PROJECT SUMMARY For several decades, clinical outcomes of allogeneic hematopoietic stem cell (HSC) transplantation have been limited by the availability of donor-matched sources of HSCs. This has motivated global improvements in donor recruitment and matching, as well as aggressive pursuit of new strategies for development of patient-derived or universally compatible hematopoietic cells. Attempts to specify human HSCs have only produced progenitors with limited lineage and engraftment potential using co-culture and expression of hematopoietic genes through modified RNAs or viral integration. Our studies show that biomechanical force caused by flow of blood through the vasculature is a critical regulator of hematopoiesis and can promote engraftment of cells with long-term hematopoietic reconstitution potential. A number of well-characterized pathways are activated by fluid shear stress in adult vascular endothelial cells, yet little is known about signaling within hemogenic endothelial cells and their precursors in embryogenesis. Our preliminary data strongly implicates initiation of blood flow as a critical determinant of energy metabolism and mitochondrial dynamics in the HSC precursor known as the hemogenic endothelium. The objective of our research is to define signaling mechanisms triggered by biomechanical force that promote definitive hematopoiesis, with the long-term goal of exploiting biophysical cues such as shear stress in directed differentiation and expansion of customized HSCs for therapeutic transplant and blood disease modeling. Specifically, we aim to identify mitochondrial adaptations induced by vascular force that promote expansion of hemogenic endothelium via utilization of reporter mouse models of HSC emergence and mitochondrial dynamics. We will identify mitochondrial features that contribute to fate selection and survival of cells with HSC potential using murine embryos and differentiation cultures of pluripotent stem cells. We will interrogate and define the intracellular signaling that drives mitochondrial remodeling in response to physiologic intensities of fluid force in hemogenic endothelium by pharmacological and genetic targeting. Further, consequences of disrupted or enhanced mitochondrial capacity will be defined during hemogenic endothelial cell fate selection and into adulthood to reveal how mitochondrial dynamics impact the hematopoietic program at its earliest stages within the vasculature. The proposed study promises to fill a major gap in our knowledge of how newly specified HSCs and their precursors produce energy and manage metabolic processes, and will provide insight into novel methods for engineering competitive self-renewing HSCs through manipulation of metabolism.