Neogenin-1 distinguishes between myeloid-biased and balanced Hoxb5⁺ mouse long-term hematopoietic stem cells

Hematopoietic stem cells (HSCs) self-renew and generate all blood cells. Recent studies with single cell transplants and lineage tracing suggest that adult HSCs are diverse in their reconstitution and lineage potentials. However, prospective isolation of these subpopulations has remained challenging. Here, we identify Neogenin-1 (NEO1) as a unique surface marker on a fraction of mouse HSCs labeled with Hoxb5, a specific reporter of long-term HSCs (LT-HSCs). We show that NEO1⁺Hoxb5⁺ LT-HSCs expand with age and respond to myeloablative stress in young mice while NEO1^-Hoxb5⁺ LT-HSCs exhibit no significant change in number. Furthermore, NEO1⁺Hoxb5⁺ LT-HSCs are more often in the G₂/S cell cycle phase compared to NEO1^-Hoxb5⁺ LT-HSCs in both young and old bone marrow. Upon serial transplantation, NEO1⁺Hoxb5⁺ LT-HSCs exhibit myeloid-biased differentiation and reduced reconstitution while NEO1^-Hoxb5⁺ LT-HSCs are lineage-balanced and stably reconstitute recipients. Gene expression analysis reveals erythroid and myeloid priming in the NEO1⁺ fraction and association of quiescence and self-renewal-related transcription factors with NEO1^- LT-HSCs. Finally, transplanted NEO1⁺Hoxb5⁺ LT-HSCs rarely generate NEO1^-Hoxb5⁺ LT-HSCs while NEO1^-Hoxb5⁺ LT-HSCs repopulate both LT-HSC fractions. This supports a model in which dormant, balanced NEO1^-Hoxb5⁺ LT-HSCs can hierarchically precede active, myeloid-biased NEO1⁺Hoxb5⁺ LT-HSCs.