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C. Jomary, H. Thomson, A. Lotery, S. Jones; Effect of Crx Expression on Differentiation of Human Adult Iris and Retinal Stem Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5780.
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© ARVO (1962-2015); The Authors (2016-present)
To assess the effect of exogenous expression of the transcription factor Crx on the differentiation of human adult iris and retinal stem cells into cells with a functional photoreceptor phenotype exhibiting light-sensitive properties.
Stem cells isolated from fresh trabeculectomy iris tissue and from post-mortem retinal ciliary body were maintained in serum-free culture, and genetically modified by electroporation to express exogenous epitope-tagged murine Crx. Changes in the expression of stem cell markers (homeodomain transcription factor Pax6, POU transcription factor Oct3/4, proliferating cell nuclear antigen), neuronal markers (nestin, neuron-specific class III β-tubulin and neurofilament), and photoreceptor-specific markers (rhodopsin, cyclic nucleotide-gated cation channel-3, blue-cone opsin, cyclic (c)GMP phosphodiesterase) expression were evaluated by immunocytochemistry and western blot analysis. Photo-transduction cascade activity was assessed by measuring light-induced hydrolysis of cGMP levels using a cGMP enzyme-linked immunoassay.
Expression of stem cell markers of proliferation and pluripotency was significantly decreased by exogenous Crx expression. Notably, the homeodomain transcription factor Pax-6, which is expressed developmentally in embryonic retina, showed decreased expression in Crx-electroporated stem cells in comparison with controls. Concomitantly, Crx induced expression of the analysed neuronal and photoreceptor-specific markers. Light-induced cGMP hydrolysis was increased in stem cells expressing exogenous Crx, and inhibition of PDE resulted in elevated cGMP levels.
Our previous investigations (Jomary & Jones, IOVS in press) into exogenous Crx expression in murine retinal stem cells demonstrated the potential for this factor to promote differentiation of these cells into cells expressing photoreceptor-specific markers and displaying light-induced sensitivity characteristic of an activatable visual phototransduction cascade. The present study extends these findings to the effect of exogenous Crx expression in human iris- and retina-derived stem cells and is consistent with the notion that Crx has a broadly-applicable ability to promote differentiation of such cells into photoreceptor phenotypes.
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