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Budd A. Tucker, Edwin M. Stone, Bonita Moses, Luan M. Streb, Jeaneen L. Andorf, Connie Kuusisto, Zara Melkoumian, Michael J. Young; Use Of A Synthetic Xeno-Free Culture Substrate For iPSC Induction And Retinal Differentiation. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2223.
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© ARVO (1962-2015); The Authors (2016-present)
The purpose of this study was to determine whether a proprietary xeno-free synthetic culture surface (Corning® SynthemaxTM Surface) could be used to aid in the production and subsequent retinal specific differentiation of clinical grade iPSCs.
iPSCs were generated using adult mouse and human dermal fibroblasts via infection with a single cre-excisable lentiviral vector driving expression of the transcription factors Oct4, Sox2, KLF4, and c-Myc. Retinal precursor cells were derived via targeted differentiation of iPSCs with exogenous delivery of dkk-1, noggin, IGF1, bFGF, aFGF, and DAPT. Western blotting, immunocytochemistry, H&E staining, and rt-PCR were used to determine reprogramming efficiency, pluripotency, and fate of undifferentiated and differentiated iPSCs.
Compared to normal tissue culture treated or ECM coated surfaces, plating on synthemax substrates resulted in enhanced cellular adhesion. Following lentiviral transduction of the transcription factors Oct4, Sox2, KLF4, and c-Myc, cells underwent prototypical morphological changes resulting in the formation of iPSC colonies large enough to manually isolate/passage at 3-4 weeks post-infection. To identify pluripotency, iPSCs grown under standard undifferentiating conditions were tested via immunocytochemistry, rt-PCR, and teratoma formation assays. As with normal ES cells, iPSCs expressed the pluripotency genes SSEA1 (mouse), SSEA3&4 (human), Oct4, Sox2, KLF4, c-Myc, and Nanog. Following transplantation into immune-compromised mice these cells proceeded to form teratomas containing tissue comprising all three germ layers. When subjected to our previously developed retinal differentiation protocol, as with cells plated on ECM coated dishes, a significant proportion of the xenofree substrate derived cells expressed the retinal progenitor cell marker Pax6 and went on to express the photoreceptor markers CRX, recoverin, and rhodopsin.
SynthemaxTM culture substrates provide an ideal surface for the xeno-free production, culture, and differentiation of adult mouse and human fibroblast derived iPSCs. These findings demonstrate the potential utility of these surfaces for the production of clinical grade retinal neurons for transplantation and induction of retinal regeneration.
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