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MIGUEL FLORES-BELLVER, Silvia Aparicio Domingo, M Natalia Vergara, Minda M. McNally, Valeria Canto Soler; Derivation and Characterization of induced-primary RPE culture (ipRPE) from 3D mini retinas. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4569.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal pigmented epithelium (RPE) derived from human induced pluripotent stem (iPS) cells is a promising source of cells for transplantation, disease modeling and drug screening. Successful generation of RPE from heterogeneous cultures of differentiating human iPS cells occurs with varying efficiency. Recently, our team established a simple and efficient strategy for inducing human iPS cells to differentiate into 3D mini retinas in vitro, with spatial and temporal features that replicate the development of the human retina in vivo, including the formation of RPE tissue. The goal of the present study was to establish a novel methodology for deriving RPE cells from the RPE tissue present in 3D mini retinas.
Similarly to the approach generally followed for deriving cultures of primary RPE cells, RPE tissue is dissected from the 3D mini retinas, dissociated into single cells and seeded on matrigel-coated plates where they form characteristic RPE monolayers. Expression of specific molecules associated with differentiation and function of the RPE cultures was evaluated by RT-PCR, immunofluorescence, and western blot (MITF, ZO-1, EZRIN, RPE65, CRALBP, LRAT, TYR, among others).
Expression of the key RPE genes evaluated in our cultures was in accordance with patterns previously reported for adult human RPE cells in vivo, as well as human primary RPE cultures, and RPE cells derived from pluripotent stem cells. Interestingly, RPE monolayers also showed formation of epithelial domes, suggesting that the cells are functional and engaged in active fluid transport, as is known to occur through tight junction in mature RPE cells.
Therefore, we propose this new pseudo human primary RPE culture, referred by us as induced-primary RPE (ipRPE), as an alternative and promising source of pure hiPSC-derived RPE cells.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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