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Diana Katsman, Daniel R. Demin, Debora B. Farber; Transfer of mRNA and Micro-RNA by Embryonic Stem Cell-Derived Microvesicles Induces Gene Expression Changes in Retinal Progenitor Müller Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5900.
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© ARVO (1962-2015); The Authors (2016-present)
To examine the morphological and gene expression changes induced in Müller cells by Embryonic Stem Cell-Derived Microvesicle (ESMV) exposure.
ESMVs were isolated from cultured mouse ESCs and added to human Müller cell culture medium, examining cells' morphology at different time points by light microscopy, RNA was isolated at 8, 24, and 48 h post-ESMV treatment and expression of transcripts important for maintenance of stem cell pluripotency (Oct4, Sox2, Nanog), early retinal transcripts (Pax6 & Rx), and stem cell specific miRNAs (mir 292 & 295) was examined by qRT-PCR; RNA was also hybridized to Agilent human gene expression arrays and Exiqon microRNA arrays. Select gene and miRNA expression changes identified by microarray analysis were verified by qRT-PCR.
ESMV-exposed Müller cells were seen as individual heterogeneous cells as compared to the uniform, spindle-like adherent cellular sheets of untreated cells. qRT-PCR results indicated that mouse Oct4 and Sox2 mRNAs were transferred from ESMVs to human Müller cells and still found at 48 h post-ESMV exposure. Moreover, up-regulation of human Oct4 in Müller cells was noted, suggesting that ESMV treatment induced endogenous Oct4 expression in Müller cells. miRNAs 292 and 295, which are not present in Müller cells, were found in ESMV-exposed cells. Increase in early retinal transcripts Pax6 and Rx was also observed in Müller cells over the 48 h post-ESMV treatment. Global gene expression analysis of ESMV-treated Müller cells compared to untreated cells at the level of mRNA and miRNA revealed the up-regulation of genes involved in induction of pluripotecy (Oct4, Lin28, Klf4, and Cyclin D2), early retinal genes (BMP7, Pax6, and Rx), genes involved in retinal protection (IL6) and regeneration (IGF2), as well as multiple extracellular matrix modifying molecules (MMP3) known to create permissive environment for tissue remodeling. Among the down-regulated genes were those promoting differentiation (DNMT3a and GATA4), inhibitory extracellular matrix components (Aggrecan), and inhibitory scar tissue components (GFAP). Several of the up-regulated miRNAs were involved in promoting pluripotency and cellular proliferation, while many down-regulated miRNAs were involved in differentiation and cell cycle arrest.
Our data demonstrated that ESMVs selectively transfer ESC mRNAs and miRNAs to Müller cells, inducing in them morphological and gene expression changes. Our results suggest that ESMVs may induce de-differentiation of Müller cells to a stem cell phenotype.
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