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Marina Riera, Anna Salas Torras, Anna Seriola, Yolanda Muñoz, Miguel A Zapata, Anna Veiga, Jose Garcia-Arumi; Differentiation and transplantation of human ESC and iPSC-derived RPE cells for the treatment of retinitis pigmentosa. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1394.
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© ARVO (1962-2015); The Authors (2016-present)
Retinitis pigmentosa (RP) is the most common form of inherited retinal dystrophies and it is characterized by progressive degeneration of photoreceptor and RPE cells. Some approaches to therapy for RP include, among others, cell transplantation. We here present our first results in the differentiation and transplantation of human RPE cells into a rat model of RP (Royal College of Surgeons rat, RCS).
Two hiPSC lines and one hESC line were differentiated into RPE in adherent culture. Cells were cultured in pluripotent basic culture media and were changed into differentiation media containing DMEM/F12, dexamethasone and glycerol phosphate for 2 weeks. Cells were continuously assessed for morphological changes. Samples were collected at different time points of differentiation; immunostaining and RT-PCR were used for assessment of several retinal markers. Differentiated cells were injected into the subretinal space of immunosuppressed RCS animals at P21. The location of transplanted human cells, their integration and expression profile were assessed by hematoxylin and eosin stain and immunohistochemistry.
RPE cells were successfully generated from the two hiPSCs and the hESC line. Differentiated cells exhibited epithelial monolayer morphology and expressed the retinal differentiation Pax6 and ZO-1 genes from day 8 of differentiation, and CHX10 and OTX2 from day 10. OCT4 (pluripotency marker) expression was completely lost after 4 days of differentiation. After 4 weeks of differentiation induction, pigmented areas were observed. Cells also showed an increased expression of the visual cycle related genes (CRALBP and RPE65), and genes essential for eye development (Dkk-1 and NOG) and phagocytosis (MERTK). Finally, differentiated cells were negative for Ki67; indicating cells were no longer proliferative. Preliminary results showed that hiPS-RPE grafts were stable, transplanted cells survived in the subretinal space and did not migrate from the eye. We are currently working on the integration and functionality of these cells in the host retina.
We have achieved adequate differentiation to RPE cells from both hiPSC and hESC and the differentiated cells were successfully transplanted into the rat model of RP. The functionality and long term follow up of the transplants need to be assessed to confirm the suitability of the strategy.
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