Project Summary Peripheral arterial disease (PAD) afflicts approximately 15% of the U.S. population over 55 years of age. Initially characterized by intermittent claudication, progressive PAD results in unrelenting rest pain and ulceration, a condition referred to as critical limb ischemia (CLI). Despite advances in percutaneous intervention or surgical treatment, up to half of these CLI patients are amputated within one year. Pathophysiologically, alleviation of ischemia through direct vessel formation would be an optimal treatment. While cell therapy with adult stem or progenitor cells has been investigated as a new revascularization therapy for PAD, clinical trials showed no or minimal effects. Meanwhile, human induced pluripotent stem cells (hiPSCs) were discovered and showed almost similar properties to human embryonic stem cells (hESCs). Because of genuine differentiation capacity to target cells, hiPSCs have emerged as a promising therapeutic option for cell therapy. We and others developed a protocol to generate hiPSCs with non-integrating episomal vectors from a small amount of peripheral blood. We further developed a system to differentiate hiPSCs into endothelial cells (hiPSC-ECs) in a clinically compatible manner and demonstrated their long-term vessel-forming effects and therapeutic effects on animal models. The ultimate goal of this project is to develop hiPSC-ECs as a therapeutic agent for treating severe peripheral artery disease. Specifically, in this proposal, we will generate hiPSCs from peripheral blood of PAD patients and normal volunteers, differentiate them into hiPSC-ECs using our established procedure. We will then determine the identity and potency of these hiPSC- ECs using both in vitro and in vivo assays, particularly focusing on whether there is similar potency between the patient-derived and normal-volunteer-derived hiPSC-ECs. If shown similarity, a safer autologous approach will be developed for this disease. Despite its incredible therapeutic potential, cell therapy with hiPSC-ECs was not published or proposed worldwide to date. Our team of investigators have all the necessary expertise and experience to move our discovery into a future clinical trial. This grant will bridge the utility of hiPSCs from bench to bedside, which, we believe, will be the most significant source of cell therapy in the near future. Moreover, this work is a first step for therapeutic application of hiPSC- EC. There are many potential diseases that need a vascular supply such as ischemic heart disease, stroke, diabetic retinopathy, diabetic neuropathy, and wound healing, which will be all candidates that benefit from this development. Thus, the impact of this study will be huge.