheet contained the corneal limbal stem cells.
Gene encoding the eye development associated transcription factor, pax6, and green fluorescence protein (GFP) were electrotransfected to mouse ES cells and the transfected cells were cultivated with G418 for 14 days. To fabricate the stratified epithelial sheet, pax6 transfected cells were cultured on the upper chambers of a six-well plate with culture inserts, under which mitomycin C (MMC)-treated 3T3 fibroblasts were cultured. We evaluated corneal limbal stem cell markers of the cells by RT-PCR and immunostaining.
The pax6 transfected cells formed a mono layer of epithelium-like cells and expressed keratin12 in vitro. They expressed a putative corneal limbal stem cell marker, the G2 subtype member of the ATP binding cassette transporter (ABCG2) and p63. The pax6 transfected cells expressed E-cadherin and CD44 as cell adhesion molecules. Expression of Proliferating Cell Nuclear Antigen (PCNA) of the cells was also detected. Moreover, pax6 transfected cells constituted the multilayered epithelial sheet when they were cocultured on the MMC-treated 3T3 fibroblasts for 7 days.
The corneal epithelium like cells shared the characteristics with the corneal limbal stem cells and they made a cell sheet possibly applicable for corneal transplantation. We considered that the stratified corneal epithelium like cell sheet by using pax6 transfected cells was a useful for future transplantation.
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