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

Haematopoietic stem cells (HSCs) reside in a perivascular niche but the specific location of this niche 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. Here, using a green fluorescent protein (GFP) knock-in for the gene Ctnnal1 in mice (hereafter denoted as alpha-catulin(GFP)), we discover that alpha-catulin(GFP) is expressed by only 0.02% of bone marrow haematopoietic cells, including almost all HSCs. We find that approximately 30% of alpha-catulin-GFP(+)c-kit(+) cells give long-term multilineage reconstitution of irradiated mice, indicating that alpha-catulin-GFP(+)c-kit(+) cells are comparable in HSC purity to cells obtained using the best markers currently available. We optically cleared the bone marrow to perform deep confocal imaging, allowing us to image thousands of alpha-catulin-GFP(+)c-kit(+) cells and to digitally reconstruct large segments of bone marrow. The distribution of alpha-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 positive (Lepr(+)) and Cxcl12(high) niche cells, and approximately 85% of HSCs were within 10 mm of a sinusoidal blood vessel. Most HSCs, both dividing (Ki-67(+)) and non-dividing (Ki-67(-)), were distant from arterioles, transition zone vessels, and bone surfaces. Dividing and non-dividing HSCs thus reside mainly in perisinusoidal niches with Lepr(+)Cxcl12(high) cells throughout the bone marrow.