ABSTRACT A leading goal of our research is identification of the key metabolic pathways directing hematopoietic stem cell (HSC) fate decisions. While early findings suggested that HSCs depend mainly on glycolysis, emerging evidence from our lab and others has shown that mitochondrial metabolism, and particularly fatty acid oxidation, is essential to HSC fate determination. We hypothesize that mitochondrial metabolism is remodeled at the initiation of the fate choice process to meet the changing needs of proper HSC function. However, our understanding of the relationship between HSC self-renewal and lipid metabolism remains limited. To identify metabolite- dependent pathways, we have used an adapted gene expression-oriented bioinformatics tool and our own metabolomics analyses. We have also established a biosensor for assessment of fatty acid oxidation activity in live cells to determine the metabolic modes which are relevant to the controlled equilibrium of HSCs. Quantitative live imaging and our single cell approaches will illuminate the processes of symmetric or asymmetric mitochondrial segregation during HSC division. Our innovative local transplantation system will allow us to monitor the migration and cell divisions of single HSCs in vivo, and our established image-guided technique of micropipette aspiration of individual cells directly from the bone marrow of live animals will enable subsequent single-cell assay. Analysis of the resulting data will yield new insights into the fate decision process of HSCs, and facilitate the development of new therapeutic strategies for shifting the division balance of HSCs toward self- renewal through metabolic manipulation. The goals of this proposal are three-fold: (1) In Aim 1, we will induce the selective consumption of metabolites localized in the mitochondria to identify metabolic targets of fatty acid metabolism that affect HSC fate; (2) in Aim 2, we will use pharmacological or genetic modulation of key genes impacting fatty acid oxidation or its downstream targets to define the metabolic crosstalk between mitochondria and the cytosol; and (3) in Aim 3 we will evaluate the coordinated process that yields HSC division symmetry in vivo, and analysis of division balance will provide insights into the in vivo relevance of fatty acid metabolisms to HSC fate choice. If successful, the proposed research will positively impact the field by providing a deeper understanding of the metabolic cues coordinating HSC fate decisions, and will suggest potential methods of shifting the division balance of HSCs toward self-renewal through metabolic manipulation to improve clinical outcomes after transplantation.