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Comparative proteomic analysis of extracellular vesicles isolated from porcine adipose tissue-derived mesenchymal stem/stromal cells.

ABSTRACT: Extracellular vesicles (EVs) isolated from mesenchymal stem/stromal cells (MSCs) contribute to recovery of damaged tissue. We have previously shown that porcine MSC-derived EVs transport mRNA and miRNA capable of modulating cellular pathways in recipient cells. To identify candidate factors that contribute to the therapeutic effects of porcine MSC-derived EVs, we characterized their protein cargo using proteomics. Porcine MSCs were cultured from abdominal fat, and EVs characterized for expression of typical MSC and EV markers. LC-MS/MS proteomic analysis was performed and proteins classified. Functional pathway analysis was performed and five candidate proteins were validated by western blot. Proteomics analysis identified 5,469 distinct proteins in MSCs and 4,937 in EVs. The average protein expression was higher in MSCs vs. EVs. Differential expression analysis revealed 128 proteins that are selectively enriched in EVs versus MSCs, whereas 563 proteins were excluded from EVs. Proteins enriched in EVs are linked to a broad range of biological functions, including angiogenesis, blood coagulation, apoptosis, extracellular matrix remodeling, and regulation of inflammation. Excluded are mostly nuclear proteins, like proteins involved in nucleotide binding and RNA splicing. EVs have a selectively-enriched protein cargo with a specific biological signature that MSCs may employ for intercellular communication to facilitate tissue repair.

SUBMITTER: Eirin A

PROVIDER: S-EPMC5081562 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Comparative proteomic analysis of extracellular vesicles isolated from porcine adipose tissue-derived mesenchymal stem/stromal cells.

Eirin Alfonso A Zhu Xiang-Yang XY Puranik Amrutesh S AS Woollard John R JR Tang Hui H Dasari Surendra S Lerman Amir A van Wijnen Andre J AJ Lerman Lilach O LO

Scientific reports 20161027

Extracellular vesicles (EVs) isolated from mesenchymal stem/stromal cells (MSCs) contribute to recovery of damaged tissue. We have previously shown that porcine MSC-derived EVs transport mRNA and miRNA capable of modulating cellular pathways in recipient cells. To identify candidate factors that contribute to the therapeutic effects of porcine MSC-derived EVs, we characterized their protein cargo using proteomics. Porcine MSCs were cultured from abdominal fat, and EVs characterized for expressio ...[more]

PMID: 27786293

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Project description:Background: Extracellular vesicles (EVs) isolated from mesenchymal stem/stromal cells (MSCs) contribute to recovery of damaged tissue in animals models of human disease. We have previously shown that EVs isolated from porcine MSCs transport mRNA and miRNA capable of modulating several cellular pathways in recipient cells, yet their proteome remained to be profiled. Using a quantitative proteomic strategy, we sought to study the protein cargo of porcine MSC-derived EVs to identify candidate molecules for mediating their therapeutic effect. Methods: Autologous MSCs were collected from abdominal fat of 3 female domestic pigs, and MSC-derived EVs were subsequently isolated, cultured, and characterized by the expression of typical MSC and EV markers. LC-MS/MS proteomic analysis was performed and proteins classified using the Panther Classification System. Functional pathway analysis was performed with DAVID 6.7. Three candidate proteins were selected for validation and their expression in EVs and MSCs confirmed by Western blot. Results: Proteomics analysis identified 5,469 protein groups in MSCs and 4,937 in EVs. Average protein intensity was higher in MSCs compared to EVs (p<0.0001). Differential expression analysis revealed 128 proteins upregulated in EVs vs. MSCs (log2 fold change>10, p<0.05), whereas 563 proteins were excluded from EVs (log2 fold change<-10, p<0.05). Biological functional analysis of proteins enriched in EVs indicated a broad distribution, with the most frequently represented categories being proteins involved in angiogenesis, blood coagulation, apoptosis, extracellular matrix remodeling, and regulation of inflammatory responses. Proteins excluded from EVs were mostly nuclear proteins and proteins involved in nucleotide binding and RNA splicing. Conclusions: The present study provides novel proteomic characterization of the biological signatures of porcine adipose MSC-derived EVs. The selective cargo that EVs shuttle may define the spectrum of their roles in mediating MSC intercellular communication.

Project description:BackgroundEquine adipose-derived mesenchymal stromal cells (e-AdMSC) exhibit attractive proregenerative properties strongly related to the delivery of extracellular vesicles (EVs) that enclose different kinds of molecules including RNAs. In this study, we investigated small RNA content of EVs produced by e-AdMSC with the aim of speculating on their possible biological role.MethodsEVs were obtained by ultracentrifugation of the conditioned medium of e-AdMSC of 4 subjects. Transmission electron microscopy and scanning electron microscopy were performed to assess their size and nanostructure. RNA was isolated, enriched for small RNAs (<200?nt), and sequenced by Illumina technology. After bioinformatic analysis with state-of-the-art pipelines for short sequences, mapped reads were used to describe EV RNA cargo, reporting classes, and abundances. Enrichment analyses were performed to infer involved pathways and functional categories.ResultsElectron microscopy showed the presence of vesicles ranging in size from 30 to 300?nm and expressing typical markers. RNA analysis revealed that ribosomal RNA was the most abundant fraction, followed by small nucleolar RNAs (snoRNAs, 13.67%). Miscellaneous RNA (misc_RNA) reached 4.57% of the total where Y RNA, RNaseP, and vault RNA represented the main categories. miRNAs were sequenced at a lower level (3.51%) as well as protein-coding genes (1.33%). Pathway analyses on the protein-coding fraction revealed a significant enrichment for the "ribosome" pathway followed by "oxidative phosphorylation." Gene Ontology analysis showed enrichment for terms like "extracellular exosome," "organelle envelope," "RNA binding," and "small molecule metabolic process." The miRNA target pathway analysis revealed the presence of "signaling pathways regulating pluripotency of stem cells" coherent with the source of the samples.ConclusionWe herein demonstrated that e-AdMSC release EVs enclosing different subsets of small RNAs that potentially regulate a number of biological processes. These findings shed light on the role of EVs in the context of MSC biology.

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Comparative proteomic analysis of extracellular vesicles isolated from porcine adipose tissue-derived mesenchymal stem/stromal cells.

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Comparative proteomic analysis of extracellular vesicles isolated from porcine adipose tissue-derived mesenchymal stem/stromal cells.

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