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A. Yuan, E. L. Farber, A. Rapoport, C. Yamashita, N. B. Akhmedov, D. B. Farber; Embyonic Stem Cell Microvesicles (ESMVs): Signaling in the Niche and Therapeutic Uses. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1730.
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© ARVO (1962-2015); The Authors (2016-present)
To characterize the RNA and protein content of ESMVs and determine whether they can be engineered to transfer exogenously expressed proteins and RNA from cell to cell.
ES cells (ESCs) expressing GFP were grown without feeders in serum free media. ESMVs were collected by differential ultracentrifugation, their RNA profiles obtained on an Agilent 2100 bioanalyzer and their total protein resolved by SDS-PAGE. Individual mRNA levels were determined by qRT-PCR and mRNA integrity was established by comparing the 5’/3’ amplicon ratios of transcripts in ESMVs to those in ESCs. Transfer of GFP to other ESCs was detected by confocal microscopy after incubation of ESMVs expressing GFP with ESCs without GFP. Transfer of ESC-specific miRNAs to mouse embryonic fibroblasts (MEFs) was detected by qRT-PCR after incubation of MEFs with ESMVs.
ESMVs' density, obtained by equilibrium density ultracentrifugation, is consistent with vesicles containing mostly lipid and protein. In addition to endogenous mRNA and proteins expressed in ESCs, we identified in ESMVs mRNA and protein expressed from a GFP transgene. The GFP mRNA levels were comparable to those of endogenous transcripts found in ESMVs. The 5’/3’ amplicon ratios for mRNAs isolated from ESMVs and ESCs were not significantly different, indicating that transcripts found inside ESMVs are protected from degradation. We also found miRNAs in ESMVs with different relative abundance to that in ESCs. Using confocal microscopy, we demonstrated the fusion and transfer of GFP by ESMVs to ESCs as early as 3 h after incubation. We also showed by qRT-PCR the transfer of miRNAs by ESMVs to growth-arrested MEFs.
ESMVs contain exogenously expressed GFP mRNA and protein in addition to endogenous proteins, mRNA, and miRNAs. Interestingly, miRNAs and GFP can be transferred to MEFs and ESCs by ESMVs. ESMVs may be important for intercellular signaling in stem cell niches and useful as therapeutic tools for transferring mRNA, protein, and miRNA to the eye without the use of viral vectors. Furthermore, ESMVs may be a basic method of intercellular communication within local tissue environments of the eye.
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