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TSG-6 Activated MSC-derived Extracellular Vesicles Present Altered micro-RNA Contents and Ameliorate the Inflammatory
Iker Martinez-Zalbidea1, Alyssa Rzasa1, Varun Puvanesarajah2
1Department of Biomedical Engineering, Rochester Institute of Technology (RIT), Rochester, NY, USA.
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.
Insights
CRISPR activation of TSG-6 in mesenchymal stem cells (MSCs) enhances their derived extracellular vesicles (EVs). These modified MSC-EVs exhibit boosted anti-inflammatory effects, improving their therapeutic potential for immunotherapy.
Area of Science:
- Biotechnology
- Immunology
- Regenerative Medicine
Background:
- Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) possess immunomodulatory capabilities.
- Enhancing the therapeutic efficacy of MSC-EVs remains a key challenge in their clinical application.
Purpose of the Study:
- To investigate the impact of CRISPR-mediated TSG-6 gene activation in MSCs on EV cargo and immunomodulatory function.
- To assess the potential of TSG-6 overexpressing MSC-EVs in modulating macrophage inflammatory responses.
Main Methods:
- CRISPR activation was used to increase TSG-6 expression in MSCs, confirmed by RT-qPCR.
- MSC-EVs were isolated and characterized using nanoparticle tracking analysis, TEM, and Western blot.
- Small RNA sequencing identified differentially expressed miRNAs in TSG-6-overexpressing MSC-EVs.
- In vitro assays evaluated the effect of these EVs on LPS-stimulated macrophage cytokine expression.
Main Results:
- CRISPR activation led to over an 1800-fold increase in TSG-6 mRNA in MSCs.
- TSG-6-overexpressing MSC-EVs showed altered miRNA cargo compared to control EVs.
- Treatment with TSG-6-overexpressing MSC-EVs significantly reduced pro-inflammatory cytokine gene expression and secretion in macrophages.
- A dose-dependent effect was observed for the anti-inflammatory impact of these EVs.
Conclusions:
- CRISPR-based TSG-6 activation effectively reprograms MSC-EV miRNA cargo.
- Elevated TSG-6 expression in MSCs enhances the anti-inflammatory properties of their derived EVs.
- This strategy presents a promising platform for augmenting MSC-EV bioactivity and immunotherapeutic efficacy.
