Differentiation Of Swine IPSC Into Rod Photoreceptors And Their Integration Into The Retina

Purpose:A two-step protocol was developed for efficient differentiation of porcine induced pluripotent stem cells (piPSC) into rod photoreceptors for transplantation into a swine model of rod photoreceptor loss.Materials and Methods:piPSC were cultured on the irradiate inactivated SNL feeder cell layer. P28, P40 and P43 piPSC were used for the differentiation. The cells were put in floating culture to form 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 markers Oct4, ABCG2, Nanog, Sox2; rod lineage markers including RCVRN, NRL, RHO, IRBP, ROM1, cone-arrestin and rod bipolar cell marker PRKCA. Differentiated cells were then infected with the IRBP-GFP lentivirus. 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.Results:Real time PCR and immunostaining demonstrated loss of expression of the stem cell specification gene OCT4, Nanog, Sox2 and induction of rod photoreceptor gene markers including RCVRN, NRL, RHO, IRBP, ROM1, cone-arrestin and rod bipolar cell marker PRKCA. Immunostaining results and statistic analysis showed among the differentiated cells,30.91±7.2% were positive for NRL,25.61±8.5% were positive for RCVRN,6.765±2.083% expressed RHO, and 2.6±0.4% expressed PRKCA. Adherent culture on laminin-fibronectin led to a higher number of RHO+ cells while Matrigel led to a morphology resembling primary cultures of rod photoreceptors and to concentration of RHO and ROM1 in outer segment-like projections. After infection by the IRBP-GFP lentivirus, about 44% of differentiated cells were GFP+, which reflected rod and cone photoreceptors. 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.Conclusions: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.