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Comparative proteomic analysis of extracellular vesicles isolated from porcine adipose tissue-derived mesenchymal
Alfonso Eirin1, Xiang-Yang Zhu1, Amrutesh S Puranik1
1Divisions of Nephrology and Hypertension, Mayo Clinic, Rochester, MN, USA.
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.
Insights
Mesenchymal stem cell extracellular vesicles (EVs) carry specific proteins that aid tissue repair. Proteomics revealed EVs are selectively enriched with factors promoting healing and intercellular communication.
Area of Science:
- Cell biology
- Regenerative medicine
- Proteomics
Background:
- Mesenchymal stem/stromal cells (MSCs) and their extracellular vesicles (EVs) promote tissue repair.
- Porcine MSC-derived EVs transport mRNA and miRNA, influencing recipient cell pathways.
- Understanding the protein cargo of EVs is crucial for elucidating their therapeutic mechanisms.
Purpose of the Study:
- To identify and characterize the protein cargo of porcine MSC-derived EVs.
- To determine candidate proteins contributing to the therapeutic effects of these EVs.
- To understand the functional implications of EV protein enrichment for tissue repair.
Main Methods:
- Porcine MSCs were cultured, and their derived EVs were isolated and characterized.
- Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used for proteomic analysis of MSCs and EVs.
- Differential expression analysis and functional pathway analysis were performed, with key proteins validated by Western blot.
Main Results:
- Proteomics identified 5,469 proteins in MSCs and 4,937 in EVs.
- 128 proteins were selectively enriched in EVs, while 563 were excluded.
- Enriched EV proteins are associated with angiogenesis, coagulation, apoptosis, extracellular matrix remodeling, and inflammation regulation.
Conclusions:
- Porcine MSC-derived EVs possess a selectively enriched protein cargo.
- This protein cargo has a distinct biological signature linked to tissue repair functions.
- EVs utilize this specific protein composition for intercellular communication to facilitate tissue regeneration.
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