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David M Gamm, Neehar Bhatia, Anna Petelinsek, Jee Min, Elizabeth E Capowski, Travis Cordie, Diana Drier, Connor Lyons, Derek Hei, Joe Phillips; cGMP production of neural retina from hiPSCs. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3170.
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© ARVO (1962-2015); The Authors (2016-present)
Clinical trials involving human ESC and iPSC-derived RPE transplantation are well underway. However, in later stages of most outer retinal degenerative diseases, photoreceptor replacement alone or in combination with RPE must be considered. The purpose of this study is to convert an established hiPSC differentiation method for deriving neural retina (including photoreceptor precursors) to current Good Manufacturing Practices (cGMP).
All hiPSC lines investigated were created using an integration-free reprogramming method. Some hiPSC lines, including those derived from homozygous HLA “super donors,” were banked under cGMP conditions. hiPSCs were then thawed and cultured using multiple cGMP-compliant methods and differentiated to optic vesicle-like structures (OVs) using our previously established 3-D protocol. ICC analysis was performed to confirm neural retinal progenitor cell (iNRPC) and photoreceptor precursor (iPRP) cell production.
Each hiPSC maintenance platform produced high, reproducible yields of OVs following retinal differentiation, with a single 6 well plate of hiPSCs giving rise to up to 400 VSX2+ OVs containing up to 24 million iNRPCs at early stages of differentiation. By day 60 of differentiation, >98% of OVs contained iPRPs as demonstrated by ICC (CRX+/RCVRN+). Cells within the OVs had >95% viability following gentle dissociation.
This study demonstrates robust production of hiPSC-derived neural retina across multiple cGMP-compliant culturing platforms. In our hands, E8/vitronectin is the most ideal hiPSC maintenance medium/substrate. Current studies are focused on full characterization of super donor hiPSC lines and their neural retinal derivatives and optimizing cryopreservation methods.
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