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M.-H. Errera, S. Thomasseau, J.-A. Sahel, O. Goureau, G. Orieux; Characterization of Stem Cells From the Ciliary Epithelium of Adult Mice and Pigs: Assay of Their Proliferation and Differentiation Potential. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3561. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Cell therapy by transplantation of retinal stem cells is a one of the future strategies to prevent visual loss with application to degenerative retinal diseases.
We isolated in vitro retinal stem cells from ciliary margin (RSCM) of adult pigs. The cells suspension was cultured in serum-free media containing growth factors (EGF, 20 ng/ml and FGF2, 10 ng/ml) during approximately one week. In order to force the differentiation in photoreceptors of RSCM, we cultured ciliary margin spheres colonies under conditions which promote cell differentiation (serum supplemented medium, withdrawal of growth factors) in presence of postnatal retinal explants.
The RSCM from adult pigs can proliferate in culture and grown to form spheres colonies of cells.The low frequency of these RSCM in the adult pig eye was respectively 0.48% (±0.33) of pigmented cells in the ciliary margin. Thus, further experiments will be necessary to induce proliferation of these cells before potential clinical evaluation.To reveal a broader proliferation potential of RSCM, we used different combination of growth factors (EGF and FGF2) and activation of Wnt pathway. We failed to stimulate proliferation of RSCM in primary neurospheres but number of secondary spheres (after passaging) was enhanced by the Wnt signaling pathway or FGF2 signaling suggesting an improvement in maintaining of the mitotic status of RSCM. Under conditions of differentiation, RSCM expressed specific markers for differentiated retinal cells essentially of inner nuclear layer such as glutamin synthase (Müller glia), Pax6 and calbindin (amacrine cells) but failed to express outer nuclear layer markers. In order forcing the capacity of RSCM to differentiate into photoreceptors, dissociated pigs neurospheres colonies were induced to differentiate in vitro and co-cultured with postnatal mice retinas. A small proportion of differentiated RSCM expressed the photoreceptors markers recoverine and R4D2 under co-culture conditions.
This study showed that RSCM appear to differentiate into retinal-specific cell types, including amacrine and bipolar cells and Müller glia or remain undifferentiated progenitors. Conditions to assay the potential of differentiation toward photoreceptors must be optimized.
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