PROJECT SUMMARY: The metabolism of nutrients has been studied using unfractionated tissues, or in vitro. An unresolved question is how nutrients are metabolized by stem cells in vivo. Our understanding of stem cell metabolism has been limited by the fact that metabolomics typically requires millions of cells, while stem cells are rare. We developed methods to profile the metabolome and to trace stable isotope labeled nutrients in hematopoietic stem cells (HSCs) and other rare cell types purified from tissues. We found that T cell progenitors in the thymus are glucose avid as compared to HSCs, myeloid and B cell restricted progenitors, in contrast to the prevailing view that HSCs are more glycolytic than hematopoietic progenitors. Stable isotope tracing experiments showed that in the bone marrow but not the thymus, glycolysis and the TCA cycle are disconnected. Hematopoietic loss of pyruvate dehydrogenase (PDH), the gatekeeper enzyme that connects glycolysis to the TCA cycle, reduced the number of double positive (DP) T cell progenitors but did not affect HSCs or other hematopoietic cell types. Loss of PDH paradoxically did not impair the TCA cycle in the thymus, but caused accumulation of pyruvate and aberrant redox balance. Cells which do not oxidize glucose in the TCA cycle are classically thought to ferment glucose through glycolysis to lactate via lactate dehydrogenase (LDH). Hematopoietic loss of LDHA, one of the two LDH isoforms, impaired development of erythroid progenitors but not HSCs, T cell progenitors or other restricted hematopoietic progenitors. The cell type specificity in the requirement of LDH and PDH in the hematopoietic system raises the question of why different stem or progenitor cell types choose to use LDH-mediated fermentation or PDH-mediated oxidation in vivo. This application’s objective is to systematically dissect the role of glycolytic as compared to oxidative metabolism in HSCs and restricted progenitors. Our hypothesis is that T cell progenitors require oxidation of glucose via PDH to regulate pyruvate levels and redox homeostasis, in contrast to HSCs, myeloid and B cell progenitors which are metabolically flexible. In Aim 1 we will test the metabolic mechanisms which mediate the effects of PDH on DP cells. In Aim 2 we will determine the cellular and metabolic effects of blocking LDHA/B or PDH alone or in combination in HSCs and restricted progenitors. In Aim 3 we will investigate the role of LDHA/B and PDH in hematopoietic and thymopoietic regeneration. These experiments will identify the contribution of glucose to metabolite pools in HSCs and progenitors in vivo, systematically test the idea that HSCs are glycolytic, and identify mechanisms by which central carbon metabolism regulates hematopoietic differentiation and regeneration. More generally our experiments will address a fundamental metabolic question by testing if stem or progenitor cells in vivo switch between glucose fermentation or oxidation, as is the t...