Transcriptome Landscape of Epithelial to Mesenchymal Transition of Human Stem Cell–Derived RPE

Abstract

Purpose: RPE injury often induces epithelial to mesenchymal transition (EMT). Although RPE-EMT has been implicated in a variety of retinal diseases, including proliferative vitroretinopathy, neovascular and atrophic AMD, and diabetic retinopathy, it is not well-understood at the molecular level. To contribute to our understanding of EMT in human RPE, we performed a time-course transcriptomic analysis of human stem cell-derived RPE (hRPE) monolayers induced to undergo EMT using 2 independent, yet complementary, model systems. Methods: EMT of human stem cell-derived RPE monolayers was induced by either enzymatic dissociation or modulation of TGF-β signaling. Transcriptomic analysis of cells at different stages of EMT was performed by RNA-sequencing, and select findings were confirmed by reverse transcription quantitative PCR and immunostaining. An ingenuity pathway analysis (IPA) was performed to identify signaling pathways and regulatory networks associated with EMT. Results: Proteocollagenolytic enzymatic dissociation and cotreatment with TGF-β and TNF-α both induce EMT in human stem cell-derived RPE monolayers, leading to an increased expression of mesenchymal factors and a decreased expression of RPE differentiation-associated factors. Ingenuity pathway analysis identified the upstream regulators of the RPE-EMT regulatory networks and identified master switches and nodes during RPE-EMT. Of particular interest was the identification of widespread dysregulation of axon guidance molecules during RPE-EMT progression. Conclusions: The temporal transcriptome profiles described here provide a comprehensive resource of the dynamic signaling events and the associated biological pathways that underlie RPE-EMT onset. The pathways defined by these studies may help to identify targets for the development of novel therapeutic targets for the treatment of retinal disease.