Interplay Between Retinal Pigment Epithelium and Other Ocular Cell Types During Differentiation From Human Embryonic Stem Cells

Abstract

Purpose: : The retinal pigment epithelium (RPE) is required for eye development (Raymond & Jackson, 1995, Curr. Biol. 5:1286). Here we examine interrelationships between genesis of human embryonic stem cell derived RPE (HESC-RPE) and the expression of markers for other ocular cell types.

Methods: : HESC RPE were produced according to Vugler et al., (2008) Exp. Neurol. 214:341. After 5 weeks, flasks containing varying numbers of pigmented foci were generated. Some pigmented / non-pigmented foci were also grown as embryoid bodies (EB) supplemented with retinoic acid and taurine. Real-time quantitative PCR (QPCR) was performed on EB and on flasks of HESC both prior to and post-pigmentation. Intravitreal injections of dissociated EB were made into P3 rats which were killed after 2 weeks. Protein expression was assesed by immunocytochemistry (ICC).

Results: : QPCR revealed a positive correlation between pigmented foci and tyrosinase expression and a negative correlation with Chx10. Other eye field transcription factors and rod/cone opsins showed little correlation with pigmentation. Expression of the lens specific alpha A-crystallin (Cryaa) increased post-RPE production. Melanopsin transcription was initiated following EB formation and was associated with increased beta III tubulin. In pigmented versus non-pigmented EB samples there was increased expression of CRX, Cryaa, and IRBP. By ICC, Crx and Otx2 were found to co-localise in both pigmented HESC-RPE and non-pigmented (Pmel17 positive) RPE precursors. We also identified non-pigmented cells which were Crx positive / Otx2 negative. Some RPE cells stained positively for Cryaa but ICC failed to reveal opsin positive cells, although false-positive staining was obtained using some opsin antibodies. Following transplantation into developing eyes, pigmented and non-pigmented cells survived in the vitreous with some integration into neural retina. No transplanted cells appeared positive for Cryaa, even when directly opposed to the host lens.

Keywords: opsins • retinal pigment epithelium • crystallins