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Monte Radeke, Carolyn Radeke, Aaron Newman, Jane Hu, Dean Bok, Lincoln Johnson, Peter Coffey; FGF2 mediated inhibition of epithelial-to-mesenchyme transitions in cultured human fetal RPE. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1178.
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Epithelial-to-mesenchymal transitions (EMT) are common in development, wound healing or stress response, and oncogenesis. With respect to RPE cells, EMT is a central feature of proliferative vitreoretinopathy, of practical consequence to cell-based replacement therapies, and potentially important in AMD. With the goal of developing approaches to its regulation, we have undertaken a whole transcriptome analysis of EMT in RPE.
To promote EMT, differentiation competent fetal RPE cells were subjected to serial passage or were plated at low density. The extent of RPE differentiation was assessed by morphological analysis and transcriptome expression profiles were determined as a function of time, passage, and /or treatment using microarray and RNA-Seq methods.
Comparison of the transcriptome profiles of minimally passaged (P0) 64 day differentiated RPE to highly passaged (P5) 64 day non-differentiated RPE reveals large-scale gene expression changes with over 35% of expressed genes having > 2-fold differences (FDR=0.1). Functional and gene set enrichment analyses indicate that the bulk of the differences are accounted for by increased expression of mesenchymal markers and promoters of the mesenchymal phenotype, and decreased expression of RPE markers and regulators of RPE differentiation. In contrast, in 3 day mitotically active P0 and P5 cultures, fewer than 2% of expressed genes are differentially regulated (> 2-fold, FDR=0.1). Among these genes, the only notable functionally-enriched class is cell cycle/division. This is consistent with our observation that P5 cells have a slightly reduced rate of mitosis and fully arrest upon reaching confluence, whereas P0 cells undergo additional rounds of cell division post-confluence. Given this difference, we tested whether FGF2 could inhibit the onset of EMT by promoting cell division. In the presence of FGF2, RPE maintained the ability to differentiate for 1-2 additional passages over control cells. Similarly, P0 RPE plated at low density in the presence of FGF2 became fully differentiated, while RPE without FGF2 underwent EMT.
Based on our transcriptome analysis, it appears that a loss of ability to override contact inhibition and establish high density cuboidal monolayers may be fundamental to RPE-to-mesenchymal transitions. Growth factors or trophic agents may prove useful in maintaining or reestablishing the RPE phenotype.
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