Martinez-Zalbidea Iker, Wagner Gabbie, Bergendahl Nea, Mesfin Addisu, Puvanesarajah Varun, Hitzl Wolfgang, Schulze Stefan, Wuertz-Kozak Karin
Department of Biomedical Engineering, Rochester Institute of Technology (RIT), Rochester, NY USA.
Medstar Orthopaedic Institute, Georgetown University School of Medicine Washington, Washington, DC USA.
Cell Mol Bioeng. 2025 Feb 5;18(1):83-98. doi: 10.1007/s12195-025-00843-4. eCollection 2025 Feb.
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 on human intervertebral disc (IVD) cells.
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.
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.
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.
The online version contains supplementary material available at 10.1007/s12195-025-00843-4.
本研究旨在通过CRISPR激活过表达TSG-6基因来提高间充质干细胞（MSC）衍生的细胞外囊泡（EVs）的治疗效果，并评估这些修饰的间充质干细胞来源的细胞外囊泡对人椎间盘（IVD）细胞的生物学活性。
用靶向TSG-6的CRISPR激活慢病毒系统转导永生化人MSC系。通过超速离心收获MSC-EVs，并通过纳米颗粒跟踪分析确定颗粒数量/大小分布。通过分析基因和蛋白质表达来评估转导激活效率。通过质谱分析EV蛋白质组含量。从接受脊柱手术的患者中分离出人IVD细胞，进行扩增，用IL-1β预刺激并与MSC-EVs共同处理。
MSC-EVs呈现出与常见EV特征一致的大小分布、形态和分子标志物。相对于对照，转导的间充质干细胞中TSG-6的表达水平显著更高（>800倍）。对间充质干细胞和细胞外囊泡的蛋白质分析表明，CRISPR激活样品中TSG-6的蛋白质表达高于对照。细胞外囊泡的蛋白质组学鉴定出35种蛋白质（包括TSG-6）在TSG-6激活的细胞外囊泡与对照细胞外囊泡中差异表达。细胞外囊泡与IL-1β预刺激的IVD细胞共同处理导致IL-8和COX-2显著下调。
我们成功构建了过表达TSG-6的间充质干细胞系。此外，我们表明从这些修饰的间充质干细胞中分离的细胞外囊泡具有减弱IVD细胞中促炎基因表达的潜力。因此，这种基因组工程方法有望提高细胞外囊泡的治疗效果。
在线版本包含可在10.1007/s12195-025-00843-4获取的补充材料。
