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

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-1ß, CCL2, CXCL10, and TNF-a) and secreted protein levels (CCL2, TNF-a, CXCL1, and MIP-3a) 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. Overall design: RNA-seq profiling of Extracellular Vesicles (EVs) obtained from 3 different populations of mesenchymal stem cells. These populations are: Wild-Type, Non-Target controls (a negative control), and TSG-6 activated (CRISPR activation of gene TNFAIP6/TSG-6). Samples are in triplicate, with miR samples labeled as follows: Non-Targets labeled: “NA1_NA2_NA3” “NB1_NB2_NB3” “NTA_EV_RNA_NTB_EV_RNA_NTC”; TSG-6 activated labeled: “TA1_TA2_TA3” “TB1_TB2_TB3” “TSGA_EV_RNA_TSGB_EV_RNA_TSGC_EV_RNA”; Wild type labeled:“WA1_WA2_WA3” “WB1_WB2_WB3” “WTA_EV_RNA_WTB_EV_RNA_WTC_EV_RNA”.