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Purpose <p>The purpose of this study was to boost the therapeutic effect of mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) by overexpressing the gene TSG-6 through CRISPR activation, and assess the biological activity of EVs from these modified MSCs <i>in vitro</i> on human intervertebral disc (IVD) cells.</p> Methods <p>An immortalized human MSC line was transduced with a CRISPR activation lentivirus system targeting TSG-6. MSC-EVs were harvested by ultracentrifugation and particle number/size distribution was determined by nanoparticle tracking analysis. The efficiency of transduction activation was assessed by analyzing gene and protein expression. EV proteomic contents were analyzed by mass spectrometry. Human IVD cells from patients undergoing spinal surgery were isolated, expanded, exposed to IL-1β pre-stimulation and co-treated with MSC-EVs.</p> Results <p>MSC-EVs presented size distribution, morphology, and molecular markers consistent with common EV characteristics. The expression level of TSG-6 was significantly higher (&gt; 800 fold) in transduced MSCs relative to controls. Protein analysis of MSCs and EVs showed higher protein expression of TSG-6 in CRISPR activated samples than controls. Proteomics of EVs identified 35 proteins (including TSG-6) that were differentially expressed in TSG-6 activated EVs vs control EVs. EV co-Treatment of IL-1β pre-Stimulated IVD cells resulted in a significant downregulation of IL-8 and COX-2.</p> Conclusions <p>We successfully generated an MSC line overexpressing TSG-6. Furthermore, we show that EVs isolated from these modified MSCs have the potential to attenuate the pro-inflammatory gene expression in IVD cells. This genomic engineering approach hence holds promise for boosting the therapeutic effects of EVs.</p>

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CRISPR-dCas9 Activation of TSG-6 in MSCs Modulates the Cargo of MSC-Derived Extracellular Vesicles and Attenuates Inflammatory Responses in Human Intervertebral Disc Cells In Vitro

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年2月5日

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  • Iker Martinez-Zalbidea,
  • Gabbie Wagner,
  • Nea Bergendahl,
  • Addisu Mesfin,
  • Varun Puvanesarajah,
  • Wolfgang Hitzl,
  • Stefan Schulze,
  • Karin Wuertz-Kozak

摘要

Purpose

The purpose of this study was to boost the therapeutic effect of mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) by overexpressing the gene TSG-6 through CRISPR activation, and assess the biological activity of EVs from these modified MSCs in vitro on human intervertebral disc (IVD) cells.

Methods

An immortalized human MSC line was transduced with a CRISPR activation lentivirus system targeting TSG-6. MSC-EVs were harvested by ultracentrifugation and particle number/size distribution was determined by nanoparticle tracking analysis. The efficiency of transduction activation was assessed by analyzing gene and protein expression. EV proteomic contents were analyzed by mass spectrometry. Human IVD cells from patients undergoing spinal surgery were isolated, expanded, exposed to IL-1β pre-stimulation and co-treated with MSC-EVs.

Results

MSC-EVs presented size distribution, morphology, and molecular markers consistent with common EV characteristics. The expression level of TSG-6 was significantly higher (> 800 fold) in transduced MSCs relative to controls. Protein analysis of MSCs and EVs showed higher protein expression of TSG-6 in CRISPR activated samples than controls. Proteomics of EVs identified 35 proteins (including TSG-6) that were differentially expressed in TSG-6 activated EVs vs control EVs. EV co-Treatment of IL-1β pre-Stimulated IVD cells resulted in a significant downregulation of IL-8 and COX-2.

Conclusions

We successfully generated an MSC line overexpressing TSG-6. Furthermore, we show that EVs isolated from these modified MSCs have the potential to attenuate the pro-inflammatory gene expression in IVD cells. This genomic engineering approach hence holds promise for boosting the therapeutic effects of EVs.