Human induced pluripotent stem cell-derived nanovesicles for the treatment of ischemic limb disease

Critical limb ischemia is an advanced stage of peripheral artery disease, characterized by claudication, ischemic pain, and ulceration. It is a severe condition associated with an increased risk of limb amputation and mortality. Although extracellular vehicles (EVs) secreted by endothelial cells (ECs) or mesenchymal stem cells (MSCs) have shown promise for the treatment of ischemic limb diseases in mice, clinical translation has been limited by the low EV yields from cultured cells. In this study, a hypo-immunogenic human induced pluripotent stem cell (hiPSC) line with β2-microglobulin knockout (^B2MKOhiPSC) was used to manufacture nanovesicles (^B2MKOhiPSCNVs), which are hypo-immunogenic as compared to wild type hiPSC manufactured nanovesicles (^WThiPSCNVs). Notably, over 9500 NVs could be produced from a single hiPSC. The zeta potential of hiPSCNVs was -16.7 mV, as measured by the ZETASIZER Nano series instrument. The NVs exhibited a mean diameter of 115.9 ± 43.5 nm, as determined by Nanosight analysis, and displayed bilayer lipid membranes under transmission electron microscopy. In vitro, both ^WThiPSCNVs and ^B2MKOhiPSCNVs protected human umbilical vein endothelial cells (HUVECs) from hypoxic injury, promoted HUVEC proliferation, and did not induce hemolysis. In vivo,^B2MKOhiPSCNVs administration significantly improved blood perfusion in ischemic limbs 14 days post-treatment. This was accompanied by a notable increase in mouse endothelial cell (EC) proliferation and neovascularization compared to C57BL mice treated with either a saline injection or ^WThiPSCNVs, without the use of immunosuppressive drugs. Furthermore, intramuscular injections of ^WThiPSCNVs did not cause adverse effects on liver or kidney function. These findings suggest that ^B2MKOhiPSC- NVs represent a promising allogeneic therapeutic approach for the treatment of ischemic limb diseases. STATEMENT OF SIGNIFICANCE: The use of hiPSCs as parental cells can easily scale-up NV production. NVs derived from β2 microglobulin-knockout hiPSCs (^B2MKOhiPSCNVs) protect endothelial cells from hypoxic injury and enhance their proliferation. In immunocompetent mice, administration of ^B2MKOhiPSCNVs significantly enhanced blood perfusion and promoted neovascularization in ischemic limbs.