Transplantation Of Es Cell-derived Retinal Cells Into The Adult Retina

Purpose: : Embryonic stem (ES) cells provide a promising source for retinal cell transplantation, a novel strategy to repair the degenerate retina. We previously demonstrated that post-mitotic photoreceptor precursors can functionally integrate into the adult retina. In this study we first sought to develop an improved protocol to increase the number of differentiated retinal progenitors and enable the feasibility of ES cell-derived retinal cell transplantation to be tested. Secondly, we sought to determine if a pure population of retinal cells would enable the integration of ES cell-derived photoreceptor precursors into the adult retina. Methods: : Using a mouse EB5 ES cell line containing a GFP reporter driven by the retinal transcription factor Rx and a serum-free floating culture method (Osakada, et al, 2008), we assessed the efficiency of retinal progenitor (Rx.GFP+) differentiation. The efficacy of photoreceptor differentiation was also assessed, with or without the selection of Rx.GFP+ cells, by immunocytochemistry. Photoreceptor development was examined by quantitative PCR in comparison with the early postnatal retina. AAV2/9.CMV.GFP labelled cells were FAC-sorted and subretinally transplanted into adult mice. Photoreceptor cell integration and retinal cell survival were evaluated 2 weeks post transplantation. Results: : The differentiation of Rx.GFP+ progenitors was significantly improved with greater seeding cell densities (3,000-27,000 cells/well). Further differentiation of these cells generated significantly more Crx+ and Rhodopsin+ photoreceptors compared with mixed populations of unselected cells. Transcriptional analysis demonstrated an increase in crx and nrl expression, photoreceptor precursor markers, until day 28. We therefore dissociated cells at this stage and 200,000 GFP+ cells were subretinally transplantated. Although GFP+ cells were present in the subretinal space, no integrated ES cell-derived photoreceptors were observed in the host retinas. Conclusions: : This method for retinal cell differentiation improves the number of retinal progenitors and produces both photoreceptor precursors and mature photoreceptors from mouse ES cells. However, the number of photoreceptor precursors present in the differentiated cell population (0.2% Nrl+ cells) is too low for successful transplantation. Therefore, methods to purify photoreceptor precursors may be required to confirm their ability to integrate into the adult retina