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

Nature Cell Biology volume 15, pages 533–543 (2013)Cite this article

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A Corrigendum to this article was published on 01 August 2013

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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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Figure 1: Quantitative image analysis of the spatial distribution of c-kit+ cells in whole longitudinal femoral sections.
Figure 2: Most HSPCs lie in direct contact with bone marrow microvessels.
Figure 3: 3D study of bone marrow microvasculature heterogeneity.
Figure 4: HSPCs directly interact with structurally and phenotypically diverse types of bone marrow microvessel.
Figure 5: c-kit+ progenitors exhibit a hypoxic status regardless of their perivascular localization and distribution in the bone marrow cavity.
Figure 6: The hypoxic status of HSPCs is independent of their localization in bone marrow regions and their cycling status.
Figure 7: Stable HSPC expression of HIF- 1α is not dictated by localization in the bone marrow.
Figure 8: HIF- 1α expression on cycling and actively proliferating HSPCs.

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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.

References

  1. Orkin, S. H. & Zon, L. I. Hematopoiesis: an evolving paradigm for stem cell biology. Cell 132, 631–644 (2008).

    Article  CAS  Google Scholar 

  2. Wang, L. D. & Wagers, A. J. Dynamic niches in the origination and differentiation of haematopoietic stem cells. Nat. Rev. Mol. Cell Biol. 12, 643–655 (2011).

    Article  CAS  Google Scholar 

  3. Adams, G. B. & Scadden, D. T. The hematopoietic stem cell in its place. Nat. Immunol. 7, 333–337 (2006).

    Article  CAS  Google Scholar 

  4. Wilson, A. et al. c-Myc controls the balance between hematopoietic stem cell self-renewal and differentiation. Genes Dev. 18, 2747–2763 (2004).

    Article  CAS  Google Scholar 

  5. Kiel, M. J. & Morrison, S. J. Uncertainty in the niches that maintain haematopoietic stem cells. Nat. Rev. Immunol. 8, 290–301 (2008).

    Article  CAS  Google Scholar 

  6. Zhang, J. et al. Identification of the haematopoietic stem cell niche and control of the niche size. Nature 425, 836–841 (2003).

    Article  CAS  Google Scholar 

  7. Xie, Y. et al. Detection of functional haematopoietic stem cell niche using real-time imaging. Nature 457, 97–101 (2009).

    Article  CAS  Google Scholar 

  8. Kiel, M. J., Yilmaz, O. H., Iwashita, T., Terhorst, C. & Morrison, S. J. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 121, 1109–1121 (2005).

    Article  CAS  Google Scholar 

  9. Sugiyama, T., Kohara, H., Noda, M. & Nagasawa, T. Maintenance of the hematopoietic stem cell pool by CXCL12-CXCR4 chemokine signalling in bone marrow stromal cell niches. Immunity 25, 977–988 (2006).

    Article  CAS  Google Scholar 

  10. Mendez-Ferrer, S. et al. Mesenchymal and haematopoietic stem cells form a unique bone marrow niche. Nature 466, 829–834 (2010).

    Article  CAS  Google Scholar 

  11. Omatsu, Y. et al. The essential functions of adipo-osteogenic progenitors as the hematopoietic stem and progenitor cell niche. Immunity 33, 387–399 (2010).

    Article  CAS  Google Scholar 

  12. Ding, L., Saunders, T. L., Enikolopov, G. & Morrison, S. J. Endothelial and perivascular cells maintain haematopoietic stem cells. Nature 481, 457–462 (2012).

    Article  CAS  Google Scholar 

  13. Parmar, K., Mauch, P., Vergilio, J. A., Sackstein, R. & Down, J. D. Distribution of hematopoietic stem cells in the bone marrow according to regional hypoxia. Proc. Natl Acad. Sci. USA 104, 5431–5436 (2007).

    Article  CAS  Google Scholar 

  14. Takubo, K. et al. Regulation of the HIF- 1α level is essential for hematopoietic stem cells. Cell Stem Cell 7, 391–402 (2010).

    Article  CAS  Google Scholar 

  15. Simsek, T. et al. The distinct metabolic profile of hematopoietic stem cells reflects their location in a hypoxic niche. Cell Stem Cell 7, 380–390 (2010).

    Article  CAS  Google Scholar 

  16. Krohn, K. A., Link, J. M. & Mason, R. P. Molecular imaging of hypoxia. J. Nucl. Med. 49 (Suppl 2), 129S–148S (2008).

    Article  CAS  Google Scholar 

  17. Brahimi-Horn, M. C. & Pouyssegur, J. Oxygen, a source of life and stress. FEBS Lett. 581, 3582–3591 (2007).

    Article  CAS  Google Scholar 

  18. Eliasson, P. & Jonsson, J. I. The hematopoietic stem cell niche: low in oxygen but a nice place to be. J. Cell Physiol. 222, 17–22 (2010).

    Article  CAS  Google Scholar 

  19. Winkler, I. G. et al. Positioning of bone marrow hematopoietic and stromal cells relative to blood flow in vivo: serially reconstituting hematopoietic stem cells reside in distinct nonperfused niches. Blood 116, 375–385 (2010).

    Article  CAS  Google Scholar 

  20. Mohyeldin, A., Garzon-Muvdi, T. & Quinones-Hinojosa, A. Oxygen in stem cellbiology: a critical component of the stem cell niche. Cell Stem Cell 7, 150–161 (2010).

    Article  CAS  Google Scholar 

  21. Suda, T., Takubo, K. & Semenza, G. L. Metabolic regulation of hematopoietic stem cells in the hypoxic niche. Cell Stem Cell 9, 298–310 (2011).

    Article  CAS  Google Scholar 

  22. Harnett, M. M. Laser scanning cytometry: understanding the immune system in situ. Nat. Rev. Immunol. 7, 897–904 (2007).

    Article  CAS  Google Scholar 

  23. Butler, J. M. et al. Endothelial cells are essential for the self-renewal and repopulation of Notch-dependent hematopoietic stem cells. Cell Stem Cell 6, 251–264 (2010).

    Article  CAS  Google Scholar 

  24. Hooper, A. T. et al. Engraftment and reconstitution of hematopoiesis is dependent on VEGFR2-mediated regeneration of sinusoidal endothelial cells. Cell Stem Cell 4, 263–274 (2009).

    Article  CAS  Google Scholar 

  25. Yang, L. et al. Identification of Lin(-)Sca1(+)kit(+)CD34(+)Flt3- short-term hematopoietic stem cells capable of rapidly reconstituting and rescuing myeloablated transplant recipients. Blood 105, 2717–2723 (2005).

    Article  CAS  Google Scholar 

  26. Pereira, J. P., An, J., Xu, Y., Huang, Y. & Cyster, J. G. Cannabinoid receptor 2 mediates the retention of immature B cells in bone marrow sinusoids. Nat. Immunol. 10, 403–411 (2009).

    Article  CAS  Google Scholar 

  27. Kopp, H. G., Avecilla, S. T., Hooper, A. T. & Rafii, S. The bone marrow vascular niche: home of HSC differentiation and mobilization. Physiology (Bethesda) 20, 349–356 (2005).

    CAS  Google Scholar 

  28. Kopp, H. G., Hooper, A. T., Avecilla, S. T. & Rafii, S. Functionalheterogeneity of the bone marrow vascular niche. Ann. NY Acad. Sci. 1176, 47–54 (2009).

    Article  CAS  Google Scholar 

  29. Li, X. M., Hu, Z., Jorgenson, M. L. & Slayton, W. B. High levels of acetylated low-density lipoprotein uptake and low tyrosine kinase with immunoglobulin and epidermal growth factor homology domains-2 (Tie2) promoter activity distinguish sinusoids from other vessel types in murine bone marrow. Circulation 120, 1910–1918 (2009).

    Article  CAS  Google Scholar 

  30. Kimura, Y. et al. c-Kit-mediated functional positioning of stem cells to their niches is essential for maintenance and regeneration of adult hematopoiesis. PLoS One 6, e26918 (2011).

    Article  CAS  Google Scholar 

  31. Yamazaki, S. et al. Nonmyelinating Schwann cells maintain hematopoietic stem cell hibernation in the bone marrow niche. Cell 147, 1146–1158.

    Article  CAS  Google Scholar 

  32. Kubota, Y., Takubo, K. & Suda, T. Bone marrow long label-retaining cellsreside in the sinusoidal hypoxic niche. Biochem. Biophys. Res. Commun. 366, 335–339 (2008).

    Article  CAS  Google Scholar 

  33. Jiang, B. H., Semenza, G. L., Bauer, C. & Marti, H. H. Hypoxia-inducible factor 1 levels vary exponentially over a physiologically relevant range of O2 tension. Am. J. Physiol. 271, C1172–C1180 (1996).

    Article  CAS  Google Scholar 

  34. Pedersen, M. et al. Stem cell factor induces HIF- 1α at normoxia in hematopoietic cells. Biochem. Biophys. Res. Commun. 377, 98–103 (2008).

    Article  CAS  Google Scholar 

  35. Kirito, K., Fox, N., Komatsu, N. & Kaushansky, K. Thrombopoietin enhances expression of vascular endothelial growth factor (VEGF) in primitive hematopoietic cells through induction of HIF- 1α. Blood 105, 4258–4263 (2005).

    Article  CAS  Google Scholar 

  36. Piccoli, C. et al. The hypoxia-inducible factor is stabilized in circulatinghematopoietic stem cells under normoxic conditions. FEBS Lett. 581, 3111–3119 (2007).

    Article  CAS  Google Scholar 

  37. Eliasson, P. et al. Hypoxia mediates low cell-cycle activity and increases the proportion of long-term-reconstituting hematopoietic stem cells during in vitro culture. Exp. Hematol. 38, 301–302 (2010).

    Article  CAS  Google Scholar 

  38. Guitart, A. V., Hammoud, M., Dello Sbarba, P., Ivanovic, Z. & Praloran, V. Slow-cycling/quiescence balance of hematopoietic stem cells is related to physiological gradient of oxygen. Exp. Hematol. 38, 847–851 (2010).

    Article  CAS  Google Scholar 

  39. Keith, B. & Simon, M. C. Hypoxia-inducible factors, stem cells, and cancer. Cell 129, 465–472 (2007).

    Article  CAS  Google Scholar 

  40. Lo Celso, C. et al. Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche. Nature 457, 92–96 (2009).

    Article  CAS  Google Scholar 

  41. Pittman, R. N. Oxygen transport and exchange in the microcirculation. Microcirculation 12, 59–70 (2005).

    Article  CAS  Google Scholar 

  42. Wang, L. et al. Identification of a clonally expanding haematopoietic compartment in bone marrow. EMBO J. 32, 219–230 (2013).

    Article  Google Scholar 

Download references

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.

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Author notes

  1. Alexei Protopopov

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

Authors and Affiliations

  1. Division of Transfusion Medicine, Department of Laboratory Medicine, Children’s Hospital Boston, 300 Longwood Avenue, Massachusetts 02115, Boston, 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, Illinois 61801, Urbana Champaign, USA

    Brendan Harley

  3. Division of Hematologic Malignancies, Dana-Farber Cancer Institute, Massachusetts 02115, Boston, 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.

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

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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. (MOV 22098 kb)

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) (MOV 19305 kb)

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). (MOV 42972 kb)

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. (MOV 17776 kb)

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). (MOV 10992 kb)

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). (MOV 9790 kb)

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). (MOV 31669 kb)

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Nombela-Arrieta, C., Pivarnik, G., Winkel, B. et al. Quantitative imaging of haematopoietic stem and progenitor cell localization and hypoxic status in the bone marrow microenvironment. Nat Cell Biol 15, 533–543 (2013). https://doi.org/10.1038/ncb2730

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