Project Abstract Vasculature is an efficient delivery system for oxygen and nutrients to organs and is essential for organ function. Therefore, vascularization is a critical consideration for regenerative medicine and tissue engineering, since oxygen and nutrient diffusion significantly limits the size of tissues grown in vitro, and perfusion is critical for in vivo engraftment and survival of engineered tissues. Many approaches have been used to generate engineered tissue with a vascular network with or without vascular flow; however, one of the major caveats to these advances is that they often use endothelial cell (EC) lines such as human umbilical vein endothelial cells (HUVECs). While the use of cell lines is ideal for proof-of-concept experiments, there is a significant body of evidence, primarily coming from animal models, demonstrating that ECs have organ-specific gene expression and function. Given that ECs within individual organ systems create organ-specific microenvironments critical for function, organ- specific ECs will be imperative to achieve long-term organ-level function for tissue engineering and regenerative medicine approaches. However, critical gaps in our knowledge exists with respect to human ECs; it is unknown if tissue-specific EC gene expression and function is present during development of human organ systems, and it is further unknown how such expression patterns are established. To begin to address these unknowns I isolated ECs and non-ECs from human fetal kidney, lung and intestine, performed bulk RNA sequencing, and used gene clustering approaches to identify organ-specific EC-enriched genes. My preliminary data has identified over 100 organ-specific EC-enriched genes for each organ analyzed. This is the first evidence to suggest organ-specific EC gene expression is established during embryonic/fetal development. My proposal is designed to: 1) Validate novel organ-enriched EC genes during human development and test organ-specific EC function using in vitro co-culture assays 2) leverage these insights to induce organ-specific patterning of human pluripotent stem cell (hPSC)-derived ECs.