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L. Zhou, D. C. Dean, T. Ezashi, R. M. Roberts, H. J. Kaplan; Photoreceptor Differentiation From Porcine Induced Pluripotent Stem Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3140.
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Because retinal regeneration is limited in higher vertebrates, stem cells are being utilized to create progenitors for use in cell transplants in models of retinal damage. We have generated a genetic porcine model of retinitis pigmentosa (RP) utilizing the P23H rhodopsin mutation, which is common in human patients with RP. As an initial step toward developing cells for transplant into this large animal model, induced pluripotent stem cells (iPSC) were generated from porcine skin fibroblasts utilizing retroviral expression of embryonic stem cell genes. These iPSC were then used in cell culture experiments for generation of cells expressing markers of photoreceptor differentiation.
A protocol for photoreceptor differentiation (Lamba et al. Proc Natl Acad Sci USA. 2006;103:12769-74) was adapted for porcine iPSC differentiation in culture. Porcine iPSC were cultured as floating aggregates in nonadherant plates in media containing the Wnt inhibitor Dkk1, the BMP inhibitor Noggin, and insulin-like growth factor (IGF-1) for three days, and then the aggregates were transferred to poly-D-lysine plus matrigel coated plates to allow for attachment of the aggregates. Twenty days after transfer to the poly-D-lysine plus matrigel coated plates, cells were fixed and immunostained.
Following the differentiation protocol, many of the adherent cells showed neuronal morphology and these cells immunostained for the general neuronal marker β III-tubulin. Additionally, approximately 50% of the adherent cells immunostained for the photoreceptor marker recoverin.
These studies demonstrate that porcine iPSC can efficiently differentiate into cells expressing a photoreceptor marker in culture. Thus, these cells should provide a source of cells for transplant into the porcine model of retinitis pigmentosa.
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