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Liang Zhou, Wei Wang, Toshihiko Ezashi, Bhanu Prakash V. Telugu, Henry J. Kaplan, Douglas C. Dean; Differentiation of Swine iPSC into Rod Photoreceptors and Their Integration into the Retina. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2224.
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A two-step protocol was developed for efficient differentiation of swine induced pluripotent stem cells (iPSC) into rod photoreceptors for transplantation into a swine model of rod photoreceptor loss.
Swine iPSC were derived from skin fibroblasts by lentiviral transduction of the stem cell specification genes POU5F1, KLF4, SOX2 and c-MYC. These iPSC were then subjected to a two-step differentiation protocol consisting of floating culture as embryoid bodies followed by three weeks of differentiation in adherent culture. We examined the effect of substratum for adhesion culture and media composition on differentiation to rod photoreceptor lineage. Real time PCR and immunostaining were used to follow iPSC differentiation and the morphology of the cells was examined as well. We analyzed expression of the stem cell marker POU5F1 and rod lineage markers including RCVRN, NRL, RHO and ROM1. Differentiated cells were then transplanted into the subretinal space of swine treated with iodoacetic acid to eliminate rod photoreceptors. Three weeks after transplantation, retinal sections were immunostained to follow engrafted cells.
Real time PCR and immunostaining demonstrated loss of expression of the stem cell specification gene POU5F1 and induction of rod photoreceptor gene markers including RCVRN, NRL, RHO and ROM1. Adherent culture on Matrigel led to a morphology resembling primary cultures of rod photoreceptors and to concentration of RHO and ROM1 in outer segment-like projections. After transplantation, RHO+ cells were evident in all retinal layers, but they were concentrated in the outer nuclear layer where photoreceptors normally reside. A portion of these transplanted cells had projections resembling outer segments.
Skin-derived swine iPSC can efficiently differentiate to express markers of rod lineage and they morphologically resemble rods in culture concentrating RHO and ROM1 into projections resembling outer segments. These cells can integrate into the outer nuclear layer following rod photoreceptor loss, and some of engrafted cells display outer segment-like projections suggesting transition to functional morphology.
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