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TISSUE-SPECIFIC STEM CELLS |
Unit of Gene Therapy and Stem Cell Biology, Jules Gonin Eye Hospital, Lausanne, Switzerland
Key Words. Radial glia • Neurogenesis • Neuron differentiation • Lentivirus • Müller cells
Correspondence: Yvan Arsenijevic, Ph.D., Unit of Gene Therapy and Stem Cell Biology, Jules Gonin Eye Hospital, 15 av. de France, 1004 Lausanne, Switzerland. Telephone: +41-21-626.82.60; Fax: +41-21-626.88.88; e-mail: yvan.arsenijevic{at}ophtal.vd.ch
Received on July 12, 2005;
accepted for publication on April 17, 2006.
First published online in STEM CELLS EXPRESS April 27, 2006.
The purpose of the present work was to generate, from retinal stem cells (RSCs), a large number of cells committed toward 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 fibroblast growth factor (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 given that 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 green fluorescent protein (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. Reverse transcription-polymerase chain reaction 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: Approximately 25%–35% of the total cells harbored photoreceptor markers. The generation of a significant number of nondifferentiated RSCs as well as differentiated photoreceptors will enable researchers to determine via transplantation studies which cells are the most adequate to integrate a degenerating retina.
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