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B. A. Tucker, I.-H. Park, H. J. Klassen, S. M. Redenti, C. Jiang, S. D. Qi, G. Q. Daley, M. J. Young; Transplantation of Adult Mouse iPS Cell-Derived Photoreceptor Precursors Restores Retinal Structure and Function in Retinal Degenerative Mice. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1233.
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To determine whether iPS cells, generated from adult mouse fibroblast, could be used to produce retinal precursors and subsequently photoreceptor cells for retinal transplantation and subsequent restoration of retinal function.
iPS cells were generated using fibroblasts isolated from the dermas of adult dsRed mice via infection with retrovirus expressing Oct4, Sox2, KLF4 and c-Myc. Retinal precursor cells were derived via targeted differentiation of iPS cells with exogenous delivery of dkk-1, noggin, IGF1, bFGF, aFGF and DAPT. Western blotting, immunocytochemistry, H&E staining, rt-PCR, microarray, in vitro Ca++ imaging, ERG and subretinal transplantation were used to determine the capacity for pluripotency, fate and functionality of undifferentiated and differentiated iPS cells.
To identify pluripotency, adult mouse iPS cells grown under standard undifferentiating conditions were tested via immunocytochemistry, rt-PCR and teratoma formation assays. As with normal mouse ES cells, iPS cells expressed the pluripotency genes SSEA1, Oct4, Sox2, KLF4, c-Myc and Nanog. Following transplantation into the eye of immune-compromised retinal degenerative mice these cells proceeded to form teratomas containing tissue comprising all three germ layers. At 33 days post-differentiation a large proportion of the cells expressed the retinal progenitor cell marker Pax6 and went on to express the photoreceptor markers, CRX, recoverin, and rhodopsin. When tested using calcium imaging these cells were shown to exhibit characteristics of normal retinal physiology, responding to delivery of neurotransmitters. Following subretinal transplantation into degenerative hosts these cells took up residence in the retinal outer nuclear layer and gave rise to increased electro retinal function as determined by ERG.
Adult fibroblast-derived iPS cells provide a viable source for the production of retinal precursors to be used for transplantation and treatment of retinal degenerative disease.
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