Multiple cell types have been proposed to create niches for haematopoietic stem cells (HSCs). However, the expression patterns of HSC maintenance factors have not been systematically studied and no such factor has been conditionally deleted from any candidate niche cell. Thus, the cellular sources of these factors are undetermined. Stem Cell Factor (SCF) is a key niche component that maintains HSCs. Using Scfgfp knock-in mice we found Scf was primarily expressed by perivascular cells throughout bone marrow. HSC frequency and function were not affected when Scf was conditionally deleted from haematopoietic cells, osteoblasts, Nestin-Cre, or Nestin-CreER-expressing cells. However, HSCs were depleted from bone marrow when Scf was deleted from endothelial cells or Leptin receptor (Lepr)-expressing perivascular stromal cells. Most HSCs were lost when Scf was deleted from both endothelial and Lepr-expressing perivascular cells. HSCs reside in a perivascular niche in which multiple cell types express factors that promote HSC maintenance.

While haematopoietic stem cells (HSCs) are commonly assumed to reside within a specialized microenvironment, or niche1, most published experimental manipulations of the HSC niche have also impacted the function of diverse restricted progenitors. This raises the fundamental question of whether HSCs1 and restricted progenitors2,3 reside within distinct, specialized niches or whether they share a common niche. Here we assess the physiological sources of the chemokine, CXCL12, for HSC and restricted progenitor maintenance. Cxcl12DsRed knock-in mice showed that Cxcl12 was primarily expressed by perivascular stromal cells and at lower levels by endothelial cells, osteoblasts, and some haematopoietic cells. Conditional deletion of Cxcl12 from haematopoietic cells or Nestin-cre-expressing cells had little or no effect on HSCs or restricted progenitors. Deletion of Cxcl12 from endothelial cells depleted HSCs but not myeloerythroid or lymphoid progenitors. Deletion of Cxcl12 from perivascular stromal cells depleted HSCs and certain restricted progenitors and mobilized these cells into circulation. Deletion of Cxcl12 from osteoblasts depleted certain early lymphoid progenitors, but not HSCs or myeloerythroid progenitors and did not mobilize these cells into circulation. Different stem/progenitor cells thus occupy distinct cellular niches in bone marrow: HSCs in a perivascular niche and early lymphoid progenitors in an endosteal niche.

SUMMARY Hematopoietic stem cells (HSCs) and multipotent hematopoietic progenitors (MPPs) are routinely isolated using various markers but remain heterogeneous. Here we show that four SLAM family markers, CD150, CD48, CD229, and CD244, can distinguish HSCs and MPPs from restricted progenitors and subdivide them into a hierarchy of functionally distinct subpopulations with stepwise changes in cell-cycle status, self-renewal, and reconstituting potential. CD229 expression largely distinguished lymphoid-biased HSCs from rarely-dividing myeloid-biased HSCs, enabling prospective enrichment of these HSC subsets. Differences in CD229 and CD244 expression resolved CD150−CD48−/lowLineage−/lowSca-1+c-Kit+ cells into a hierarchy of highly-purified MPPs that retained erythroid and platelet potential but exhibited progressive changes in mitotic activity and reconstituting potential. Use of these markers, and reconstitution assays, showed that conditional deletion of Scf from endothelial cells and perivascular stromal cells eliminated the vast majority of bone marrow HSCs, including nearly all CD229−/low HSCs, demonstrating that quiescent HSCs are maintained by a perivascular niche.