• A Corrigendum to this article was published on 20 July 2016

Abstract

Bone marrow endothelial cells (BMECs) form a network of blood vessels that regulate both leukocyte trafficking and haematopoietic stem and progenitor cell (HSPC) maintenance. However, it is not clear how BMECs balance these dual roles, and whether these events occur at the same vascular site. We found that mammalian bone marrow stem cell maintenance and leukocyte trafficking are regulated by distinct blood vessel types with different permeability properties. Less permeable arterial blood vessels maintain haematopoietic stem cells in a low reactive oxygen species (ROS) state, whereas the more permeable sinusoids promote HSPC activation and are the exclusive site for immature and mature leukocyte trafficking to and from the bone marrow. A functional consequence of high permeability of blood vessels is that exposure to blood plasma increases bone marrow HSPC ROS levels, augmenting their migration and differentiation, while compromising their long-term repopulation and survival. These findings may have relevance for clinical haematopoietic stem cell transplantation and mobilization protocols.

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Acknowledgements

We thank G. Karsenty and M. A. Lichtman for fruitful discussions and for critically reviewing the manuscript. We thank S. Méndez-Ferrer and M. Argueta Hernandez for assistance in studies involving MSPCs and nervous system elements. We thank Z. Porat for technical assistance with ImageStream analysis and R. Rotkopf for assistance with statistical data analysis. This study was partially supported by the Ministry of Science, Technology & Space, Israel and the DKFZ, Germany, grants from the Israel Science Foundation (851/13), the Ernest and Bonnie Beutler Research Program of Excellence in Genomic Medicine and EU FP7-HEALTH-2010 (CELL-PID #261387) (T.L.). Confocal studies were supported by the European Research Council Advanced Grant 339409, ‘AngioBone’ (R.H.A.). Intravital multiphoton studies were supported by NIH grants EB017274 and HL100402 (C.P.L. & D.T.S.).

Author information

Affiliations

  1. Department of Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel

    • Tomer Itkin
    • , Shiri Gur-Cohen
    • , Guy Ledergor
    • , Idan Milo
    • , Alexander Kalinkovich
    • , Aya Ludin
    • , Karin Golan
    • , Eman Khatib
    • , Anju Kumari
    • , Orit Kollet
    • , Guy Shakhar
    •  & Tsvee Lapidot
  2. Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA

    • Joel A. Spencer
    • , Yookyung Jung
    •  & Charles P. Lin
  3. Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA

    • Joel A. Spencer
    • , Yookyung Jung
    •  & Charles P. Lin
  4. Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts 02138, USA

    • Amir Schajnovitz
    •  & David T. Scadden
  5. Harvard Stem Cell Institute, Cambridge, Massachusetts 02114, USA

    • Amir Schajnovitz
    •  & David T. Scadden
  6. Center for Regenerative Medicine and Cancer Center, Massachusetts General Hospital, Boston, Massachusetts 02114, USA

    • Amir Schajnovitz
    •  & David T. Scadden
  7. Max Planck Institute for Molecular Biomedicine, Department of Tissue Morphogenesis and Faculty of Medicine, University of Münster, D-48149 Münster, Germany

    • Saravana K. Ramasamy
    • , Anjali P. Kusumbe
    •  & Ralf H. Adams
  8. Internal Medicine Department, Tel-Aviv Sourasky Medical Center, Tel-Aviv 64239, Israel

    • Guy Ledergor
  9. Department of Genetic Medicine, Weill Cornell Medical College, New York, New York 10065, USA

    • Michael G. Poulos
    • , Jason M. Butler
    •  & Shahin Rafii

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Contributions

T.I. designed and performed experiments, analysed data and wrote the manuscript; S.G-.C. helped in the design and execution of experiments and analysed data; J.A.S., A.S., and Y.J. designed and performed intravital related experiments, analysed data and helped with writing the manuscript; S.K.R. and A.P.K. designed and performed confocal related experiments and analysed data; G.L., I.M., M.G.P., A.K., A.L., and O.K. helped with experiments; K.G. participated in CFU-F and CFU-Ob related experiments; E.K. participated in metabolic ROS experiments, including plasma penetration and NAC treatment; A.K. participated in mice genotyping; G.S. helped and guided some intravital related experiments; J.M.B. and S.R. helped in design of endothelial-related studies; R.H.A helped and guided in design of confocal and in vivo endothelial related experiments; D.T.S. helped and guided design of in vivo intravital live imaging experiments and wrote the manuscript; and T.L. and C.P.L. guided and designed the research and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Charles P. Lin or Tsvee Lapidot.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary information

    This file contains Supplementary Table 1, a methods related antibodies table, displaying all the antibodies applied in the manuscript, their clone/catalogue #, the company they were acquired from and their application in the presented study.

Videos

  1. 1.

    A Z-stack of two-photon images captured inside Sca-1-EGFP (green) mouse calvarium

    Blood vessels were labeled using Qtracker (red). Sca-1 positive small diameter BVs are found adjacently to calcified bone surface (second harmonic signal, blue). Note the disappearance of EGFP labeled BVs in the depth of the marrow, away from the endosteal surface, while still observing EGFP labeled round single hematopoietic cells. Scale bar indicates 25 µm.

  2. 2.

    A Z-stack of two-photon images captured inside nestin-EGFP (green) mouse calvarium

    Blood vessels were labeled using Qtracker (red). Nestin positive small diameter BVs are found adjacently to calcified bone surface (second harmonic signal, blue). Mice were injected with Di-Acetyl-LDL (pink) 2 hours prior to intravital images acquisition. Note the exclusive uptake of LDL by the large diameter sinusoids which are all negative for GFP. Scale bar indicates 15 µm.

  3. 3.

    A representative intravital live video of in vivo permeability measurements via perfusion and leakage of Rhodamine-Dextran (70 kDa) (red) in bone marrow vasculature of nestin-GFP (green) mouse calvarium

    Calcified bone and blood flow reflectance (in blue) were visualized by second harmonic signal. Rhodamine diffusion signal was collected from defined regions that were set immediately adjacent to the BV of interest (i.e. positive or negative for nestin expression). The average intensity within the regions of interest was measured over time and the slope calculated. The slope value indicated for the blood vessel permeability. Scale bar indicates 100 µm.

  4. 4.

    An intravital video showing only the red channel (in grey) of Supplementary video 3, allowing the measurements of Dextran diffusion

    Regions of interest for signal acquisition are defined by yellow borders. Note the higher diffusion in the region adjacent to the sinusoid relatively to the region adjacent to the nestin+ BV.

  5. 5.

    A representative intravital video showing PB circulating labeled RBCs (in yellow), flowing at high speed thru a small-diameter endosteal arteriole, and dramatically decelerating once entering a larger sinusoidal BV. Blood vessels are labeled with Rhodamine-Dextran (500 kDa) (red).

  6. 6.

    A representative intravital video of DiD-labeled HSPCs (red) that were injected into nestin-GFP (green) mice and monitored for their trafficking in different types of BVs

    Note that the monitored cell arrives at the endosteal area (blue) via a nestin+ BV then slows down, arrests and crawls, only when it reaches the downstream sinusoid. Scale bar indicates 50 µm.

  7. 7.

    A three dimensional reconstruction of Z-stack planes of intravital 2 photon images taken in the calvarium of nestin-GFP (green) mouse that was injected with Dil-labeled BM MNC (blue) and DiD-labeled BM HSPC (red).

    The video represents a network of blood vessels (grey) in the BM and adjacently adhering or extravasating cells. Note the occurrence of these events only in the large diameter nestin- sinusoids.

  8. 8.

    A representative intravital video of Dil-labeled BM MNC (blue) and DiD-labeled BM HSPC (red) that were injected into nestin-GFP (green) mice and monitored for their trafficking in different types of BVs.

    Note two extravasation events (in the middle of the screen) of MNCs (blue) via BM sinusoids (gray) into the marrow. Scale bar indicates 25 µm.

  9. 9.

    A representative intravital video of Dil-labeled BM MNC (blue) and DiD-labeled BM HSPC (red) that were injected into nestin-GFP (green) mice and monitored for their trafficking in different types of BVs.

    Note extravasation event (in the upper right side of the screen) of HSPC (red) via BM sinusoids (gray) into the marrow. Scale bar indicates 25 µm.

  10. 10.

    A Z-stack of two-photon images captured inside nestin-EGFP (green) mouse calvarium

    A Z-stack of intravital images captured inside nestin-EGFP (green) mouse calvarium with labeled blood vessels (Angiosense 750EX, red). Video represents the environment of the location of cellular trafficking and extravasation (sinusoid). Note the relative proximity to a nestin+ BV. Scale bar indicates 25 µm.

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DOI

https://doi.org/10.1038/nature17624

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