Screening for RPE to Neural Retina Reprogramming using CRISPR Edited VSX2-specific fluorescent reporter hiPSC

Abstract

Purpose : Neural Retina (NR) detachment causes human retinal pigment epithelium (RPE) to undergo an epithelial to mesenchymal transition that leads to fibrosis. In contrast, surgical removal of the NR induces a spontaneous regenerative response in newt RPE that results in functional recovery of both the RPE and NR. Here we test the hypothesis that human RPE has the capacity to reprogram to VSX2 positive NR progenitors if given the appropriate stimulation

Methods : A NR progenitor reporter was created in human induced pluripotent stem cells (hiPSC) by replacing the stop codon of VSX2 with a P2A fused to cyan fluorescent protein (CFP) using CRISPR/Cas9-mediated homology directed repair (HDR). G418 resistant hiPSC clones were screened for homologous recombination by PCR. The created heterozygous VSX2/CFP reporter line was selected for retinal cup (RC) differentiation to verify CFP expression in NR progenitors. This transgenic line was also differentiated into mature RPE, which was verified by RT-qPCR, immunofluorescence (IF) for RPE mature markers, Na⁺/K⁺ATPase distribution, and phagocytosis. Mature hiPSC-derived RPE was treated with combinations of factors previously shown to support RPE→NR reprogramming in newt, Xenopusor embryonic chick models. These factors included: EGTA, FGF2, SB431542 and XAV939. RPE→NR was verified by real-time CFP expression, IF for VSX2 and RT-qPCR analysis of genes preferentially expressed in NR and RPE. Following optimization of factors for RPE→NR reprogramming, we repeated the reprogramming protocol on commercially hiPSC-derived RPE (iCell^©) and from TJP1 P34 NIH-RPE and verified VSX2 expression by IF and RT-qPCR

Results : 8.3% (6/72) of G418 clones exhibited the expected PCR bands (sequence verified) of the VSX2/CFP targeting locus. The RCs exhibited CFP expression starting at 30 days of differentiation, and these RCs expressed all major retinal cell types by 166 days of differentiation. The VSX2/CFP.hiPSC-derived RPE exhibited mature RPE markers, polarized Na⁺/K⁺ATPase, and phagocytosis. Treatment of the RPE with a cocktail of EGTA, FGF2, SB431542 and XAV939 led to the onset of CFP expression. The best combination of factors induced VSX2 expression in 3 independent hiPSC derived RPE lines: (1) the VSX2/CFP.hiPSC, (2) iCell^©RPE, and (3) TJP1 P34 NIH-RPE

Conclusions : hiPSC-derived RPE can undergo NR reprogramming upon treatment with EGTA, FGF2, SB431542 and XAV939

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.