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. 2014 Nov 1;551(1):55-64.
doi: 10.1016/j.gene.2014.08.041. Epub 2014 Aug 23.

MicroRNA and mRNA cargo of extracellular vesicles from porcine adipose tissue-derived mesenchymal stem cells

Alfonso Eirin  1 , Scott M Riester  2 , Xiang-Yang Zhu  1 , Hui Tang  1 , Jared M Evans  3 , Daniel O'Brien  3 , Andre J van Wijnen  2 , Lilach O Lerman  4
Affiliations

MicroRNA and mRNA cargo of extracellular vesicles from porcine adipose tissue-derived mesenchymal stem cells

Alfonso Eirin et al. Gene. .

Abstract

Mesenchymal stromal/stem cells (MSCs) are clinically useful for cell-based therapy, but concerns regarding their ability to replicate limit their human application. MSCs release extracellular vesicles (EVs) that mediate at least in part the paracrine effects of the parental cells. To understand the molecular basis of their biological properties, we characterized the RNA cargo of EVs from porcine adipose-tissue derived MSCs. Comprehensive characterization of mRNA and miRNA gene expression using high-throughput RNA sequencing (RNA-seq) revealed that EVs are selectively enriched for distinct classes of RNAs. For example, EVs preferentially express mRNA for transcription factors (e.g. MDFIC, POU3F1, NRIP1) and genes involved in angiogenesis (e.g. HGF, HES1, TCF4) and adipogenesis (e.g. CEBPA, KLF7). EVs also express Golgi apparatus genes (ARRB1, GOLGA4) and genes involved in TGF-β signaling. In contrast, mitochondrial, calcium signaling, and cytoskeleton genes are selectively excluded from EVs, possibly because these genes remain sequestered in organelles or intracellular compartments. RNA-seq generated reads for at least 386 annotated miRNAs, but only miR148a, miR532-5p, miR378, and let-7f were enriched in EVs compared to MSCs. Gene ontology analysis indicates that these miRNAs target transcription factors and genes that participate in several cellular pathways, including angiogenesis, cellular transport, apoptosis, and proteolysis. Our data suggest that EVs transport gene regulatory information to modulate angiogenesis, adipogenesis, and other cell pathways in recipient cells. These observations may contribute to development of regenerative strategies using EVs to overcome potential complications of cell-based therapy.

Keywords: Exosomes; Extracellular vesicles; Gene expression; Mesenchymal stem cells; Microvesicles; Next generation sequencing (NGS); RNASeq; miRNA.

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Figures

Figure 1
Figure 1
A: MSCs displayed a fibroblast-like, spindle-shaped morphology, expressed MSC markers, and transdifferentiated into osteocytes, chondrocytes, and adipocytes. B: Transmission (left) and scanning (right) electron microscopy demonstrated that cultured MSC release substantial amounts of EVs. C: EVs expressed microvesicle, exosome, and MSC markers.
Figure 2
Figure 2
A: The 100 most highly expressed genes in MSCs and EVs account for 46% of all mapped transcripts. B: mRNAs expressed at >100 RPKM encode mostly proteins involved in translation. C: 30.2% of all annotated genes are expressed at levels >1 RPKM. D: Pie chart of data in panel C, expression (RPKM) of the 100 most abundant genes relative to other genes, and differential expression of genes in MSCs and EVs.
Figure 3
Figure 3
Only 3.5% of genes expressed at levels greater than 1 RPKM changed by more than 4-fold when comparing MSCs and EVs, while a smaller subset showed a >10-fold-change in mRNA expression. B: mRNA RPKM (top) and fold-change (bottom) showing that EVs preferentially expressed transcription factors, Golgi apparatus genes, and genes involved in TGF-β signaling.
Figure 4
Figure 4
mRNA RPKM (left) and fold-change (right) showing that mitochondrial (A), calcium signaling (B), and cytoskeleton (C) genes were selectively depleted from EVs.
Figure 5
Figure 5
A: Only 4 miRs were enriched in EVs. B: miR148a, miR532-5p, miR378, and let-7f total reads in MSCs and EVs.
See this image and copyright information in PMC

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