Project Summary/Abstract Immediately after fertilization, metabolism in the early embryo undergoes drastic remodeling. While studies have thoroughly characterized pre-implantation embryonic development, measurements of metabolism in the embryo post-implantation, from gastrulation to birth have been limited by scarcity of tissue and inaccessibility of the embryo. in vitro differentiation of human embryonic stem cells provides an unlimited source of tissue and a platform to study the metabolic transitions that occur within the rapidly expanding embryo. Using directed differentiation of human embryonic stem cells into pancreatic epithelium, this proposal aims to combine metabolic interrogation of cell proliferation and biomass accumulation with developmental biology. These studies will blend recent conceptual advances in cancer metabolism with an in vitro differentiation platform to understand how metabolism instructs and directs cell fate decisions in the developing fetus. Experiments will first focus on cellular redox state, testing whether changes in NAD+/NADH ratio as human embryonic stem cells undergo differentiation into pancreatic epithelium are necessary to support differentiation into the pancreatic lineage. Then, kinetic tracing studies using isotopically labeled nutrient sources will quantify rates of metabolic flux to determine how changes in overall metabolism contribute to differentiation. Finally, metabolites that have been identified to undergo large fluctuations in abundance throughout differentiation will be studied to determine whether their accumulation is necessary for successful pancreatic differentiation. This proposal is designed to explain the roles of metabolism in the developing pancreas as it continuously differentiates and expands in a changing nutrient environment. Results from each of these aims can be used to improve in vitro differentiation of stem cells by providing new mechanisms to guide successful differentiation into a desired cell type. By providing new insight into metabolic regulation of stem cell fate, this work will enhance the ability to generate curative stem cell-based therapies for patients suffering from lost or dysfunctional tissues, especially type 1 diabetes. These therapies hold great promise for improving health and quality of life for millions of patients. These experiments will be carried out in the laboratory of Professor Matthew Vander Heiden within the Koch Institute for Integrative Cancer Research at Massachusetts Institute of Technology. Training under this fellowship will include presentation of this data at institutional and public conferences, publication of this work in peer-reviewed scientific journals, and regular meetings with professor Vander Heiden to prepare the applicant for success as a tenure track research faculty member.