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.

Proteomic and phosphoproteomic analyses identify liver-related signaling in retinal pigment epithelial cells during EMT

“…Although enzymatic dissociation is a less commonly used approach for inducing RPE EMT, it is becoming increasing relevant to understand the molecular events associated with dissociation because enzymatic treatment is being used more commonly in preparation of RPE cells for transplantation and high-content screening studies. Overall, the perturbations we observed overlap well with two recently published studies examining transcriptional changes in hiPSC-RPE after TGNF treatment, including one from our group, which also examined dissociation (Boles et al, 2020;Sripathi et al, 2021). Each study reports changes to the cytoskeleton and extracellular matrix organization, among many others.…”

“…Proteomics and phosphoproteomics analyses of early hiPSC-RPE cell responses to TGF-b and tumor necrosis factor alpha (TNF-a) (TGNF) and enzymatic dissociation show particularly strong overlap in the phosphoproteome at 1 h As reported previously by our group (Sripathi et al, 2021), hiPSC-RPE cells exhibit many canonical characteristics of EMT when co-treated with TGF-b and TNF-a or when dissociated from their culture substrate and neighboring RPE cells by proteo-collagenolytic enzymes (AccuMAX). These include increased transcription of SNAI1, SNAI2, ZEB1, TWIST1, VIM, and CTNNB1 and decreased transcription of RPE and epithelial markers such as RPE65, TYR, CDH1, and BEST1 (Sripathi et al, 2021). By 72 h, protein abundance, localization, and phosphorylation changes can be observed widely throughout the culture population in E-cadherin, Erk1 and Erk2, and Twist-related protein 1 (Figure 1A; Figure S1).…”

“…Transcriptomics studies have begun to define some of the gene expression changes associated with RPE EMT (Boles et al, 2020;Sripathi et al, 2021;Tangeman et al, 2021). However, because kinase/phosphatase signaling can occur rapidly in response to stimuli, and changes in protein phosphorylation provide one of the earliest signatures of EMT induction, understanding the mechanisms of EMT requires study of the process at the protein level.…”

Proteome Landscape of Epithelial-to-Mesenchymal Transition (EMT) of Retinal Pigment Epithelium Shares Commonalities With Malignancy-Associated EMT

“…However, proteogenomic studies often show significant discordance between RNA and protein expression levels ( 49 ). To determine whether EMT-related protein abundance changes correlate with changes in the corresponding mRNA expression levels, we compared our proteomic RPE-EMT results with data that we previously generated through an RNA-Seq study ( 46 , 50 ). We compared the significantly altered 7647 genes that we identified from our transcriptome analysis with the current proteome analysis.…”

Fine Tuning of an Oxidative Stress Model with Sodium Iodate Revealed Protective Effect of NF-κB Inhibition and Sex-Specific Difference in Susceptibility of the Retinal Pigment Epithelium

“…For EMT-related genes, BAY 651942 did not suppress the mRNA levels of EMT-TFs, Snai1 , Snai2 , Zeb1 , and Zeb2 , compared with vehicle, except Snai1 at 60 mg/kg BW ( Figure 2 c). Since these EMT-TFs are generally upregulated quickly within hours after triggering stimuli such as TGFβ/TNFα treatment and disruption of cell-cell contacts [ 46 ], they would return to the baseline by 7 days later, and therefore it was predicted that significant changes of the mRNA levels might not be observed for these genes at this time point. In contrast, the upregulation of EMT markers, Acta2 (gene for α-smooth muscle actin (α-SMA)) and Vim (gene for vimentin), induced by NaIO 3 was significantly suppressed by BAY 651942 particularly at 30 mg/kg BW compared with vehicle ( Figure 2 c).…”