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T. Inoue, B.L. K. Coles, Y. Inoue, R. Bremner, R. Kageyama, C.M. Craft, Y. Tano, R.R. McInnes, D. van der Kooy; Increased OTX2/CRX and Decreased CHX10 Induce Optimal Photoreceptor Differentiation From Adult Human Retinal Stem Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3249.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Adult mammalian retinal stem cells (RSCs) can give rise to all of the retinal cell types, but only a minority differentiates into photoreceptors in vitro. Our focus is to drive RSC progeny down the photoreceptor lineage. Methods: To enhance the number of photoreceptors that differentiate from human RSCs, we transferred critical genes such as Crx, Nrl, NeuroD, Otx2 or Chx10–dominant negative constructs (Chx10VP16) into RSCs in vitro. It has been shown that Crx, Nrl, NeuroD or Otx2 are important for photoreceptor development, while Chx10 maintains retinal cells in an immature state. GFP positive human RSC clonal sphere colonies were differentiated in vitro. Furthermore, we performed human RSC transplantation into mouse retinal explants. Results: 40% of the Otx2–expressing virus–infected human RSC progeny differentiated as photoreceptor compared to 30% of Crx–, 14% of Nrl–, 18% of NeuroD–, 35% of CHX10–VP16–, and 15% of the control–virus infected cells. To assay for synergistic effects of these genes, a double expression vector of Otx2/Crx and a triple expression vector of Otx2/Crx/Chx10VP16 were constructed, and then 75% of the Otx2/Crx/Chx10VP16–overexpressing hRSC progeny differentiated as photoreceptors compared to 55% of Otx2/Crx–virus infected RSC progeny. Transplanted human RSC progeny integrated into the explants, and 88% of Otx2/Crx/Chx10VP16 overexpressing human RSC progeny adopted a photoreceptor cell fate compared to 24% of GFP only transfected cells. Conclusions: We found that the best gene combination for enhancement of photoreceptor development among human RSC progeny, was the overexpression of the genes Otx2 and Crx and the suppression of Chx10. These genes may be useful for treatment of photoreceptor disease via human RSC therapies.
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