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Anil Upreti, Phuong T. Lam, Jared Austin Tangeman, Brad Wagner, Davide Ortolan, Ruchi Sharma, Kapil Bharti, Katia Del Rio-Tsonis, Michael L Robinson; Reprogramming human induced pluripotent stem cell (hiPSC)-derived RPE into neural retina progenitors (NRPs). Invest. Ophthalmol. Vis. Sci. 2020;61(7):2503.
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© ARVO (1962-2015); The Authors (2016-present)
Human neural retina (NR) damage (from trauma or disease), causes human RPE (hRPE) to undergo an epithelial to mesenchymal transition (EMT) that ultimately leads to fibrosis. In contrast, surgical removal of the NR induces a spontaneous regenerative response in newt RPE that ultimately results in functional recovery of both the RPE and NR. Here we describe a small molecule screening strategy that led to the identification of U0126, a MEK1/2 inhibitor that facilitates human RPE to neural retina progenitor reprogramming.
Human RPE derived from the PGP1 retina reporter hiPSC line was used to screen two small molecule libraries. PGP1 contains three fluorescent reporter genes (Cerulean, eGFP and mCherry) driven by the endogenous VSX2, BRN3b, and RCVRN promoters, respectively. RPE maturity was verified by gene expression, immunofluorescence (IF), Transepithelial resistance (TER), and phagocytosis activities. Using fluorescence readout as an indicator, the mature PGP1-RPE was used in high-throughput screening to identify the factor(s) that can induce RPE-NR reprogramming. Compounds that elicited Cerulean expression, indicative of VSX2 positive NRPs, were retested on PGP1-RPE grown on transwell inserts by fluorescence-activated cell sorting (FACS), IF, and RNA-Seq to evaluate RPE reprogramming activity.
The small molecule screen of PGP1-RPE identified the MEK1/2 inhibitor, U0126 as a potent inducer of VSX2-Cerulean expression both on extracellular matrix coated plates and on vitronectin coated transwell inserts. FACS analysis of PGP1-RPE grown on transwells inserts revealed that 52.6% of the U0126 treated cells expressed Cerulean following 15 days of treatment. U0126-treated PGP1-RPE cells also expressed VSX2 protein. RNA-Seq analysis of U0126-treated PGP1-RPE revealed significant down-regulation of multiple genes characteristic of RPE (RPE65, BEST1, and MITF), and genes related to EMT/fibrosis (FN1, CLDN, COL1A, TNF).
Treatment of hiPSC-derived RPE with U0126 induces VSX2 expression and suppresses genes characteristic of both mature RPE and EMT/fibrosis. These results suggest that suppressing human RPE fibrosis may encourage reprogramming toward NR fate.
This is a 2020 ARVO Annual Meeting abstract.
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