Mitochondrial activity-driven hematopoietic stem cell fate and lympho-myeloid lineage choice is first established in the aorta-gonad-mesonephros

Mitochondrial metabolism determines bone marrow hematopoietic stem cell (HSC) heterogeneity and influences long-term repopulation potential. However, the origin of this heterogeneity and how it regulates HSC phenotype is unclear. We show that during the endothelial-to-hematopoietic transition (EHT) in the mouse embryo, dynamic changes in mitochondrial activity drive the production of mature HSCs with differing potencies. Lowering mitochondrial activity in the AGM by pharmacological or genetic means activates Wnt signaling to promote HSC expansion. Further, mitochondrial membrane potential (MMP) gives rise to functional heterogeneity in the definitive HSC pool. In-vitro and in-vivo functional assays, and single-cell transcriptomics on AGM HSCs showed that MMP ^low HSCs are myeloid-biased, with enhanced differentiation potential, while MMP ^high HSCs are lymphoid-biased with diminished differentiation potential. Mechanistically, low mitochondrial activity upregulates Phosphoinositide 3-kinase (PI3K) signaling to promote HSC differentiation. These insights into the initiation of metabolic heterogeneity could be leveraged to isolate HSCs to efficiently generate desired lineages.