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B. A. Tucker, S. Redenti, I.-H. Park, G. Q. Daley, M. J. Young; Generation of Retinal Precursors From Murine ips Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5149.
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Retinal degenerative diseases, such as Retinitis pigmentosa (RP) and age-related macular degeneration (AMD), are debilitating incurable disorders characterized by photoreceptor cell death. As the intrinsic regenerative capacity of the CNS is extremely limited, therapies for these diseases focusing on cellular replacement are required. A range of data suggests that the use of stem cells to achieve such a goal is now feasible. The purpose of this study was to determine whether induced pluripotent stem (iPS) cells, generated from adult mouse fibroblast ubiquitously expressing DsRed protein, could be used to produce retinal precursors and subsequently photoreceptor cells for retinal transplantation.
iPS cells were generated using adult DsRed mouse fibroblasts via infection with retrovirus expressing Oct4, Sox2, KLF4 and c-Myc. Retinal precursor cells were derived via targeted differentiation of DsRed-iPS cells via exogenous delivery of dkk-1, noggin, IGF1, bFGF and DAPT. Western blotting, ICC, H&E staining, rt-PCR, microarray and in vitro Ca++ imaging were used to determine pluripotency, fate and functionality of undifferentiated and differentiated iPS cells.
To identify pluripotency, adult mouse DsRed-iPS cells grown under standard undifferentiating conditions were tested via immunocytochemistry and teratoma formation assays. As with normal mouse ES cells, DsRed-iPS cells expressed the pluripotency genes SSEA1, OCT3/4, Sox2, KLF4, c-Myc and Nanog. Following transplantation into the eye of immune-compromised retinal degenerative mice these cells proceeded to form large teratomas that contained tissue comprising all three germ layers, including the neural retina. DsRed-iPS cells were subsequently subjected to a series of differentiation conditions to induce development and production of retinal specific cell types. At 30 days post-differentiation a large proportion of the cells expressed the retinal progenitor cell markers Pax6 and Chx10. Of these, cells were identified that expressed markers of many retinal cell types including retinal ganglion (NF200, and MAP1B), Muller glia (GFAP and GS) and photoreceptors (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.
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 disorders such as RP and AMD.
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