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

Nature volume 508, pages 269–273 (2014)Cite this article

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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 ( p O 2 ) in the bone marrow of live mice. Using two-photon phosphorescence lifetime microscopy, we determined the absolute p O 2 of the bone marrow to be quite low (<32 mm Hg) despite very high vascular density. We further uncovered heterogeneities in local p O 2 , with the lowest p O 2 (∼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 p O 2 values change markedly after radiation and chemotherapy, pointing to the role of stress in altering the stem cell metabolic microenvironment.

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Figure 1: BM vascular density and oxygenation.
Figure 2: Variation in BM p O 2 according to vessel diameter and distance from the endosteal surface.
Figure 3: BM p O 2 after cytoreductive therapy and at sites of HSPC homing.
Figure 4: BM vascular mapping and the effects of cellularity on local p O 2 after cytoreductive conditioning.

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Change history

  • 10 April 2014

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

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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.).

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Authors and Affiliations

  1. Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School, Boston, 02114, Massachusetts, 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, 02114, Massachusetts, 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, 02155, Massachusetts, USA

    Joel A. Spencer

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

    Francesca Ferraro, Alyssa Klein, Rushdia Yusuf & David T. Scadden

  5. Harvard Stem Cell Institute, Cambridge, 02138, Massachusetts, USA

    Francesca Ferraro, Alyssa Klein, Rushdia Yusuf, David T. Scadden & Charles P. Lin

  6. Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, 02138, Massachusetts, USA

    Francesca Ferraro, Alyssa Klein, Rushdia Yusuf & David T. Scadden

  7. Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, 19104, Pennsylvania, USA

    Emmanuel Roussakis & Sergei A. Vinogradov

  8. Department of Anatomy, Stem Cell Unit, College of Medicine, King Saud University, Riyadh 11461, Saudi Arabia,

    Walid Zaher

  9. Department of Biomedical Engineering, Boston University, Boston, 02215, Massachusetts, 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

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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.

Corresponding author

Correspondence to Charles P. Lin.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Schematic of the two-photon intravital imaging and 2PLM system.

BP, bandpass filter; DAQ, data acquisition device; f, focal length; Galvo, galvanometer mirror; L, lens; LP, longpass filter; PBS, polarizing beamsplitter; PMT, photomultiplier tube; λ/2, half-wave plate. L13 and L14 have a focal length of 5 cm.

Extended Data Figure 2 In vitro calibration measurements.

The p O 2 values of a solution of PtP-C343 are plotted as a function of the phosphorescent lifetime measurements recorded by our microscope. p O 2 was determined by an oxygen microsensor inserted into the solution. The solid line is the published calibration curve for the same batch of PtP-C343 recorded by a different laboratory24.

Extended Data Figure 3 Schematic of PtP-C343 platinum porphyrin oxygen sensor.

The core porphyrin, PEG chains, C343 moieties and dendrimer are denoted in red, green and blue, respectively.

Extended Data Figure 4 PtP-C343 leakage into extravascular space within the BM.

Two-photon intravital image of PtP-C343 fluorescence ∼60 min after intravenous administration. Scale bar, ∼100 µm.

Extended Data Figure 5 HSC homing to bone marrow.

Scatter plot of HSC (lineagelowc-kit+Sca-1+CD150+CD48−) homing to nestin+ vessels and bone (endosteum) in lethally irradiated recipients (9.5 Gy) 2 days after adoptive transfer (n = 19 cells from 3 mice).

Extended Data Figure 6 Vascular leakage after cytoreductive conditioning.

a–c, Intravital images of BM in untreated controls (a), lethally irradiated (b) and busulphan-treated (c) mice 2 days after conditioning reveals increased permeability and decreased contrast of BM vessels. Rhodamine B–dextran (red) and bone SHG (blue) signal is shown. Scale bar, ∼50 µm.

Extended Data Figure 7 In vivo immunostaining and blood flow analysis after cytoreductive conditioning.

a, Intravital BM image of anti-Sca-1 immunostaining (red), nestin-GFP (green), blood vessels (blue) and bone (SHG, grey) demonstrating Sca-1+ nestin-GFP vessels. Scale bar, ∼50 µm. b, Standard deviation image from 600 frames of a confocal video sequence showing the path of RBC flow (green) and bone signal (SHG, blue) on day 2 after lethal irradiation (9.5 Gy). Each pixel displays the standard deviation of the corresponding pixel location across all 600 frames of the video. Scale bar, ∼100 µm.

Extended Data Figure 8 Cytoreduction of the BM after myeloablative or myelosuppressive conditioning.

BM cellularity as a function of treatment day is plotted for untreated (n = 3 mice), sub-lethally irradiated (4.5 Gy, n = 3 mice per time point), lethally irradiated (9.5 Gy, n = 3 mice per time point) and busulphan-treated (n = 3 mice per time point) mice.

Extended Data Figure 9 Revised model of local oxygen tension in the BM.

Instead of a poorly perfused hypoxic zone near the endosteum, we detected an opposite gradient, with the peri-sinusoidal region being more hypoxic than the endosteal region, which is perfused with small arteries. After irradiation, the sinusoids are greatly dilated and the blood flow is maintained, whereas the reduced oxygen consumption due to declining BM cellularity causes the p O 2 to become elevated in the entire marrow space, with no defined spatial gradients.

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Spencer, J., Ferraro, F., Roussakis, E. et al. Direct measurement of local oxygen concentration in the bone marrow of live animals. Nature 508, 269–273 (2014). https://doi.org/10.1038/nature13034

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