Therapeutic ability of extracellular vesicles derived from angio-miRNA-modified mesenchymal stromal cells in ischemic animal models

BackgroundRegenerative therapy using extracellular vesicles (EVs) is expected to be an option for supportive therapy of chronic limb-threatening ischemia. In this study, we examined the angiogenic potential of EVs derived from genetically modified MSCs, focusing on the angio-miRNAs in these vesicles. MethodsBone marrow mesenchymal stromal cells (BM-MSCs) were transfected with a lentiviral vector containing specific angio-miRNAs (miRNA-126, -135b, or -210). EVs were separated from the culture medium of BM-MSCs via ultracentrifugation. The quality and quantity of the EVs were evaluated using nanoparticle tracking analysis, fluorometry, and an ExoScreen assay. Tube formation assays were conducted using human umbilical vein endothelial cells to assess the angiogenic potency of the EVs in vitro. The EVs were injected into the ischemic hind limb muscles of mice. The grades of limb ischemia, blood perfusion, and muscle tissues were analyzed to assess the angiogenic potency of the EVs in vivo. ResultsAngio-miRNAs were overexpressed in MSCs transfected with a lentiviral vector. Tube formation was significantly enhanced when endothelial cells were cultured with the EVs (P < 0.05). Compared to non-contact co-culture with BM-MSCs, EVs derived from modified BM-MSCs significantly promoted tube formation in endothelial cells (P < 0.05). The severity of limb ischemia on day 7 was alleviated when a combination of two or three EVs derived from modified BM-MSCs was injected compared with native EVs. Toe blood perfusion on day 14 significantly recovered when a combination of two or three EVs derived from modified BM-MSCs was injected (P < 0.05). Tissue analysis revealed that the number of newly formed endothelial cells in ischemic muscles increased when a combination of two or three EVs derived from modified BM-MSCs was injected. ConclusionsThe use of a combination of EVs derived from genetically modified MSCs stimulated angiogenesis both in vitro and in vivo.