Molecular Characterization and Transplantation of Human Retinal Progenitor Cells

Abstract

Purpose: : Work in pigs and rodents has shown that retinal progenitor cells exhibit markers representative of mature photoreceptors following transplantation to the degenerating retina. The purpose of the present study was to characterize early and late passage human retinal progenitor cells (hRPCs) based on the protein expression, co-culture GFP-positive hRPCs in-vitro and transplant in-vivo to assess integration and differentiation.

Methods: : hRPCS were isolated from 14-18 week embryonic retinas, and expanded in serum-free media supplemented with EGF and b-FGF. Immunoblot and immunocytochemistry assays were performed on undifferentiated and differentiated hRPCs of early and late passage. In-vitro, GFP positive hRPCs were co-cultured with retinal explants obtained from neonatal and adult rho -/- mice. In-vivo studies were carried out by transplanting GFP positive cells to the sub-retinal space of neonatal Scid rd1 and rho -/- mice. Mice were sacrificed at 7-21 days and retinas assessed histologically for integration and differentiation of grafted cells.

Results: : Immunocytochemistry results showed a temporal decrease in the percentage of Ki-67 positive cells from early to late passage. Immunoblot analysis showed an overall decrease in Sox2, nestin, recoverin, and CRX expression from early to late passage. Upon both in-vitro and in-vivo transplantation, GFP positive hRPCs were seen to integrate with the host tissue and extend their processes through the retinal layers. A sub-population of integrated hRPCs with photoreceptor-like morphology expressed rhodopsin.

Conclusions: : The molecular characterization of hRPCs indicates a decrease in the expression of immature markers from early to late passage. Our transplantation studies show that these cells have the potential to integrate into host retinal tissue and undergo differentiation into rod-like cells.

Keywords: transplantation • immunohistochemistry • differentiation