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Catherine Jomary, Stephen E. Jones, Mandeep Singh, Paul Wong, Robert MacLaren; Generation Of Photoreceptor Phenotypes From Induced Nrl-gfp Mouse Hair Follicle-derived Epithelial Stem Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3181.
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© ARVO (1962-2015); The Authors (2016-present)
Hair follicles provide a readily accessible potential donor source of autologous cells for photoreceptor transplantation, if these cells could be reprogrammed. Previous work with the nrl.gfp transgenic mouse has shown that the post-mitotic rod precursor represents the optimal stage for transplantation into a degenerate retina The aim of the present study was to induce rod photoreceptor precursors from hair follicle-derived epithelial stem cells harvested from the nrl.gfp mouse. This was achieved by modulating the microenvironment in vitro using retina-specific conditioned media and a retinal explant co-culture system.
Epithelial stem cells were isolated from the hair follicle bulge region of whiskers from 2 week old nrl.gfp mice by mechanical dissection, enriched by clonal expansion, and subcultured in retina-conditioned media or with retinal explants. Changes in the mRNA and protein expression of stem cell markers (homeodomain transcription factor Pax6, POU transcription factor Oct3/4) and putative skin stem cell markers (K15, alpha 6 integrin), neuronal markers (nestin, neuron-specific class III ß-tubulin and neurofilament), and photoreceptor-specific markers (rhodopsin, cyclic nucleotide-gated cation channel-3, blue-cone opsin, cyclic GMP phosphodiesterase) were evaluated by immunocytochemistry, Western blotting, and reverse transcription-polymerase chain reaction.
Isolated stem cells from the nrl.gfp mouse hair follicle bulges were successfully expanded by clonal growth. Media derived from cultured retinal cells or retinal explant co-culture systems were found to be effective in inducing differentiation of skin stem cells into rod photoreceptor-like cells. Expression of stem cell markers of proliferation and pluripotency was decreased. Significantly, cells expressed bright GFP de novo together with photoreceptor-specific markers.
The present study demonstrates that hair follicle epithelial stem cells can be reprogrammed into photoreceptor-like cells when exposed to a retina-specific microenvironment. Since the induced expression of GFP is driven by the nrl transcription factor, these cells may be at the optimal developmental stage for retinal repair by autologous transplantation.
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