Experimental Transplantation of Corenal Epithelial Cells Derived From Mouse Embryonic Stem (ES) Cells Transfected With Pax6 Gene

Abstract

Purpose: : ES cells differentiate into three germinal layers. We previously reported transplantation of corneal epithelial cells derived from mouse ES cells that had been cultured on type IV collagen onto damaged mouse cornea. Herein, we induced higher purified epithelial cells from ES cells by pax6 gene transfection and transplanted them onto damaged cornea.

Methods: : The eye development associated transcription factor, pax6 gene+green fluorescence protein (GFP) was electrotransfected to ES cells and the transfected cells were cultivated with G418 for ten days. These cells were transplanted onto a mouse cornea damaged by n–heptanol treatment. One hour and 24 hours after the transplantation, reconstitution of the cornea epithelial layers was assessed and their expressions of keratin and adhesion molecules were evaluated.

Results: : The pax6 transfected cells formed a mono layer of epithelium–like cells and expressed keratin12 by RT–PCR and immunostaining, suggesting their differentiation into corneal epithelial cells in vitro. When the pax6 transfected cells were transplanted onto the damaged cornea, they formed bi–layers of epithelial cells that were GFP positive 24 hours after transplantation. The injury induced detachement of corneal epithelial layers caused swelling of corneal stroma, whereas transplantation of the pax6 transfected cells reduced the swelling and the inflammatory cell infiltration of the corneal stroma was subsided. The pax6 transfected cells expressed keratin12 and E–cadherin on the grafted site. Contamination of other germ cells was not detected and development of teratoma was not recognized.

Conclusions: : The highly purified corneal epithelial cells derived from the pax6 transfected ES cells kept alive on the injured cornea. The cells restored the damaged cornea histologically and functioned as native corneal epithelial cells because they suppressed corneal epithelial damage induced inflammation. The ES derived epithelial cells may become applicable for clinical transplantation.

Keywords: cornea: epithelium • transplantation • cell adhesions/cell junctions