PAX6 Isoforms, along with Reprogramming Factors, Differentially Regulate the Induction of Cornea-specific Genes

Abstract

Purpose : We developed the transplantation of cultured autologous oral mucosal epithelial sheets for eyes with bilateral limbal stem cell deficiencies. This approach has shown some promise; however, the reconstructed ocular surface does not allow a full, long-term improvement of visual acuity, and is prone to problems associated with neovascularization. Presumably, this is due to the incomplete corneal phenotype of the reconstructed epithelium, owing at least to the fact that the oral mucosal epithelium lacks corneal epithelial specific genes, such as PAX6 and KRT12. To reduce such limitations in our ability to reconstruct and maintain the ocular surface, we tested the function of two PAX6 isoforms in the human oral mucosal epithelial cells.

Methods : Two PAX6 isoforms (PAX6a and PAX6b), along with Yamanaka four factors (OCT4, SOX2, KLF4, c-MYC), were transduced into human oral mucosal epithelial cells by lentiviruses. Three days after transduction, we measured the expression of corneal epithelium specific genes, keratin 3 (KRT3) and keratin 12 (KRT12). Reporter assay and single-cell gene expression analysis were also performed.

Results : The highest induction of KRT12 was obtained by the combinational transduction of PAX6b, OCT4 and KLF4. KRT3 was induced by PAX6a solely and it was enhanced when combined with KLF4 transduction. Reporter assay showed that PAX6a targeted the upstream region of KRT3 gene. Single-cell gene expression analysis revealed that KRT12 induction required high expression of transgenes. KRT3 and KRT12 were preferentially induced in surface ectoderm-derived cells when compared to the other cell source.

Conclusions : Two PAX6 isoforms differentially and cooperatively regulate the expression of genes specific to the structure and function of the corneal epithelium, particularly KRT3 and KRT12.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.