In vitro Corneal Epithelial Cell Induction With High Purity by Pax6 Gene Transfection of Mouse Embryonic Stem Cells

Abstract

Purpose: : Corneal transplantation is widely performed for treating patients with ocular trauma and those with ocular diseases, such as Stevens–Johnson syndrome and ocular cicatrical pemphigoid. We have reported corneal epithelial cell differentiation from mouse embryonic stem (ES) cells that had been cultured on type four collagen. Here, we introduced pax6 gene that is a homeobox transcription factor acknowledged to have a critical and evolutionarily conserved role in eye development to mouse ES cells for yielding highly purified corneal epithelial cells.

Methods: : Undifferentiated mouse ES cells were transfected with pax6 cDNA by using electroporation method, followed by selection procedure with G418. Some of the cells were used for limiting dilution culture. The cells were characterized by using RT–PCR and immunostaining.

Results: : After limiting dilution procedure, epithelium–like–cells appeared in the culture wells. RT–PCR revealed that these cells expressed a corneal epithelium specific marker, cytokeratin12. These cells, but not undifferentiated ES cells, expressed mRNAs of cell adhesion molecules including E–cadherin, integrinα4, integrinß7, ß–catenin, and CD44. The cells expressed cytokeratin12 protein and E–cadherin protein. They were free from contamination of other germ layers and outgrowth of tumor cells was not recognized in culture.

Conclusions: : Pax6 gene transfection into mouse ES cells leads to differentiation of corneal epithelial cells that were cytokeratin12 positive. They expressed not only a specific marker of cornea but also some adhesion molecules. The adhesion molecules were induced only on pax6 transfected cells and they may contribute to cell–to–cell adhesion after corneal transplantation.

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