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A. Yuan, A. Rapoport, S. Kar, D. Tejada, C. K. Yamashita, N. B. Akhmedov, D. B. Farber; Characterization of Embryonic Stem Cell Microvesicles. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1672.
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Microvesicles are membrane-derived vesicles released into the extracellular space by activated or apoptotic eukaryotic cells. It has been postulated that microvesicles may serve a role in intercellular communication. We characterized the RNA and protein content of ESMVs derived from a mouse ES cell line expressing green fluorescent protein to determine if exogenously expressed RNA and protein may be delivered by ESMVs.
ES cells were grown without feeders in serum free media. ESMVs were collected and pelleted by differential ultracentrifugation or equilibrium density ultracentrifugation. ESMV RNA profiles were obtained on an Agilent 2100 bioanalyzer and also by 15% denaturing PAGE for small RNAs. RT-PCR was performed to detect specific transcripts. MicroRNA microarrays were performed with Ambion Bioarrays.
ESMVs from GFP-expressing ES cells contain exogenously expressed GFP and RNA. We were able to visualize GFP directly in the vesicles by fluorescence microscopy. SDS-PAGE analysis of the protein content in ESMVs revealed several discreet bands representing the most abundant proteins. We are currently identifying these major protein bands by mass spectroscopy. In addition to protein, ESMVs amazingly contain a copious supply of RNA. The total RNA yield from ESMV is 21.0±2.1 ng/cm2 of ES cells grown to 70% confluence collected over a 24 hour period. Interestingly, we were able to detect exogenously expressed GFP mRNA in the ESMVs in addition to endogenous transcripts such as the transcription factor, Oct4. Because ESMVs contained such an abundant supply of RNA, we hypothesized that they might be able to regulate gene expression in neighboring cells by transferring microRNAs. Thus we looked for a ubiquitously expressed microRNA, miR-16, by RT-PCR and confirmed its presence in ES cells and ESMVs. To profile the microRNAs in ESMVs, we performed microRNA array analysis. Our preliminary results suggest that certain microRNAs are preferentially packaged into ESMVs. Finally, using equilibrium density ultracentrifugation, we determined that the density of ESMVs is 1.18 g/cm3 to 1.29 g/cm3, consistent with vesicles containing mostly lipid and protein.
Our isolated ESMVs contain exogenously expressed GFP and RNA in addition to endogenous proteins and RNA. ESMVs may function as physiological liposomes and may be used to deliver proteins and RNA to cells in vivo. We discovered microRNAs in ESMVs and thus, one important role of ESMVs during development might be in intercellular gene expression regulation via the action of microRNAs.
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