Project Summary Ischemic cardiovascular diseases are the leading causes of morbidity and mortality in the USA. Despite advancement in therapeutics, treating patients with severe conditions are still far from optimal. Recently, cell therapy emerged as a promising option for those advanced cases for which no interventional or surgical therapy is able to effectively revascularize the ischemic areas. Human pluripotent stem cells (hPSCs), which include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), have emerged as a promising candidate for vascular regeneration as they have strong target cell differentiation capacity as well as paracrine effects. Thus, investigators have developed various protocols to differentiate hPSCs into endothelial cell (EC)-lineage cells. We have developed a fully defined, xenogeneic ingredient-free cell culture system that can generate purified functional endothelial cells (ECs) at high yield. We further demonstrated that these hPSC-derived ECs (hPSC-ECs) have robust and prolonged vessel-forming activities in vivo. However, one of the caveats of this approach is that their contribution is mainly restricted to the capillary level without pericytes. For optimal vascularization, more stable and larger vessels are also necessary. In previous cell therapy studies, this aspect was virtually unaddressed. Therefore, we recently generated human PSC-derived SMCs (hPSC-SMCs) by using a defined culture system as well and observed their contribution to vessel formation as vascular pericytes and SMCs. Another important barrier for cell therapy is short-term survival of the transplanted cells. To overcome this problem, we and others have investigated bioengineered cell therapy and demonstrated its effectiveness for cell survival and function. However, uneven and localized distribution of the injected cells emerged as another problem. Recently, we have developed a novel biodegradable hybrid copolymer consisting of gelatin and poly glycerol sebacate (PGS), which was further made into a microbead form with alginate. We refer to this co- polymer as AlGPM. This hybrid polymer is biodegradable and elicits minimal inflammatory responses. Moreover, its microbead form promotes wide and homogeneous distribution of encapsulated cells in vivo. Accordingly, in this study, we will address two unmet needs of the current cell therapy for ischemic vascular disease. First, we will use both hPSC-ECs and hPSC-SMCs to induce formation of not only bare capillaries but also pericyte-covered capillaries and SMC-covered arterioles. Second, we will develop a new biomaterial that can enhance cell survival and distribution in vivo to maximize stable vessel formation and therapeutic effects. Specifically, we will investigate whether a combination of these two cell types with AlGPM hydrogel microbeads is able to exert the optimal effects on vascular regeneration. The long-term goal of this study is to develop clinically applicab...