Transcription Factor Overexpression Drives Reliable Differentiation of Retinal Pigment Epithelium From Human Induced Pluripotent Stem Cells. Transcription Factor Overexpression Drives Reliable Differentiation of Retinal Pigment Epithelium From Human Induced Pluripotent Stem Cells

We report a robust, scalable method to generate iPSC-RPE using doxycycline-inducible expression of eye field transcription factors OTX2, PAX6 and MITF paired with RPE-permissive culture media. Doxycycline addition induces exogenous expression of these transcription factors in patient- and wildtype iPSCs to efficiently produce monolayers of RPE with characteristic morphology and gene expression. Further, these RPE monolayers display functionality features including light absorption via pigmentation, polarity-driven fluid transport, and phagocytosis. With this method, we achieve a highly efficient and easily scalable differentiation without the need for mechanical isolation or enrichment methods, generating RPE cultures applicable for in vitro studies. Overall design: We analyzed samples from 3 different stages of the differentiation for each cell line, with 3 replicates each, resulting in 18 samples total.