Deep imaging of bone marrow shows non-dividing stem cells are mainly perisinusoidal

Hematopoietic stem cells (HSCs) reside in a perivascular niche but the location remains controversial(1). HSCs are rare and few can be found in thin tissue sections(2,3) or upon live imaging(4), making it difficult to comprehensively localize dividing and non-dividing HSCs. We discovered that α-catulin(GFP/+) was expressed by only 0.02% of bone marrow hematopoietic cells, including virtually all HSCs. One in 3.5 α-catulin-GFP(+)c-kit(+) cells gave long-term multilineage reconstitution of irradiated mice, indicating that α-catulin-GFP(+)c-kit(+) cells contain HSCs with a purity comparable to the best markers available. We were able to optically clear the bone marrow to perform deep confocal imaging, making it possible to image thousands of α-catulin-GFP(+)c-kit(+) cells and to digitally reconstruct large segments of bone marrow. The distribution of α-catulin-GFP(+)c-kit(+) cells indicated that HSCs were more common in central marrow than near bone surfaces and in the diaphysis relative to the metaphysis. Nearly all HSCs contacted Leptin Receptor(+) and Cxcl12(high) niche cells. Approximately 85% of HSCs were within 10μm of a sinusoidal blood vessel. Most HSCs were distant from arterioles, transition zone vessels, and bone surfaces. This was true of Ki-67(+) dividing HSCs and Ki-67(−) non-dividing HSCs. Dividing and non-dividing HSCs thus reside mainly in perisinusoidal niches with Leptin Receptor(+)Cxcl12(high) cells throughout the bone marrow.