Experimental Transplantation of Corneal Epithelium Induced by PAX6 Gene Trnsfection to Mouse Embryonic Stem (ES) Cell

Abstract

Abstract: : Purpose: Embryonic stem (ES) cells can differentiate into any cell type and may be suitable for use in allogeneic corneal transplantation because cornea is an immune privileged organ. To determine whether transfection with the eye development–associated transcription factor, pax6, gene of ES cells leads to their preferential differentiation to corneal epithelium, we transfected pax6 gene into mouse ES cells, and the ES–derived epithelium was transplanted onto damaged mouse cornea. Methods: Pax6 cDNA was ligated to a stable expression vector having green fluorescence protein (GFP) gene and was electrotransfected to ES cells. G418 resistant cells were recovered for subsequent analysis and transplantation. Corneal injury was developed by n–heptanol treatment of corneal epithelium of mice. The corneal epithelial cells derived from pax6 transfected ES cells were transplanted onto a damaged mouse cornea. Reconstitution of corneal epithelium was evaluated by confocal laser microscopy. Results: The ES cells transfected with pax6 have differentiated exclusively, if not all, into epithelial cells and formed a sheet of the epithelial cells. These cells expressed mRNA for cytokeratins and Pax6. Thus the cells were applicable for corneal transplantation. When the pax6–transfected cells were transplanted onto corneal lesion, they overlaid the corneal surface for at least 12 hours and formed a mono–layer and bi–layers of epithelial cells. GFP positive ES–originated cells were detected on the recipient cornea by confocal laser microscope, suggesting that the transfected ES–derived epithelial cells have kept alive on the cornea. Contamination of other germ cells was not detected. Conclusions:We have successfully transfected pax6 into mouse ES cells, that led to the differentiation of ES cells into corneal epithelial cells exclusively. Pax6 gene transfection and subsequent selection made purified corneal epithelial cells available for the transplantation, that were free from contamination of other germ cells. Thus, the ES–derived epithelial cells were applicable for transplantation of corneal injury in mice. ES cells may become an unlimited donor source of corneal epithelial cells for clinical transplantation.

Keywords: cornea: epithelium • transplantation • regeneration