Technical Report | Published:

Quantitative imaging of haematopoietic stem and progenitor cell localization and hypoxic status in the bone marrow microenvironment

Nature Cell Biology volume 15, pages 533543 (2013) | Download Citation

  • A Corrigendum to this article was published on 01 August 2013

This article has been updated

Abstract

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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Change history

  • 26 June 2013

    In the version of this Technical Report originally published, reference to a study (L. Wang et al. Identification of a clonally expanding haematopoietic compartment in bone marrow. EMBO J. 32, 219–230; 2013) was inadvertently omitted. This has now been added as reference 42, and a paragraph has been added to the Discussion section. Furthermore, the e-mail address for the co-corresponding author was incorrect. These errors have now been corrected in the HTML and PDF versions of the Technical Report.

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Acknowledgements

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.

Author information

Author notes

    • Alexei Protopopov

    Present address: Institute for Applied Cancer Science, The University of Texas MD Anderson Cancer Center, Texas 77030, USA

Affiliations

  1. Division of Transfusion Medicine, Department of Laboratory Medicine, Children’s Hospital Boston, 300 Longwood Avenue, Boston, Massachusetts 02115, USA

    • César Nombela-Arrieta
    • , Gregory Pivarnik
    • , Beatrice Winkel
    • , Kimberly J. Canty
    • , Shin-Young Park
    • , Jiayun Lu
    •  & Leslie E. Silberstein
  2. Department for Chemical and Biomolecular Engineering, University of Illinois, Urbana Champaign, Illinois 61801, USA

    • Brendan Harley
  3. Division of Hematologic Malignancies, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA

    • John E. Mahoney
    •  & Alexei Protopopov

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Contributions

C.N-A. designed and performed experiments, analysed data and wrote the manuscript. G.P., B.W., K.J.C., S-Y.P. and J.L. performed experiments. B.H. participated in the design of the study. J.E.M. and A.P. provided technical help. L.E.S. designed the study and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to César Nombela-Arrieta or Leslie E. Silberstein.

Supplementary information

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    Supplementary Information

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    Supplementary Table 1

    Supplementary Information

Videos

  1. 1.

    Interaction of c-kit cells with bone marrow microvasculature.

    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.

  2. 2.

    In vivo labelling of intravascular populations.

    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)

  3. 3.

    Vascular compartment of the femoral diaphysis.

    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).

  4. 4.

    Bone marrow sinusoidal microvasculature.

    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.

  5. 5.

    Arteriolar to sinusoidal transition in endosteal regions of the diaphysis.

    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).

  6. 6.

    Vascular compartment of the femoral metaphysis.

    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).

  7. 7.

    Endosteal microvascular network of femoral metaphysis.

    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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DOI

https://doi.org/10.1038/ncb2730