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 () in the bone marrow of live mice. Using two-photon phosphorescence lifetime microscopy, we determined the absolute of the bone marrow to be quite low (<32 mm Hg) despite very high vascular density. We further uncovered heterogeneities in local , with the lowest (∼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 values change markedly after radiation and chemotherapy, pointing to the role of stress in altering the stem cell metabolic microenvironment.
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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.).
The authors declare no competing financial interests.
Extended data figures and tables
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.
The values of a solution of PtP-C343 are plotted as a function of the phosphorescent lifetime measurements recorded by our microscope. 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.
The core porphyrin, PEG chains, C343 moieties and dendrimer are denoted in red, green and blue, respectively.
Two-photon intravital image of PtP-C343 fluorescence ∼60 min after intravenous administration. Scale bar, ∼100 µm.
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).
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.
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 to become elevated in the entire marrow space, with no defined spatial gradients.
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.
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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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