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Camille Yvon, Conor Ramsden, Amanda-Jayne Francis Carr, Matthew Smart, Michael Powner, Amelia Lane, Britta Nommitse, Lyndon da Cruz, Pete Coffey; Generation of neuroretina from induced pluripotent stem cells and effects of Sonic Hedgehog. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3572. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Induced pluripotent stem cells (iPSCs) have the potential to provide robust models for the study of human retinogenesis and treatment therapies for retinal diseases. iPSC differentiation into neural retina (NR) can be driven in vitro by lineage-determining transcription factors. However, there exists a great source of variability between published protocols. We sought to determine if iPSCs could generate optic vesicle like structures or NR, by varying culture conditions of Matrigel™ and Sonic Hedgehog (Shh).
We compared culture conditions for NR differentiation using two batches of Matrigel™ (M1 and M2), and variable concentrations of Shh trapped in the Matrigel coating the plate. Early changes in retinal development were monitored by immunocytochemistry and quantitative polymerase chain reaction.
We report the generation of optic vesicle and cup structures from iPSCs, using our modified protocol. It was found that embryoid bodies cultured in M2 showed higher expression of retinal development association transcription factors such as Pax6, Rax, Otx2 and Chx10 compared to M1. Embryoid bodies cultured in elevated Shh concentrations induced a greater percentage of organized structures and increased expression of eye field transcription factors.
Our modified protocol using Shh delivered via plate coating provides practical and efficient ways of generating NR from iPSCs, thus helping to optimise the differentiation protocol.
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