High Yield of Cells Committed to the Photoreceptor Fate from Expanded Mouse Retinal Stem Cells

¹ Unit of Gene Therapy and Stem Cell Biology, Jules Gonin Eye Hospital, Lausanne, Switzerland

^* To whom correspondence should be addressed. E-mail: yvan.arsenijevic{at}ophtal.vd.ch.

	Abstract

The purpose of the present work was to generate, from retinal stem cells (RSCs), a large number of cells committed towards the photoreceptor fate in order to provide an unlimited cell source for neurogenesis and transplantation studies. We expanded RSCs (at least 34 passages) sharing characteristics of radial glial cells, and primed the cells in vitro with FGF-2 for 5 days, after which cells were treated with the B27 supplement to induce cell differentiation and maturation. Upon differentiation, cells expressed cell type-specific markers corresponding to neurons and glia. We show by immunocytochemistry analysis that a subpopulation of differentiated cells was committed to the photoreceptor lineage since these cells expressed the photoreceptor proteins recoverin, peripherin, and rhodopsin in a same ratio. Furthermore, cells infected during the differentiation procedure with a lentiviral vector expressing GFP under the control of either the rhodopsin promoter or the interphotoreceptor retinoid-binding protein (IRBP) promoter, expressed GFP. FGF-2 priming increased neuronal differentiation while decreasing glia generation. RT-PCR analyses revealed that the differentiated cells expressed photoreceptor-specific genes such as Crx, rhodopsin, peripherin, IRBP, and phosphodiesterase-. Quantification of the differentiated cells showed a robust differentiation into the photoreceptor lineage: around 25-35% of the total cells harboured photoreceptor markers. The generation of a significant number of non-differentiated RSCs as well as differentiated photoreceptors will enable to determine via transplantation studies which cells are the most adequate to integrate a degenerating retina.

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