Project Summary Ischemic cardiovascular diseases are the leading cause of morbidity and mortality. Underlying pathophysiology of these diseases is associated with loss or dysfunction of blood vessels and/or impaired new vessel formation (neovascularization). While cell therapy has emerged as a promising option to form blood vessels, the effects of adult stem cells are uncertain and embryonic or induced pluripotent stem cells (ESCs/iPSCs) are potentially tumorigenic. To avoid these problems, a new approach has been developed using lineage- or cell-type specific transcription factors (TFs) for direct conversion or reprogramming of somatic cells into other lineage cells. We have attempted this direct reprogramming toward endothelial cells (ECs) using combinations of seven TFs and found for the first time that ETV2, alone, is sufficient to convert human fibroblasts into ECs. However, since we used a lentiviral vector, these reprogrammed ECs (rECs) have limited clinical applicability. The direct reprogramming approach allows two therapeutic strategies: cell-based therapy or direct in vivo reprogramming. For clinical application, both approaches require a safer delivery vector to minimize the possibility of genomic integration. Thus, we developed an adenoviral-ETV2 (Ad-ETV2) vector and generated rECs (Adeno-rECs). Another important barrier for cell therapy is short-term survival of the transplanted cells. To overcome this problem, we have been investigating bioengineered cell therapy. In this study, first, we will generate an optimal construct combining these Adeno-rECs with novel biomaterials. 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 response. Furthermore, its microbead form promotes wide distribution of encapsulated cells after injection. The composition of AlGPM will be optimized to promote cell survival and maximize function of rECs. We will then determine the neovascularization and therapeutic effects of the selected AlGPM microbeads encapsulating rECs using ischemic animal models. Second, we will determine whether local injection of viral particles of ETV2 into animal models can directly reprogram somatic cells into endothelial cells and promote vascular regeneration and tissue repair in vivo. Moreover, by using various transgenic mice, we will genetically track the fate of somatic cells toward ECs in vivo. Together, the goal of this project is to develop clinically applicable vascular regenerative therapy using direct reprogramming approaches and bioengineering technologies.