PROJECT SUMMARY The availability of donor-matched sources of hematopoietic stem cells (HSCs) continues to limit access to and outcomes following allogeneic HSC transplant. Unmet need for improved HSC sources has motivated global improvements in donor recruitment and matching, as well as enterprising attempts to develop patient-derived or universally compatible hematopoietic cells. At present, specification of HSCs in a dish has been inefficient, and most methods using co-culture and expression of hematopoietic genes only produce progenitors with limited lineage and engraftment potential. 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. In our prior funding period, we found that initiation of blood flow is a critical determinant of energy metabolism and mitochondrial dynamics in the HSC precursor known as the hemogenic endothelium. The objective of our current research is to define metabolic adaptations 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 define the contribution of mitochondrial maturation to development of the hemogenic endothelium that gives rise to HSCs. We will employ a combination of methods that provide single-cell resolution of metabolic activity, mitochondrial ultrastructure, and hematopoietic function. Our first aim is designed to address the effects of interrupting mitochondrial maturation on commitment of hemogenic endothelial precursors to the hematopoietic fate via pharmacological targeting, biomimetic modeling with in vitro platforms, and cardiac mutant mouse models. In our second aim, we leverage pilot data from complementary datasets that support a role for the mitochondrial permeability transition pore (mPTP) in differentiation of arterial endothelium. We address how biphasic regulation of mPTP opening over the continuum of the endothelial-to-hematopoietic transition (EHT) dictates acquisition of hematopoietic fate. Consequences of disrupted or enhanced mPTP activity will be defined during EHT by assessing indicators of metabolic and hematopoietic capacity. The proposed study will address a major deficiency in our understanding of how mitochondrial maturation contributes to transition of endothelial to hematopoietic fate and promises to inspire novel methods for generation of HSCs in vitro by metabolic reprogramming.