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Mariacarmela Allocca, Ana Santos-Carvalho, Budd A. Tucker, Sara Qi, Caio V. Regatieri, Caihui Zhang Zhang, Konrad Hochedlinger, Claudia Cavadas, Michael J. Young; Characterization And Differentiation In Photoreceptors Of Retina-derived Induced Pluripotent Stem Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):321.
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Our lab and others have shown that induced pluripotent stem cells (iPS cells) obtained from fibroblasts may provide a source of photoreceptors for cell based therapies. One limitation of this approach is the low yield of photoreceptors obtained. Recently, it has also been showed that iPS cells retain a transient transcriptional and epigenetic memory of their cell of origin which affects their potential to differentiate in specific cell types. The purpose of this work was to characterize the pluripotency and the differentiation into photoreceptors of iPS cells lines obtained from murine retinas.
Retinas from "reprogrammable" mice were cultured in the presence of doxycycline to induce the expression of the four reprogramming factors c-Myc, Klf4, Oct-4 and Sox-2 and to produce iPS colonies. The pluripotency of these colonies and epigenetic memory were tested by evaluating the expression of pluripotency and photoreceptor markers by immunocytochemistry, western blot and RT-PCR analysis. Pluripotency was also evaluated by teratoma assay. The ability of iPS cells to differentiate into photoreceptors was evaluated in vitro by immunocytochemistry. The percentage of photoreceptors positive for recoverin, rhodopsin and Cone-Rod homeobox (CRX) was determined.
Retina-derived iPS cells expressed the pluripotency markers c-Myc, Klf4, Oct-4, Sox-2 and Nanog. In the teratoma assay we observed the presence of tissues from all three germ layers. The iPS cells line from retinas also expressed photoreceptor markers. The in vitro differentiation of the retina-derived iPS cells resulted in photoreceptor shaped cells and 80-90% of total number of cells expressed positive staining for photoreceptor markers (CRX, recoverin and rhodopsin). We also observed an increase in expression of photoreceptor markers compared with the undifferentiated iPS cell colonies. Moreover, the expression of pluripotency markers Oct-4 and Nanog decreased.
The iPS cells obtain from the murine retina were pluripotent and differentiated into photoreceptors with a very high efficiency.
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