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S. Balasubramanian, I. Ahmad; Directed Differentiation of Adult Limbal Stem Cells Into Photoreceptors. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1278.
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Corneal epithelium is a highly specialized with basal and stratified squamous epithelial cells, that are renewed throughout life from a stem cell population which resides in a specialized niche called limbus. We have demonstrated that these cells possess vestigial neural potential and in the absence of BMP signaling differentiate along neural lineage (Zhao et al; 2002, Dev. Biol. 250: 317). The limbal epithelium-derived neural progenitors can give rise to neurons with functional properties and demonstrate the capacity to differentiate into photoreceptors (Zhao et al; 2008, Stem cells, 4: 939) thus raising the possibility of using them as a reagent for autologous cell therapy for photoreceptor degeneration. We have begun examining the culture conditions and intrinsic mechanisms for enhancing efficiency of directed differentiation of limbal epithelium stem cells into rod photoreceptors.
Limbal epithelium from Nrl-GFP transgenic mice was dissociated and cultured in the presence of EGF+FGF2 to generate neurospheres. Neurospheres were transferred to poly-D-lysine and laminin coated glass coverslips and co cultured with the PN1 retinal cells for 5-7 days. Sister wells were cultured in identical conditions, supplemented with small molecules to perturb specific pathways (e.g., Notch, Wnt, Shh) and regulators (e.g., HDAC). Differentiation along the rod lineage was screened by Nrl GFP expression in conjunction with RT-PCR and immunocytochemical analyses of rod-specific regulators and markers.
We did not observe Nrl-GFP expression in limbal epithelium cells at the time of dissociation or in neurospheres. Nrl-GFP expression was observed in neurospheres when co-cultured with PN1 cells. RT-PCR and immunocytochemical analysis of cells in neurospheres showed the expression of regulators (Otx2, NeuroD1, Crx and Nrl) and markers (rhodopsin, rhodopsin kinase, recoverin) of rod photoreceptor differentiation.
Our results demonstrate that limbal stem cells can be reprogrammed along retinal lineage and under the influence of neonatal retinal cells can directly differentiate into rods by recruiting normal mechanism of photoreceptor development.
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