# TSG-6 Activated MSC-derived Extracellular Vesicles Present Altered micro-RNA Contents and Ameliorate the Inflammatory Phenotype of Macrophages in Vitro

## TL;DR

This study shows that increasing TSG-6 in MSC-derived EVs using CRISPR improves their ability to reduce inflammation in macrophages.

## Contribution

CRISPR-based TSG-6 activation is introduced as a novel method to enhance the anti-inflammatory properties of MSC-EVs.

## Key findings

- CRISPR activation of TSG-6 in MSCs led to an 1800-fold increase in TSG-6 mRNA.
- TSG-6-overexpressing MSC-EVs reduced pro-inflammatory cytokine gene expression and secreted protein levels in macrophages.
- EVs from TSG-6-overexpressing MSCs showed 15 differentially expressed miRNAs compared to control EVs.

## 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 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 (Standard dosage: 1000 particle/cell, n = 11; Alternative dosages: 500, 1000, or 2000 particles/cell, n = 6), 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.

The online version contains supplementary material available at 10.1007/s10753-025-02398-y.