Restoration of mesenchymal RPE by transcription factor-mediated reprogramming

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.