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

Rheumatology and Immunology Medicine Immunology Life Sciences Orthopedics Cell Biology
CRISPR-dCas9 TSG-6 Mesenchymal Stem Cells Extracellular Vesicles Intervertebral Disc Cells Inflammatory Responses Genomic Engineering

Intervertebral Disc Degeneration (IVDD) is one of the leading causes of low back pain worldwide, significantly impacting patients’ quality of life. IVDD is often accompanied by inflammatory responses, leading to the degradation of the extracellular matrix (ECM) and structural damage. Although cell therapies, such as mesenchymal stem cells (MSCs), have been explored as novel treatments for IVDD, the microenvironment of degenerated intervertebral discs poses challenges to cell survival and engraftment. In recent years, extracellular vesicles (EVs) secreted by MSCs have garnered widespread attention due to their anti-inflammatory and tissue-repairing properties. However, the therapeutic efficacy of EVs is limited by donor variability, heterogeneity, and insufficient potency. Therefore, enhancing the therapeutic potential of EVs through genetic engineering has become a research focus.

The CRISPR-Cas9 gene-editing technology provides a powerful tool for regulating gene expression. By combining “dead” Cas9 (dCas9) with a transcriptional activation domain, researchers can activate the expression of specific genes without altering the DNA sequence. This study aims to activate the Tumor Necrosis Factor-Stimulated Gene 6 (TSG-6) in MSCs using CRISPR-dCas9 technology and evaluate the biological activity of EVs secreted by these modified MSCs on human intervertebral disc cells in vitro.

Source of the Study

This research was conducted by a team from Rochester Institute of Technology, Georgetown University School of Medicine, University of Rochester Medical Center, and other institutions. The primary authors include Iker Martinez-Zalbidea, Gabbie Wagner, and Karin Wuertz-Kozak. The paper was published online on February 5, 2025, in the journal Cellular and Molecular Bioengineering, titled “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.”

Research Process and Results

1. MSC Culture and CRISPR Activation

The researchers used an immortalized human adipose-derived MSC line (ASC52Telo) and activated the TSG-6 gene using the Synergistic Activation Mediator (SAM) CRISPR system. The specific steps included: - Cell Culture: ASC52Telo cells were cultured in basal medium supplemented with 10% fetal bovine serum at a density of 5000 cells/cm². - CRISPR Activation: Three different single-guide RNAs (sgRNAs) targeting the promoter region of TSG-6 were used. The dCas9-VP64 and MS2-p65-HSF1 transcriptional activation complexes were introduced into the cells via a lentiviral system. After antibiotic selection, TSG-6-activated MSCs and non-targeting controls (NTC) were obtained.

2. EV Isolation and Characterization

EVs were isolated from TSG-6-activated MSCs and non-targeting controls. The specific steps included: - EV Isolation: Conditioned medium was collected after 48 hours of cell culture, and EVs were isolated using ultracentrifugation. - EV Characterization: Nanoparticle Tracking Analysis (NTA) was used to measure the size distribution of EVs, Transmission Electron Microscopy (TEM) was employed to observe EV morphology, and an antibody array was used to detect EV markers (e.g., CD63, TSG101).

The results showed that the isolated EVs exhibited typical cup-shaped morphology, with particle sizes mainly distributed between 100-300 nm, and expressed multiple EV markers, indicating the successful isolation of high-quality EVs.

3. Impact of TSG-6 Activation on EV Cargo

Quantitative proteomics analysis was performed to compare the protein composition of EVs from TSG-6-activated MSCs and non-targeting controls. The results revealed significantly higher expression of TSG-6 protein in TSG-6-activated EVs compared to controls (log2 fold change = 5.72). Additionally, differential expression of 35 other proteins was identified, including cytokines, growth factors, and matrix metalloproteinase inhibitors.

4. Anti-Inflammatory Effects of EVs on Human Intervertebral Disc Cells

Degenerated intervertebral disc cells were isolated from patients undergoing spinal surgery, and inflammation was induced using IL-1β. TSG-6-activated EVs and non-targeting control EVs were co-treated with IL-1β to assess their effects on the expression of inflammatory markers (e.g., IL-8, COX-2).

The results showed that TSG-6-activated EVs significantly downregulated the expression of IL-8 and COX-2, demonstrating their anti-inflammatory effects. However, compared to control EVs, TSG-6-activated EVs did not exhibit a significant advantage in anti-inflammatory efficacy.

Conclusions and Significance

This study successfully activated the TSG-6 gene in MSCs using CRISPR-dCas9 technology and demonstrated the anti-inflammatory potential of EVs secreted by these modified MSCs. Although TSG-6-activated EVs did not significantly outperform control EVs in anti-inflammatory efficacy, this research provides important experimental evidence for enhancing the therapeutic potential of EVs through genetic engineering. Future studies could explore the simultaneous activation of multiple genes or optimize EV production processes to improve their therapeutic effects.

Research Highlights

  1. Innovative Genetic Engineering Approach: The first application of CRISPR-dCas9 technology for gene regulation in MSC-derived EVs, providing a new research direction for EV-based therapies.
  2. EV Characterization and Functional Validation: Comprehensive characterization of the physical and biological properties of EVs through multiple experimental methods, along with validation of their anti-inflammatory effects.
  3. Clinical Application Potential: This study offers a potential gene-engineered EV therapy for inflammatory diseases such as IVDD.

Additional Valuable Information

This study also revealed the broad impact of TSG-6 activation on EV cargo through proteomics analysis, providing new insights into the functional mechanisms of EVs. Additionally, the research team plans to further explore the potential applications of EVs in immunomodulation and tissue repair in the future.

Through this study, we not only see the immense potential of genetic engineering in EV-based therapies but also gain important experimental foundations for future clinical applications. It is hoped that research in this field will bring breakthroughs in the treatment of more inflammatory and degenerative diseases.