Retinal degeneration (RD) results from photoreceptor apoptosis. Cell transplantation, one potential therapeutic approach, requires expandable stem cells that can form mature photoreceptors when differentiated. Freshly dissociated primary retinal cells from postnatal day 2-6 (PN2-6) mouse retina can give rise, post-transplantation, to photoreceptors in adult recipients. Unfortunately, incorporation rates are low; moreover, photoreceptor potential is lost if the same PN2-6 cells are cultured prior to transplantation. We investigated the identity of the cells forming photoreceptors post-transplantation, using FACS sorted primary postnatal day (PN) 3-5 Rho-eGFP retinal cells. Higher integration rates were achieved for cells that were expressing Rho-eGFP at PN3-5, indicating that post-mitotic photoreceptor precursors already expressing rhodopsin form the majority of integrating rods. We then investigated improvement of cell culture protocols for retinal progenitor cells (RPCs) derived from PN3-5 retinal cells in vitro. We succeeded in improving RPC survival and growth rates 25-fold, by modifying retinal dissociation, replacing N2 supplement with B27 supplement minus retinoic acid (B27-RA) and coating flasks with fibronectin. However, levels of rhodopsin and similar photoreceptor-specific markers still diminished rapidly during growth in vitro, and did not re-appear after in vitro differentiation. Similarly, transplanted RPCs, whether proliferating or differentiated, did not form photoreceptors in vivo. Cultured RPCs upregulate genes such as Sox2 and nestin, markers of more primitive neural stem cells. Use of these cells for RD treatment will require identification of triggers that favour terminal photoreceptor differentiation and survival in vitro prior to transplantation.

Vision impairment caused by degeneration of photoreceptors, termed retinitis pigmentosa, is a debilitating condition with no cure presently available. Cell-based therapeutic approaches represent one treatment option by replacing degenerating or lost photoreceptors. In this study the potential of transplanted primary retinal cells isolated from neonatal mice to integrate into the outer nuclear layer (ONL) of adult mice and to differentiate into mature photoreceptors was evaluated. Retinal cells were isolated from retinas of transgenic mice ubiquitously expressing enhanced green fluorescence protein (EGFP) at either postnatal day (P) 0, P1 or P4 and transplanted into the subretinal space of adult wild-type mice. One week to 11 months post-transplantation experimental retinas were analyzed for integration and differentiation of donor cells. Subsequent to transplantation some postnatal retinal cells integrated into the ONL of the host and differentiated into mature photoreceptors containing inner and outer segments as confirmed by immunohistochemistry and electron microscopy. Notably, the appearance of EGFP-positive photoreceptors was not the result of fusion between donor cells and endogenous photoreceptors. Retinal cells isolated at P4 showed a significant increase in their capacity to integrate into the ONL and to differentiate into mature photoreceptors when compared with cells isolated at P0 or P1. As cell suspensions isolated at P4 are enriched in cells committed towards a rod photoreceptor cell fate it is tempting to speculate that immature photoreceptors may have the highest integration and differentiation potential and thus may present a promising cell type to develop cell replacement strategies for diseases involving rod photoreceptor loss.