Characterization and Immunomodulatory Properties of Extracellular Vesicles Isolated from Bone Marrow-Derived Mesenchymal Stem Cells

Introduction: The bioactive properties of mesenchymal stem cells (MSCs) may be in part due to production of extracellular vesicles (EVs). These EVs decrease intraarticular inflammation, making them a candidate for treatment of osteoarthritis (OA). Our objective was to characterize EVs isolated from equine bone marrow-derived MSCs (BM-MSCs) and investigate their immunomodulatory properties. We hypothesized that EVs from BM-MSCs conditioned with interferon-gamma (IFN-γ) would have superior immunomodulatory properties.

Materials and Methods: BM-MSCs were cultured and conditioned with or without IFN-γ. The supernatant was collected for isolation of EVs using ultracentrifugation. Nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) characterized their size, concentration, and shape. Western blot determined if EV markers were present. T cell proliferation assay with BM-MSCs or EVs was performed. Statistical analysis was performed with a mixed-effect model (p ˂0.05).

Results: EVs were visible on TEM as small (<500 nm) membrane-enclosed vesicles. The mean (± SEM) concentration of EV samples derived from IFN-γ preconditioned BM-MSCs (6.75 × 1010 ± 3.06 × 1010) was higher than samples from unconditioned cells (6.04 × 1,010 ± 3.85 × 1,010), however, this difference was not significant. Western blot confirmed the presence of EV markers. T-cell proliferation was suppressed by co-culture with BM-MSCs, however, EVs, did not exhibit a similar suppressive effect.

Discussion/Conclusion: Isolation of EVs via ultracentrifugation was simple and practical. Future Results will determine if the immunomodulatory properties of EVs would make them beneficial for the treatment of OA. EVs could be a standardized off-the- shelf biotherapeutic, with potential for decreased immunogenicity compared with MSCs.

Acknowledgements: This study was funded by the Raymond Firestone & Raker Tulleners Fund. The authors have no conflict of interest.

Publication History

Article published online:
26 October 2022