Abstract
Purpose: :
The aim of this study was to evaluate the potential of rat hair follicle stem cells to transdifferentiate into photoreceptor-like cells, following modulation of the microenvironment using retinal-specific conditions or after genetic modification using the Crx transcription factor.
Methods: :
Epithelial stem cells were isolated from the hair follicle bulge region by mechanical dissection, enriched by clonal expansion, and either subcultured in retinal-conditioned media, or 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) 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 (c)GMP phosphodiesterase) were evaluated by immunocytochemistry, Western blotting, and quantitative reverse transcription-polymerase chain reaction.
Results: :
Isolated stem cells from the hair follicle bulge were successfully expanded by clonal growth without a feeder layer. Both media derived from cultured retinal cells and exogenous expression of Crx by genetic modification were found to be effective in inducing a photoreceptor -like phenotype. Expression of stem cell markers of proliferation and pluripotency was decreased. Concomitantly, expression of neuronal and photoreceptor-specific markers was up-regulated.
Conclusions: :
The present study suggests that rat hair follicle epithelial stem cells are capable of differentiation into photoreceptor phenotype cells ex vivo when either exposed to retinal-specific microenvironment or genetically modified with Crx. The present study extends our previous findings that exogenous Crx expression can induce mouse and human retina-derived stem cells into functional photoreceptor cells and is consistent with the notion that Crx has a broadly-applicable ability to promote differentiation of such cells into photoreceptor phenotypes.
Keywords: photoreceptors • retina • gene/expression