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Adult blood stem cell localization reflects the abundance of reported bone marrow niche cell types and their combinations.

The exact localization of hematopoietic stem cells (HSCs) in their native bone marrow (BM) microenvironment remains controversial, since multiple cell types have been reported to physically associate with HSCs. Here, we comprehensively quantified HSC localization with up to 4 simultaneous (9 total) BM components, in 152 full-bone sections from different bone types and three HSC reporter lines. We find adult femoral α-catulin-GFP+ or Mds1GFP/+Flt3Cre HSCs proximal to sinusoids, Cxcl12 stroma, megakaryocytes and different combinations of those populations, but not bone, adipocytes, peri-/arteriolar or Schwann cells. Despite micro-anatomical differences in femurs and sterna, their adult α-catulin-GFP+ HSCs have similar distribution. Importantly, their microenvironmental localizations are not different from that of random dots, reflecting the relative abundance of imaged BM populations rather than active enrichment. Despite their functional heterogeneity, dormant label-retaining (LR) and non-LR hematopoietic stem and progenitor cells (HSPCs) all have indistinguishable localization from α-catulin-GFP+ HSCs. In contrast, cycling juvenile BM HSCs preferentially locate close to Cxcl12 stroma, and further from sinusoids/megakaryocytes. We expect our study to help resolving existing confusion regarding the exact localization of different HSC types and their physical association with described BM populations and their tissue-wide combinations.