Epithelial to mesenchymal transition of human stem cell-derived retinal pigment epithelium cells modulates expression of axonal guidance molecules

Abstract

Purpose : RPE injury often induces epithelial to mesenchymal transition (EMT). RPE dysfunction due to the progression of EMT has been implicated in age-related degeneration and related retinal diseases. To better understand the molecular mechanisms of RPE EMT, we performed transcriptome analysis of human stem cell-derived RPE induced to undergo EMT by enzymatic dissociation into and by altered transforming growth factor (TGF-β) signaling.

Methods : Human RPE monolayers were differentiated from iPS-cells as previously published methods described (Maruotti et al, 2013 and 2015). For inducing EMT, RPE monolayer culture preparations were dissociated to single cells or treated with transforming growth factor beta (TGF-β) and TNF-α. RNA-Seq libraries were constructed and sequenced. Reads were aligned to NCBI build 37. 2 using Tophat (v2. 1. 0). Cuffquant and Cuffnorm (Cufflinks v2. 2. 1) were used to quantify expression levels and calculate normalized FPKM values. Biological pathway analysis was performed using Ingenuity Pathway Analysis (IPA), Gene Set Enrichment Analysis (GSEA) and validated by qRT-PCR and immunostaining.

Results : EMT-related transcription factors such as Snail family zinc finger (SNAI1, SNAI2), Zinc-finger E-box-binding (ZEB), basic- loop-helix transcription 1 (TWIST1) and transcription factor-3 (TCF3), and further downstream signaling genes including α-smooth muscle actin (ACTA2), vimentin (VIM), fibronectin (FN1) and N-Cadherin (CDH2) were significantly up-regulated. RPE-specific genes such as PMEL17, MITF, BEST1, TYR, RLBP1, L-RAT and RPE65 were dramatically down-regulated during RPE EMT. IPA and GSEA analysis uncovered many altered canonical signaling pathways. A particularly novel finding was that one of the pathways showing the most significant expression changes was the axon guidance signaling pathway, in which there was widespread dysregulation of multiple semaphorins, plexins and netrins during the progression of RPE EMT.

Conclusions : Temporal transcriptome analysis of RPE EMT enables the identification of key signaling events that regulate RPE EMT and suggests targets that can be used to develop strategies to inhibit RPE EMT. Additionally, defining the transcriptional networks involved in human RPE cells undergoing EMT may provide novel therapeutic targets for treatment of retinal diseases.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.