High content screen for small molecules that inhibit epithelial to mesenchymal transition in human stem cell-derived RPE cells

Abstract

Purpose : Epithelial to mesenchymal transition (EMT) of the RPE, which can leads to RPE dysfunction and retinal degeneration, has been implicated in diseases such as PVR and AMD. As a possible therapeutic approach to these diseases, we have been pursuing in vitro high content screening (HCS) to identify molecules that inhibit RPE-EMT.

Methods : Based on gene and protein expression studies using human stem cell-derived models of RPE EMT, we found that within 3 hours after EMT induction there are significant increases in SNAI1 and decreases in BEST1 expression. Utilizing CRISPR/Cas9 engineering, we are generating a dual knock-in fluorescent reporter stem cell line for SNAI1(GFP) and BEST1 (tdTomato). Both reporters are preceded by a 2A self-cleaving peptide (P2A) sequence, so the endogenous proteins are expressed without alteration. After differentiating the reporter line to RPE monolayers, RPE-EMT is induced by 1) dissociation followed by re-plating into 1536-well plates or 2) treatment of RPE monolayers in 384-well plates with TGF-β/TNF-α. Twelve hours prior to EMT induction, compound libraries to be screened were dispensed using an acoustic liquid handler. After 72 hours, images were acquired and analyzed to determine the percentage of cells that express the reporter fluorescent proteins, the intensity of the reporters’ fluorescence per cell, and other morphological characteristics.

Results : With the BEST1-tdTomato line that we generated, dissociation and TGF-β/TNF-α induced RPE EMT show decreased tdTomato expression at 24-72 hrs. In an initial screen for inhibitors of RPE EMT, 19 hits were identified, and these hits are being characterized in a secondary screen by qPCR to assess their effects on a variety of other EMT and RPE markers. One of the active molecules we identified is BAY 65-1942, an inhibitor of IKKβ. Expression of a core set of down-regulated visual cycle genes was significantly restored by BAY 65-1942 treatment, and elevation of EMT regulators was significantly suppressed.

Conclusions : Our initial screen has identified bioactive compounds that can inhibit EMT in hRPE cells and maintain their “RPE-ness.” We hope that this new dual reporter screening system will provide a robust platform for the discovery of novel inhibitors of RPE EMT, and aid in the understanding and treatment of RPE EMT.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.