Beta cell replacement is a primary goal for therapy in T1D. Embryonic stem (ES)-derived ß cells can maintain glucose tolerance in immunodeficient mice, yet there is a major knowledge gap regarding the details of durable metabolic output of these cells in vivo. Thus, the long-term goal of this projectis to determine the optimal metabolic environment for ß-cell function and resolution of T1D. Mitochondrial mass and function are essential to maintain ß cell bioenergetic function and survive extrinsic cytotoxic stressors. We will utilize genetically encoded mitochondrial biosensors, metabolomic profiling, cellular respirometry, and live cell imaging to define crucial parameters underlying ES-derived beta cell mitochondrial function and compare these outputs to those of mature human islets. We will also use targeted mitochondrial therapeutics to compensate for metabolic demands of engraftment and function of ES-derived ß cells post-transplant, including pharmacologic agents to induce mitochondrial biogenesis or reduce mitochondrial reactive oxygen species delivered by transplantable scaffolds. Further, the functional potential of ß cells derived from different ES lines and induced pluripotent stem (iPS) cells are highly variable, so we will expand our studies in the future to understand if failures in ß cell maturation, function, and engraftment are driven by mitochondrial dysfunction. Mitochondrial function is driving force for ß cell competence, so a deeper understanding of mitochondrial signals and approaches to improve mitochondrial function could enhance the potential of ES and iPS cells to achieve the promise of ß cell replacement in T1D.