Haematopoietic stem cell (HSC) niches, although proposed decades ago1, have only recently been identified as separate osteoblastic and vascular microenvironments2,3,4,5,6. 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–enhancer7. 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.
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We appreciate scientific support from R. Krumlauf, W. Neaves and encouragement from G. Lu and W. Shen. We are grateful to D. Scadden and P. Kulesa for discussion. We thank D. Rowe for providing the Col2.3-GFP line. We thank R. Yu, L. Ma, Q. Qiu and W. Wang for technological advice, M. Hembree, A. Box, H. Marshall, E. Rendenbaugh, C. Cooper and M. Smith for technical support, S. Beckham for histology assistance, J. Perry and J. Ross for comments, and K. Tannen for proofreading. R.A.F. was supported by DOD grant W81XWH-04-1-0801 and by a DRIF Award from the University of Maryland. L.L. is supported by Stowers Institute for Medical Research.
Author Contributions Y.X. and T.Y. developed the initial idea, designed and performed experiments. W.W. contributed to EVISC instrumentation and real-time imaging. X.C.H. helped on data analysis, troubleshooting and immunostaining for Ncad LacZ. D.S. assisted in real-time imaging. J.P., J.H., J.W., T.J. and S.Z. assisted in some experiments. J.S., K.P. and R.A. performed quantification of fluorescent intensity. H.L. contributed to statistics. D.M. and R.A.F. contributed to bioluminescent imaging. J.G. assisted in data analysis and manuscript writing. L.L. contributed to overall supervision, experimental design and manuscript writing.
Supplementary Movie 1. Real-time imaging of bone marrow cells in the trabecular bone area of GFP-tg mice.
Supplementary Movie 2. A homed (under irradiated conditions) GFP-HSC at the endosteal surface.
Supplementary Movie 3. A homed (under non-irradiated conditions) GFP-HSC at the central marrow region.
Supplementary Movie 4. This movie seems to show (with speculation) how cells derived from bone marrow (stromal cells?) were received by large cells that located on the bone surface, and were placed on the bone surface.
Supplementary Movie 5. Three-dimensional view of a homed GFP-HSC attaching to an N-cadherin+ osteoblast.
Supplementary Movie 6. A homed GFP-HSC located at the tip of a nook structure formed between two bone structures (revealed by DIC).
Supplementary Movie 7.
Supplementary Movie 8.
About this article
Cellular and Molecular Life Sciences (2018)