Mitochondrial activity dictates definitive hematopoietic stem cell production and lympho-myeloid lineage segregation during mouse embryogenesis [scRNAseq]

Mitochondrial metabolism determines bone marrow hematopoietic stem cell (HSC) heterogeneity, and influences long-term blood repopulation potential. However, when and how this mitochondrial regulation of definitive HSCs originates is unexplored. We show that dynamic changes in mitochondrial activity during endothelial to hematopoietic transition (EHT) drives the production of mature HSCs in the mouse embryo. Pharmacological and genetic manipulations show that reduced mitochondrial activity activates Wnt signalling to promote expansion of mature HSCs in the AGM. Further, single cell transcriptomics and functional assays uncovered mitochondrial membrane potential (MMP) driven functional heterogeneity within the mature HSC pool. MMPlow HSCs are myeloid-biased and exhibit enhanced differentiation potential. Contrarily, MMPhigh HSCs are lymphoid-biased with diminished differentiation potential. Mechanistically, low mitochondrial activity upregulates PI3K signalling to fuel embryonic HSC differentiation. We provide insights into the metabolic regulation of HSC origin and function, that can be leveraged to direct HSC fate decisions for clinical interventions.