Intravenous transplantation of human muse cells improves blood perfusion in a mouse model of limb ischemia

Abstract

Abstract Introduction Critical limb ischemia (CLI) is the end-stage of peripheral artery disease caused by atherosclerosis and inflammation. Despite advances in treatment of CLI, substantial number of patients with CLI suffer from severe pain, ulceration, and gangrene. Although the advent of stem cell-based therapy has opened a new avenue for the treatment of various diseases, accumulating evidence suggests current cell-based therapies are safe, but their efficacy is modest in CLI patients. Multilineage-differentiating stress enduring (Muse) cells were first reported by one of the authors and colleagues as endogenous pluripotent-like stem cells residing in the bone marrow, peripheral blood and connective tissue of many organs. Previous studies have demonstrated that systemic administration of exogenous Muse cells improves functional recovery after ischemic organ injury such as myocardial infarction. However, their therapeutic potential in CLI remains unclear. Objective The goal of this study is to elucidate the efficacy and safety of exogenous Muse cell transplantation with animal models of CLI. Methods Muse cells were isolated from human bone marrow-derived mesenchymal stem cells (MSCs) by fluorescence-activated cell sorting with anti-SSEA-3 antibody. To establish an animal model of CLI, 12–14-week-old male BALB/c mice were anesthetized and subjected to left femoral artery and vein resection. At 24 hours after establishment of hindlimb ischemia, mice were randomly divided into 5 groups and treated as follows: Group 1, intravenous injection of PBS as control group; group 2, intravenous injection of 3 × 104 SSEA3-negative MSCs (non-Muse MSCs); group 3, intravenous injection of 3 × 104 Muse cells; group 4, intramuscular injection of 3 × 104 Muse cells into ischemic limb; group 5, intramuscular injection of 2 × 105 mouse bone marrow-derived mononuclear cells (BM-MNCs) into ischemic limb (n=5 for each group). Hindlimb blood flow was evaluated by laser Doppler flowmetry for 14 days and expressed as the ratio of ischemic to non-ischemic hindlimb. Results We found that intramuscular injection of xenogeneic Muse cells without immunosuppression significantly improved the blood flow in the ischemic hindlimb compared to control at day 7 and 14 (0.67 vs. 0.52, p=0.0002 and 0.74 vs. 0.53, p<0.0001, respectively) similar to intramuscular injection of allogenic BM-MNCs. In addition, we also found that intravenous injection of xenogeneic Muse cells without immunosuppression significantly improved the blood flow in the ischemic hindlimb compared to control at day 7 and 14 (0.72, p<0.0001 and 0.67, p=0.004, respectively). In contrast, intravenous injection of xenogeneic non-Muse MSCs did not improve blood flow in the ischemic hindlimb. Conclusion Our result suggests that Muse cell-based regenerative therapy could be a novel, less invasive, cost-effective, and subsequently more effective treatment for CLI patients. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Grant-in-Aid for Scientific Research (KAKENHI)