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Restoration of mesenchymal RPE by transcription factor-mediated reprogramming

ABSTRACT: TGFbeta-mediated epithelial-to-mesenchymal transition (EMT) is a major component of the wound healing response and a negative determinant of retinal pigment epithelial (RPE) differentiation after periods of sustained sub-confluent culture or repetitive passage. Inhibition of TGFbeta signaling using receptor kinase inhibitors forestalls the onset of passage-dependent EMT and can restore the capacity to differentiate to cells that previously underwent the mesenchymal switch [Radeke et al., 2015, Genome Med. 7:58]. However, even with the sustained inhibition of mesenchymal gene expression using TGFbeta signaling inhibitors the cells eventually lose the capacity to attain a characteristic phenotype. This suggests that there are additional mechanisms at play that contributeto the prevention of RPE differentiation after protracted periods of wound stimulus and mitosis. In this study we investigate the non-TGFbeta-mediated processes that contribute to the demise of the RPE phenotype after extended periods of proliferative wound response. Using comparative transcriptomics, we show that with increasing passage there is a downregulation of RPE genes, misregulation of cell cycle genes, a decline in proliferative potential that cannot be prevented or reversed by inhibition of TGFbeta signaling using the TGFbeta receptor kinase inhibitor A-83-01. Importantly, among the RPE genes with decreased expression are several transcription factors known to be critical for RPE development. Exogenous expression of MYCN and OTX2 in conjunction with A-83-01 treatment restored the ability of passage 7 RPE to differentiate. Taken together, these results demonstrate that the loss of capacity to differentiate as a result of chronic wound stimulus is a product of both TGFbeta pathway-dependent increases in mesenchymal gene expression and a TGFbeta pathway-independent loss of RPE programming.

ORGANISM(S): Homo sapiens

PROVIDER: GSE78740 | GEO | 2016/12/01

SECONDARY ACCESSION(S): PRJNA313410

REPOSITORIES: GEO

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Project description:Age-related macular degeneration (AMD) is a leading cause of blindness. Most vision loss occurs following the transition from a disease of deposit formation and inflammation to a disease of neovascular fibrosis and/or cell death. Here, we investigate how protracted wound stimulus leads to seminal changes in gene expression and the onset of a self-sustained state of wound response in retinal pigmented epithelial (RPE) cells. Using a human fetal RPE cell culture model and a systems level transcriptome analysis, we show that prolonged subconfluent culture resulting from repeated passage, leads to terminal acquisition of a mesenchymal-like phenotype post-confluence accompanied by altered expression of >40% of the transcriptome. In contrast, at subconfluence <5% of transcripts have >2-fold expression changes after repeated passage. Protein-protein interaction analysis reveals a core set of genes comprising two interconnected modules with functions pertaining to wound response and cell division. Among the wound response genes are the TGF-beta pathway activators: TGFB1, TGFG2, INHBA, INHBB, GDF6, CTGF, and THBS1. Small molecule inhibition of TGFBR1/ACVR1B mediated signaling both forestalls and reverses the passage-dependent loss of epithelial potential. Moreover, a disproportionate number of RPE wound response genes have altered expression in neovascular and geographic AMD; including key members of the TGF-beta pathway. In conclusiton, in RPE cells the switch to a terminal mesenchymal-like state following protracted or repeated wound stimulus is driven by activation of a self-perpetuating TGF-beta feedback loop. Targeted inhibition of TGF-beta signaling may be an effective approach towards retarding AMD progression and producing RPE cells in quantity for research and cell based therapies. Transcriptome profiles were determined from 44 human fetal RPE cultures of varying passage number, seeding density, culture maturity, and/or growth factor or small molecule treatment. Probes were labeled with Cy3 or Cy5 and Agilent whole genome microarrays were hybridized with a pair of Cy3 and Cy5 probes. After background subtraction and LOWESS correction to adjust for dye-dependent effects the net intensity values were determined as described in the Data Processing section and the entire data set was quantile normalized. The values reported in the Series Matrix are the quantile normalized net intensity values of the gene specific probes.

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Restoration of mesenchymal RPE by transcription factor-mediated reprogramming

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