This has been performed to determine that how Critical limb-threatening ischemia (CLTI) is the end stage of peripheral artery disease. CLTI presents a significant risk for lower extremity amputation, especially in diabetic patients. The MarrowStim PAD Kit for the Treatment of Critical Limb Ischemia in Subjects with Severe Peripheral Arterial Disease (MOBILE) trial demonstrated that diabetic patients were refractory to intramuscular injection of autologous bone marrow-derived mononuclear cells in preventing amputations. Allogeneic mesenchymal stromal cells (MSCs) isolated from young, healthy donors may be more effective. We used a novel diabetic mouse model of CLTI to assess intramuscular injection of human MSCs or three-dimensional cultures of MSCs (spheroids) in promoting limb perfusion and skeletal muscle regeneration and function. A polygenic diabetic mouse (TALLYHO/JngJ; Jackson Laboratory, Bar Harbor, Me) was used to create a CLTI model by ligation and excision of the femoral artery in one hindlimb.