Letter | Published:

Direct measurement of local oxygen concentration in the bone marrow of live animals

Nature volume 508, pages 269273 (10 April 2014) | Download Citation

This article has been updated

Abstract

Characterization of how the microenvironment, or niche, regulates stem cell activity is central to understanding stem cell biology and to developing strategies for the therapeutic manipulation of stem cells1. Low oxygen tension (hypoxia) is commonly thought to be a shared niche characteristic in maintaining quiescence in multiple stem cell types2,3,4. However, support for the existence of a hypoxic niche has largely come from indirect evidence such as proteomic analysis5, expression of hypoxia inducible factor-1α (Hif-1α) and related genes6, and staining with surrogate hypoxic markers (for example, pimonidazole)6,7,8. Here we perform direct in vivo measurements of local oxygen tension (pO2) in the bone marrow of live mice. Using two-photon phosphorescence lifetime microscopy, we determined the absolute pO2 of the bone marrow to be quite low (<32 mm Hg) despite very high vascular density. We further uncovered heterogeneities in local pO2, with the lowest pO2 (9.9 mm Hg, or 1.3%) found in deeper peri-sinusoidal regions. The endosteal region, by contrast, is less hypoxic as it is perfused with small arteries that are often positive for the marker nestin. These pO2 values change markedly after radiation and chemotherapy, pointing to the role of stress in altering the stem cell metabolic microenvironment.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Change history

  • 10 April 2014

    Gene Hif-1 was corrected to Hif-1α in the first paragraph.

References

  1. 1.

    , & The HSC niche concept has turned 31. Ann. NY Acad. Sci. 1192, 12–18 (2010)

  2. 2.

    , & Metabolic regulation of hematopoietic stem cells in the hypoxic niche. Cell Stem Cell 9, 298–310 (2011)

  3. 3.

    , & Oxygen in stem cell biology: a critical component of the stem cell niche. Cell Stem Cell 7, 150–161 (2010)

  4. 4.

    & From stem cells to cancer stem cells: HIF takes the stage. Curr. Opin. Cell Biol. 24, 232–235 (2012)

  5. 5.

    Quantitative proteomics reveals posttranslational control as a regulatory factor in primary hematopoietic stem cells. Blood 107, 4687–4694 (2006)

  6. 6.

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

  7. 7.

    et al. Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1. Nature Med. 10, 858–864 (2004)

  8. 8.

    , , , & Distribution of hematopoietic stem cells in the bone marrow according to regional hypoxia. Proc. Natl Acad. Sci. USA 104, 5431–5436 (2007)

  9. 9.

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

  10. 10.

    et al. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 121, 1109–1121 (2005)

  11. 11.

    et al. Osteoblastic cells regulate the haematopoietic stem cell niche. Nature 425, 841–846 (2003)

  12. 12.

    & Dynamic niches in the origination and differentiation of haematopoietic stem cells. Nature Rev. Mol. Cell Biol. 12, 643–655 (2011)

  13. 13.

    The ultrastructure of the hemopoietic environment of the marrow: a review. Exp. Hematol. 9, 391–410 (1981)

  14. 14.

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

  15. 15.

    et al. Quantitative imaging of haematopoietic stem and progenitor cell localization and hypoxic status in the bone marrow microenvironment. Nature Cell Biol. 15, 533–543 (2013)

  16. 16.

    et al. Arteriolar niches maintain haematopoietic stem cell quiescence. Nature 502, 637–643 (2013)

  17. 17.

    , , & Modeling pO2 distributions in the bone marrow hematopoietic compartment. I. Krogh’s model. Biophys. J. 81, 675–684 (2001)

  18. 18.

    , , & Modeling pO2 distributions in the bone marrow hematopoietic compartment. II. Modified Kroghian models. Biophys. J. 81, 685–696 (2001)

  19. 19.

    , , , & In vivo cell tracking with video rate multimodality laser scanning microscopy. IEEE J. Sel. Topics Quantum Electron. 14, 10–18 (2008)

  20. 20.

    , & Design of metalloporphyrin-based dendritic nanoprobes for two-photon microscopy of oxygen. J. Porphyr. Phthalocyanines 12, 1261–1269 (2008)

  21. 21.

    et al. Oxygen microscopy by two-photon-excited phosphorescence. ChemPhysChem 9, 1673–1679 (2008)

  22. 22.

    , , & An optical method for measurement of dioxygen concentration based upon quenching of phosphorescence. J. Biol. Chem. 262, 5476–5482 (1987)

  23. 23.

    et al. Dendritic phosphorescent probes for oxygen imaging in biological systems. ACS Appl. Mater. Interfaces 1, 1292–1304 (2009)

  24. 24.

    et al. Two-photon high-resolution measurement of partial pressure of oxygen in cerebral vasculature and tissue. Nature Methods 7, 755–759 (2010)

  25. 25.

    et al. Simultaneous two-photon imaging of oxygen and blood flow in deep cerebral vessels. Nature Med. 17, 893–898 (2011)

  26. 26.

    et al. Three-dimensional mapping of oxygen tension in cortical arterioles before and after occlusion. Biomed. Opt. Express 4, 1061–1073 (2013)

  27. 27.

    et al. Quantification of longitudinal tissue pO2 gradients in window chamber tumours: impact on tumour hypoxia. Br. J. Cancer 79, 1717–1722 (1999)

  28. 28.

    et al. Total body irradiation causes profound changes in endothelial traffic molecules for hematopoietic progenitor cell recruitment to bone marrow. Blood 99, 4182–4191 (2002)

  29. 29.

    et al. In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment. Nature 435, 969–973 (2005)

  30. 30.

    et al. Two-photon microscopy of oxygen: polymersomes as probe carrier vehicles. J. Phys. Chem. B 114, 14373–14382 (2010)

  31. 31.

    , , , & Neural stem and progenitor cells in nestin-GFP transgenic mice. J. Comp. Neurol. 469, 311–324 (2004)

  32. 32.

    et al. Two new ‘protected’ oxyphors for biological oximetry: properties and application in tumor imaging. Anal. Chem. 83, 8756–8765 (2011)

Download references

Acknowledgements

We thank S. Sakadzic for helpful discussion on setting up the 2PLM experiment. This work was supported by the US National Institutes of Health grant HL097748, EB017274 (to C.P.L.), HL097794, HL096372 and EB014703 (to D.T.S.).

Author information

Affiliations

  1. Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA

    • Joel A. Spencer
    • , Emmanuel Roussakis
    • , Juwell Wu
    • , Judith M. Runnels
    • , Walid Zaher
    • , Luke J. Mortensen
    • , Clemens Alt
    • , Raphaël Turcotte
    •  & Charles P. Lin
  2. Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA

    • Joel A. Spencer
    • , Juwell Wu
    • , Judith M. Runnels
    • , Walid Zaher
    • , Luke J. Mortensen
    • , Clemens Alt
    • , Raphaël Turcotte
    •  & Charles P. Lin
  3. Department of Biomedical Engineering, Tufts University, Medford, Massachusetts 02155, USA

    • Joel A. Spencer
  4. Center for Regenerative Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA

    • Francesca Ferraro
    • , Alyssa Klein
    • , Rushdia Yusuf
    •  & David T. Scadden
  5. Harvard Stem Cell Institute, Cambridge, Massachusetts 02138, USA

    • Francesca Ferraro
    • , Alyssa Klein
    • , Rushdia Yusuf
    • , David T. Scadden
    •  & Charles P. Lin
  6. Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts 02138, USA

    • Francesca Ferraro
    • , Alyssa Klein
    • , Rushdia Yusuf
    •  & David T. Scadden
  7. Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA

    • Emmanuel Roussakis
    •  & Sergei A. Vinogradov
  8. Stem Cell Unit, Department of Anatomy, College of Medicine, King Saud University, Riyadh 11461, Saudi Arabia

    • Walid Zaher
  9. Department of Biomedical Engineering, Boston University, Boston, Massachusetts 02215, USA

    • Raphaël Turcotte
  10. Département de Physique, Génie Physique et Optique and Centre de Recherche de l’Institut Universitaire en Santé Mentale de Québec, Université Laval, Québec City, Québec G1J 2G3, Canada

    • Daniel Côté

Authors

  1. Search for Joel A. Spencer in:

  2. Search for Francesca Ferraro in:

  3. Search for Emmanuel Roussakis in:

  4. Search for Alyssa Klein in:

  5. Search for Juwell Wu in:

  6. Search for Judith M. Runnels in:

  7. Search for Walid Zaher in:

  8. Search for Luke J. Mortensen in:

  9. Search for Clemens Alt in:

  10. Search for Raphaël Turcotte in:

  11. Search for Rushdia Yusuf in:

  12. Search for Daniel Côté in:

  13. Search for Sergei A. Vinogradov in:

  14. Search for David T. Scadden in:

  15. Search for Charles P. Lin in:

Contributions

J.A.S. designed and built the microscope, designed experiments, conducted research, collected and analysed data and wrote the manuscript; F.F. designed experiments, conducted research, collected and analysed data, and wrote the manuscript; E.R. synthesized the PtP-C343 oxygen probe; A.K. helped conduct research and collected and analysed data; J.W., J.M.R., W.Z., L.J.M., R.T. and R.Y. helped conduct research; C.A. and D.C. helped build the microscope; S.A.V. synthesized the PtP-C343 oxygen probe and wrote the manuscript; D.T.S. designed experiments and wrote the manuscript; C.P.L. designed experiments, sponsored the project and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Charles P. Lin.

Extended data

Supplementary information

Videos

  1. 1.

    Intravital video (30 frames/sec) of blood flow in lethally irradiated mouse from Extended Data Figure 7b.

    Intravital video (30 frames/sec) of blood flow in lethally irradiated mouse from Extended Data Figure 7b.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature13034

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.