ATP2B1 expression identifies human hematopoietic stem cells across ontogeny with superior repopulation and self-renewal capacity

Long-term hematopoietic stem cells (LT-HSC) maintain lifelong hematopoiesis while preserving the stem cell compartment through self-renewal. The human LT-HSC compartment is molecularly and functionally heterogeneous and also varies across ontogeny. Dissecting the molecular basis for this variation is impeded by the absence of immunophenotypic markers to resolve LT-HSC heterogeneity. Here, we identified ATPase plasma membrane Ca²⁺transporting 1 (ATP2B1/PMCA1) as a novel cell surface marker that is heterogeneously expressed by CD49f⁺LT-HSC across ontogeny. ATP2B1 immunophenotypic expression stratified human CD49f⁺LT-HSC from fetal liver (FL), neonatal cord blood (CB) and adult mobilized peripheral blood (mPB) sources into functionally distinct subpopulations in single-cell (sc) clonogenic assays. CD49f⁺ATP2B1⁺LT-HSC exhibited superior long-term repopulation and self-renewal capacitiesin vivocompared to CD49f⁺ATP2B1^–LT-HSC. Molecular profiling by scMultiome and immunofluorescence microscopy point to enrichment of an HSC self-renewal program that includes the TFEB-endolysosomal axis in CD49f⁺ATP2B1⁺LT-HSC. Our study provides a new tool to dissect the heterogeneous molecular programs in LT-HSC across ontogeny.