Project Summary Human pluripotent stem cell derived endothelial cells (hPSC-ECs) and pericytes are an ideal autologous cell source for use in vascular therapies. ECs line the inner layer of the vasculature and pericytes provide support to small diameter vessels in order to regulate vascular permeability. Derivation of these cells from hPSCs requires time sensitive delivery of growth factors and small molecules in order to induce vascular fate. Characterization of these cells is often limited to 2D in vitro assays and cell surface protein verification. As we move towards therapeutic use, functional characterization is necessary to ensure their translational efficiency and efficacy. Hypoxia has been shown to be a key regulator of EC fate, neovascularization, and regeneration. However, its role in vascular stabilization through pericyte recruitment has not been studied. Here, thorough analysis of hPSC-ECs and pericytes in hypoxic environments we aim to: (1) Study network kinetics of hPSC- ECs in 3D low oxygen environments, (2) study hPSC-ECs and hPSC-pericyte interactions in hypoxia in vitro, and (3) Study hPSC-EC-pericyte interactions in a mouse oxygen induced retinopathy model. These aims require a multidisciplinary approach, interfacing stem cell and vascular biology with tissue engineering. Successful completion of these aims will broaden our understanding of hPSC-ECs and pericytes behavior towards therapeutic modalities.