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TSG-6 activated MSC-derived Extracellular Vesicles present altered micro-RNA contents and ameliorate the inflammatory phenotype of macrophages in vitro

ABSTRACT: Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have shown promising immunomodulatory properties; however, strategies to enhance their therapeutic potential remain limited. Here, we employed CRISPR activation of the gene TSG-6 in MSCs to evaluate the impact of elevated TSG-6 on micro-RNA (miRNAs) EV cargo and immunomodulatory function in an in vitro macrophage model. CRISPR-mediated gene activation was confirmed by RT-qPCR, demonstrating more than an 1800-fold increase in TSG-6 mRNA compared to controls. EVs were isolated from TSG-6 overexpressing MSCs and thoroughly characterized by nanoparticle tracking analysis, transmission electron microscopy, and Western blot, confirming their typical size distribution, morphology, and surface markers. Small RNA sequencing of these EVs revealed 15 differentially expressed miRNAs relative to EVs from control MSCs. When THP-1–derived macrophages were stimulated with LPS and treated with TSG-6-overexpressing MSC-EVs, a marked reduction in pro-inflammatory cytokine gene expression (IL-1β, CCL2, CXCL10, and TNF-α) and secreted protein levels (CCL2, TNF-α, CXCL1, and MIP-3α) was observed. Taken together, these findings demonstrate that CRISPR-based TSG-6 activation reprograms MSC-EV miRNA cargo (as well as their protein cargo, as previously shown), which can boost their anti-inflammatory effects. These findings underscore the promise of CRISPR-activation as a novel platform for boosting the bioactive properties of MSC-EVs and enhancing immunotherapeutic efficacy.

ORGANISM(S): Homo sapiens

PROVIDER: GSE294611 | GEO | 2025/04/18

REPOSITORIES: GEO

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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.

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TSG-6 activated MSC-derived Extracellular Vesicles present altered micro-RNA contents and ameliorate the inflammatory phenotype of macrophages in vitro

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