The existence of a haematopoietic stem cell niche as a spatially confined regulatory entity relies on the notion that haematopoietic stem and progenitor cells (HSPCs) are strategically positioned in unique bone marrow microenvironments with defined anatomical and functional features. Here, we employ a powerful imaging cytometry platform to perform a comprehensive quantitative analysis of HSPC distribution in bone marrow cavities of femoral bones. We find that HSPCs preferentially localize in endosteal zones, where most closely interact with sinusoidal and non-sinusoidal bone marrow microvessels, which form a distinctive circulatory system. In situ tissue analysis reveals that HSPCs exhibit a hypoxic profile, defined by strong retention of pimonidazole and expression of HIF- 1α, regardless of localization throughout the bone marrow, adjacency to vascular structures or cell-cycle status. These studies argue that the characteristic hypoxic state of HSPCs is not solely the result of a minimally oxygenated niche but may be partially regulated by cell-specific mechanisms.
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We are grateful to D. Rossi, L. Purton and C. P. Lin for critical reading of the manuscript and thank the Compucyte Corporation team for helpful advice. C.N-A. was a recipient of Human Frontiers in Science Program long-term fellowship 00194/2008-L. This work was financially supported in part by a seed grant from the Harvard Stem Cell Institute. L.E.S. is supported by grants P01 HL095489 and R01 HL093139, and contract HHSN268201000009C from the National Heart Lung and Blood Institute, USA.
3D reconstruction of confocal optical sections of thick bone marrow slices stained for c-kit (green) and the vascular marker Laminin (red). Multiple examples of vessel-adjacent c-kit+ cells can be visualized.
The movie depicts a series of sequential optical sections on the z-axis followed by the 3D reconstruction of a thick bone marrow femoral slice, 2 min post-injection of CD45-PE. Intravascular populations (blood vessels in green labelled with Laminin) are specifically stained with CD45 (red)
3D reconstruction of confocal optical sections of a thick slice of a murine femur stained with the panvascular marker Laminin (green) and the arterial marker Sca-1 (red).
3D reconstruction of multiphoton optical sections of a femoral diaphysis stained for Laminin (green). The central sinus, as well as an underlying central artery, can be visualized running longitudinally across the diaphysis. Bone is visualized in blue, as revealed by second harmonic generation.
High-magnification 3D reconstruction of the endosteal vessels shown in Supplementary Video 4. Arrow depicts the arteriolar to sinusoidal transition in close proximity to endosteal surfaces (bone shown in blue).
3D reconstruction of the vascular network of a femoral metaphysis. All bone marrow vascular structures are marked by Laminin (green), while the arterial network is positive for Sca-1 (red).
3D reconstruction of the vascular network of endosteal regions of a femoral metaphysis. Arrow depicts the arteriolar to sinusoidal transition in close proximity to endosteal surfaces (bone shown in blue).
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