Detection of functional haematopoietic stem cell niche using real-time imaging

Haematopoietic stem cell (HSC) niches, although proposed decades ago(1), have only recently been identified as separate osteoblastic and vascular microenvironments(2-6). Their interrelationships and interactions with HSCs in vivo remain largely unknown. Here we report the use of a newly developed ex vivo real- time imaging technology and immunoassaying to trace the homing of purified green-fluorescent-protein- expressing (GFP(+)) HSCs. We found that transplanted HSCs tended to home to the endosteum (an inner bone surface) in irradiated mice, but were randomly distributed and unstable in non- irradiated mice. Moreover, GFP(+) HSCs were more frequently detected in the trabecular bone area compared with compact bone area, and this was validated by live imaging bioluminescence driven by the stem- cell- leukaemia ( Scl) promoter enhancer(7). HSCs home to bone marrow through the vascular system. We found that the endosteum is well vascularized and that vasculature is frequently localized near N-cadherin(+) pre-osteoblastic cells, a known niche component. By monitoring individual HSC behaviour using real- time imaging, we found that a portion of the homed HSCs underwent active division in the irradiated mice, coinciding with their expansion as measured by flow assay. Thus, in contrast to central marrow, the endosteum formed a special zone, which normally maintains HSCs but promotes their expansion in response to bone marrow damage.