Enhancing Therapeutic Efficacy and Reducing Cell Dosage in Stem Cell Transplantation Therapy for Ischemic Limb Diseases by Modifying the Cell Injection Site

In conventional stem cell transplantation therapies for ischemic limb diseases, stem cells are generally transplanted into the ischemic region (IR), and most of the transplanted cells undergo hypoxia-mediated cell death. Due to massive cell death, the therapeutic efficacy is reduced and a high dose of stem cells is necessitated for the therapies. In this study, we investigated whether the therapeutic efficacy can be improved and the cell dosage can be reduced in the therapy for limb ischemia simply by modifying the stem cell injection site to a site where cell engraftment is improved and blood vessel sprouting is efficiently stimulated. Human mesenchymal stem cells (hMSCs) cultured under hypoxic condition, which simulates cells transplanted to IR, underwent extensive cell death in vitro. Importantly, cell death was significantly attenuated when hMSCs adhered first under normoxic condition for 24 h and then were exposed to hypoxic condition, which simulates cells transplanted to the border zone (BZ) in the upper thigh and migrated to IR. hMSCs, at doses of 2 × 10⁵ or 2 × 10⁶ cells, were injected into the IR or BZ of 5-week-old female athymic mice after ischemic hindlimb induction. Compared with human mesenchymal stem cell (hMSC) transplantation to the IR of mouse ischemic limbs, transplantation to the BZ significantly enhanced cell engraftment and paracrine factor secretion, which effectively stimulated vessel sprouting, enhanced blood perfusion in IR, and enabled the cell dosage reduction. Therefore, modification of the stem cell transplantation site would improve the current stem cell therapies for ischemic limb diseases in terms of cell dosage reduction and therapeutic efficacy enhancement.