Distinct bone marrow blood vessels differentially regulate haematopoiesis

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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Figure 1: Sca-1 and nestin distinguish less permeable arterial bone marrow blood vessels, which sustain ROSlow HSC.
Figure 2: Leaky sinusoids are the exclusive site for cellular trafficking.
Figure 3: Plasma penetration through leaky endothelium dictates HSPC trafficking and development.
Figure 4: Reducing endothelial barrier integrity hampers stem cell maintenance.

References

  1. 1

    Rafii, S., Butler, J. M. & Ding, B. S. Angiocrine functions of organ-specific endothelial cells. Nature 529, 316–325 (2016)

    ADS  PubMed  PubMed Central  CAS  Google Scholar 

  2. 2

    Lapidot, T., Dar, A. & Kollet, O. How do stem cells find their way home? Blood 106, 1901–1910 (2005)

    PubMed  CAS  Google Scholar 

  3. 3

    Morrison, S. J. & Scadden, D. T. The bone marrow niche for haematopoietic stem cells. Nature 505, 327–334 (2014)

    ADS  PubMed  PubMed Central  CAS  Google Scholar 

  4. 4

    Kusumbe, A. P., Ramasamy, S. K. & Adams, R. H. Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone. Nature 507, 323–328 (2014)

    ADS  PubMed  PubMed Central  CAS  Google Scholar 

  5. 5

    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)

    PubMed  CAS  Google Scholar 

  6. 6

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

    ADS  PubMed  PubMed Central  CAS  Google Scholar 

  7. 7

    Colmone, A. et al. Leukemic cells create bone marrow niches that disrupt the behavior of normal hematopoietic progenitor cells. Science 322, 1861–1865 (2008)

    ADS  PubMed  CAS  Google Scholar 

  8. 8

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

    ADS  PubMed  CAS  Google Scholar 

  9. 9

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

    ADS  PubMed  PubMed Central  Google Scholar 

  10. 10

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

    ADS  PubMed  PubMed Central  CAS  Google Scholar 

  11. 11

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

    ADS  PubMed  PubMed Central  CAS  Google Scholar 

  12. 12

    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)

    PubMed  PubMed Central  CAS  Google Scholar 

  13. 13

    Isern, J. et al. The neural crest is a source of mesenchymal stem cells with specialized hematopoietic stem cell niche function. Elife 3, e03696 (2014)

    PubMed  PubMed Central  Google Scholar 

  14. 14

    Ito, K. et al. Reactive oxygen species act through p38 MAPK to limit the lifespan of hematopoietic stem cells. Nature Med. 12, 446–451 (2006)

    PubMed  CAS  Google Scholar 

  15. 15

    Miyamoto, K. et al. Foxo3a is essential for maintenance of the hematopoietic stem cell pool. Cell Stem Cell 1, 101–112 (2007)

    PubMed  CAS  Google Scholar 

  16. 16

    Tesio, M. et al. Enhanced c-Met activity promotes G-CSF-induced mobilization of hematopoietic progenitor cells via ROS signaling. Blood 117, 419–428 (2011)

    PubMed  CAS  Google Scholar 

  17. 17

    Golan, K. et al. S1P promotes murine progenitor cell egress and mobilization via S1P1-mediated ROS signaling and SDF-1 release. Blood 119, 2478–2488 (2012)

    PubMed  PubMed Central  CAS  Google Scholar 

  18. 18

    Zhao, M. et al. Megakaryocytes maintain homeostatic quiescence and promote post-injury regeneration of hematopoietic stem cells. Nature Med. 20, 1321–1326 (2014)

    PubMed  CAS  Google Scholar 

  19. 19

    Bruns, I. et al. Megakaryocytes regulate hematopoietic stem cell quiescence through CXCL4 secretion. Nature Med. 20, 1315–1320 (2014)

    PubMed  CAS  Google Scholar 

  20. 20

    Nakamura-Ishizu, A., Takubo, K., Fujioka, M. & Suda, T. Megakaryocytes are essential for HSC quiescence through the production of thrombopoietin. Biochem. Biophys. Res. Commun. 454, 353–357 (2014)

    PubMed  CAS  Google Scholar 

  21. 21

    Nombela-Arrieta, C. 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)

    PubMed  CAS  Google Scholar 

  22. 22

    Ono, N. et al. Vasculature-associated cells expressing nestin in developing bones encompass early cells in the osteoblast and endothelial lineage. Dev. Cell 29, 330–339 (2014)

    PubMed  PubMed Central  CAS  Google Scholar 

  23. 23

    Ludin, A. et al. Monocytes-macrophages that express α-smooth muscle actin preserve primitive hematopoietic cells in the bone marrow. Nature Immunol. 13, 1072–1082 (2012)

    CAS  Google Scholar 

  24. 24

    Yamazaki, S. et al. Nonmyelinating Schwann cells maintain hematopoietic stem cell hibernation in the bone marrow niche. Cell 147, 1146–1158 (2011)

    PubMed  CAS  Google Scholar 

  25. 25

    Papayannopoulou, T., Priestley, G. V., Nakamoto, B., Zafiropoulos, V. & Scott, L. M. Molecular pathways in bone marrow homing: dominant role of α4β1 over β2-integrins and selectins. Blood 98, 2403–2411 (2001)

    PubMed  CAS  Google Scholar 

  26. 26

    Winkler, I. G. et al. Vascular niche E-selectin regulates hematopoietic stem cell dormancy, self renewal and chemoresistance. Nature Med. 18, 1651–1657 (2012)

    PubMed  CAS  Google Scholar 

  27. 27

    De Bock, K. et al. Role of PFKFB3-driven glycolysis in vessel sprouting. Cell 154, 651–663 (2013)

    PubMed  CAS  Google Scholar 

  28. 28

    Vandekeere, S., Dewerchin, M. & Carmeliet, P. Angiogenesis revisited: an overlooked role of endothelial cell metabolism in vessel sprouting. Microcirculation 22, 509–517 (2015)

    PubMed  Google Scholar 

  29. 29

    Spencer, J. A. et al. Direct measurement of local oxygen concentration in the bone marrow of live animals. Nature 508, 269–273 (2014)

    ADS  PubMed  PubMed Central  CAS  Google Scholar 

  30. 30

    Broxmeyer, H. E. et al. Rapid mobilization of murine and human hematopoietic stem and progenitor cells with AMD3100, a CXCR4 antagonist. J. Exp. Med. 201, 1307–1318 (2005)

    PubMed  PubMed Central  CAS  Google Scholar 

  31. 31

    Heissig, B. et al. Recruitment of stem and progenitor cells from the bone marrow niche requires MMP-9 mediated release of Kit-ligand. Cell 109, 625–637 (2002)

    PubMed  PubMed Central  CAS  Google Scholar 

  32. 32

    Dar, A. et al. Rapid mobilization of hematopoietic progenitors by AMD3100 and catecholamines is mediated by CXCR4-dependent SDF-1 release from bone marrow stromal cells. Leukemia 25, 1286–1296 (2011)

    PubMed  PubMed Central  CAS  Google Scholar 

  33. 33

    Kobayashi, K. et al. Stromal cell-derived factor-1α/C-X-C chemokine receptor type 4 axis promotes endothelial cell barrier integrity via phosphoinositide 3-kinase and Rac1 activation. Arterioscler. Thromb. Vasc. Biol. 34, 1716–1722 (2014)

    PubMed  CAS  Google Scholar 

  34. 34

    Itkin, T. et al. FGF-2 expands murine hematopoietic stem and progenitor cells via proliferation of stromal cells, c-Kit activation, and CXCL12 down-regulation. Blood 120, 1843–1855 (2012)

    PubMed  CAS  Google Scholar 

  35. 35

    Zhao, M. et al. FGF signaling facilitates postinjury recovery of mouse hematopoietic system. Blood 120, 1831–1842 (2012)

    PubMed  PubMed Central  CAS  Google Scholar 

  36. 36

    Murakami, M. et al. The FGF system has a key role in regulating vascular integrity. J. Clin. Invest. 118, 3355–3366 (2008)

    PubMed  PubMed Central  CAS  Google Scholar 

  37. 37

    De Smet, F. et al. Fibroblast growth factor signaling affects vascular outgrowth and is required for the maintenance of blood vessel integrity. Chem. Biol. 21, 1310–1317 (2014)

    PubMed  CAS  Google Scholar 

  38. 38

    Houlihan, D. D. et al. Isolation of mouse mesenchymal stem cells on the basis of expression of Sca-1 and PDGFR-α. Nature Protocols 7, 2103–2111 (2012)

    PubMed  CAS  Google Scholar 

  39. 39

    Wei, J. et al. Glucose uptake and Runx2 synergize to orchestrate osteoblast differentiation and bone formation. Cell 161, 1576–1591 (2015)

    PubMed  PubMed Central  CAS  Google Scholar 

  40. 40

    Fujita, K. et al. Vitamin E decreases bone mass by stimulating osteoclast fusion. Nature Med. 18, 589–594 (2012)

    PubMed  CAS  Google Scholar 

  41. 41

    Gur-Cohen, S. et al. PAR1 signaling regulates the retention and recruitment of EPCR-expressing bone marrow hematopoietic stem cells. Nature Med. 21, 1307–1317 (2015)

    PubMed  Google Scholar 

  42. 42

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

    ADS  PubMed  CAS  Google Scholar 

  43. 43

    Arai, F. et al. Tie2/angiopoietin-1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche. Cell 118, 149–161 (2004)

    PubMed  CAS  Google Scholar 

  44. 44

    Sugimura, R. et al. Noncanonical Wnt signaling maintains hematopoietic stem cells in the niche. Cell 150, 351–365 (2012)

    PubMed  PubMed Central  CAS  Google Scholar 

  45. 45

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

    ADS  PubMed  CAS  Google Scholar 

  46. 46

    Bear, M. D. et al. Alpha-Catulin co-localizes with vimentin intermediate filaments and functions in pulmonary vascular endothelial cell migration via ROCK. J. Cell. Physiol. https://doi.org/10.1002/jcp.25185 (2015)

  47. 47

    Acar, M. et al. Deep imaging of bone marrow shows non-dividing stem cells are mainly perisinusoidal. Nature 526, 126–130 (2015)

    ADS  PubMed  PubMed Central  CAS  Google Scholar 

  48. 48

    Mantel, C. R. et al. Enhancing hematopoietic stem cell transplantation efficacy by mitigating oxygen shock. Cell 161, 1553–1565 (2015)

    PubMed  PubMed Central  CAS  Google Scholar 

  49. 49

    Shen, H. et al. An acute negative bystander effect of γ-irradiated recipients on transplanted hematopoietic stem cells. Blood 119, 3629–3637 (2012)

    PubMed  PubMed Central  CAS  Google Scholar 

  50. 50

    Hu, L. et al. Antioxidant N-acetyl-l-cysteine increases engraftment of human hematopoietic stem cells in immune-deficient mice. Blood 124, e45–e48 (2014)

    PubMed  PubMed Central  CAS  Google Scholar 

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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.).

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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.

Corresponding authors

Correspondence to Charles P. Lin or Tsvee Lapidot.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Properties of distinct types of bone marrow blood vessels and cells in their microenvironment.

a, Flow cytometry quantitative analysis of VE-cadherin and ZO-1 MFIs by BMEC sub-populations. Mean ± s.e.m., n = 9 mice from three independent experiments. Two-tailed Student’s t-test; ***P < 0.005. b, Representative confocal image showing CD31 (red) and Sca-1+ (green) arterial blood vessels on proximity to endosteal regions in the metaphysis and representative confocal image of endosteal regions in the metaphysis showing Sca-1+ (green) arterial blood vessels, αSMA+ (blue) pericytes, and OPN (red) for endosteal borders. Scale bars indicate 200 μm. c, Frequencies of Sca-1+ arterial blood vessels distribution among zones representing growing distances from the endosteum in the calvaria and femur. d, Average diameters of distinct types of blood vessels in the clavarial and femoral marrow. e, f, Representative images of arterial blood vessel (green, left) and of sinusoidal blood vessels (green, right) indicating how the frequency of ROShigh cells around these blood vessels was scored. The grey-masked areas surrounding the blood vessels indicate the region of distance <20 μm from the blood vessels. Odd numbers (1, 3, and 5) tag the nuclei of cells (blue) found in the region of interest, while even numbers (2, 4, and 6) tag ROShigh (red) cells in the region of interest. ImageJ cell counter plugin was used to analyse and score the number of total cells and ROShigh cells in the region of interest. Yellow numbers in the centre of the blood vessels indicate how many ROShigh cells are scored out of total cells. Scale bar indicates 20 μm. g, Frequency of ROShigh cells scored among total bone marrow cells found in proximity (<20 μm) to different bone marrow blood vessels. Mean ± s.e.m., n = 24 bone marrow sections were analysed from n = 6 mice. Two-tailed Student’s t-test; ***P < 0.005. hk, White arrowheads indicate for SLAM HSPC h, Representative confocal images with ROS probe (red) of ROSlow/−, CD150+ (pink)/CD48 (blue) SLAM HSPC, found away (>20 μm) from Sca-1+ (green) endosteal blood vessels, neighbouring a megakaryocyte. Yellow dashed line indicates sinusoidal borders. Scale bar indicates 20 μm. i, Representative confocal images of ROShigh (red), CD150+ (pink) and CD48 (blue) SLAM-HSPC, found away (>20 μm) from Sca-1+ (green) endosteal blood vessels, surrounded by mature haematopoietic cells. Yellow dashed line indicates sinusoidal borders. Scale bar indicates 20 μm. j, Representative confocal images of cells with ROShigh (red) levels among CD150+ (pink) and CD48 (blue) SLAM-HSPC neighbouring (<20 μm) Sca-1+ (green) the endosteal arteriole. Scale bar indicates 20 μm. k, Representative tile scan confocal images of bone marrow merged Z-stalk showing (I) CD31+ blood vessels (blue) and their neighbouring CD150+ (green) CD48/Lin (red) negative SLAM HSPC. (II) Cells nuclei are visualized (green) together with CD48/Lin (red) and CD31+ blood vessels (blue). Scale bars indicate 30 μm.

Extended Data Figure 2 Different populations of nestin-expressing bone marrow cells are associated with nestin-expressing arterioles.

a, Representative fluorescence images of Sca-1+ (green) blood vessels and their neighbouring NG2+ (red) MSPCs. NG2+ MSPCs were either negative (yellow arrow) or positive (white arrow) for Sca-1 expression. Scale bar indicates 20 μm. b, Representative fluorescence images of Sca-1+ (red) blood vessels and nestin-GFP labelling (green) blood vessels and MSPCs (white arrows). Scale bar indicates 20 μm. c, Representative fluorescence images of nestin+ (green) blood vessels and VE-cadherin (red) staining, showing that nestin+ blood vessel structures are co-stained with VE-cadherin while neighbouring sinusoids are VE-cadherin+/nestin. Scale bar indicates 20 μm. d, Representative fluorescence images of nestin+ (green) blood vessels and their neighbouring NG2+ (red) MSPCs. NG2+/nestin+ MSPCs surrounded NG2/nestin+ aBMECs with elongated nuclei (white arrow). Scale bar indicates 20 μm. e, Representative fluorescence images of large- and small-diameter nestin+ (green) blood vessels and blood vessels positive for LDL (red) uptake, indicating that nestin+ labels arteries and arterioles but not sinusoids. Scale bar indicates 20 μm. f, Representative flow cytometry histogram plots for gated BMECs, showing nestin-GFP expression on BMEC subpopulation which is Sca-1+ or nestin-GFP expression by Sca-1+/− BMEC subpopulation. Mean ± s.e.m., n = 6 mice from three independent experiments. g, Representative ImageStream images of CD45-CD31+Sca-1nestin sBMECs and CD45CD31+Sca-1+nestin+ aBMECs, CD45CD31Sca-1+/−nestin+ MSPCs, and CD45+CD31Sca-1+/−nestin+ haematopoietic cells. h, Representative confocal tile scan of nestin-GFP (green) femur stained with αSMA (red). Scale bar indicates 200 μm. i, Representative confocal images of endosteal regions in the metaphysis showing αSMA (red) enwrapped nestin+ (green) andCD31+ (white) arterial blood vessels branching into smaller endosteal nestin+CD31+ arterioles which are not associated with αSMA+ pericytes. Endosteal nestin+ blood vessels are surrounded by nestin+ MSPCs. Scale bars indicate 50 μm. j, k, Representative confocal images of diaphysial area (j) and metaphysial area (k) showing GFAP (red, Schwann cell marker) fibres associated with Sca-1+ (green) arterial blood vessel (j) or with Sca-1+ endosteal arterioles (k). Scale bar indicates 50 μm (j) and 100 μm (k). l, Bone marrow cells were incubated with 20 ng ml−1 TGFβ1 or vehicle for 2 h. ROS MFI levels in bone marrow SLAM HSPCs were determined by flow cytometry quantitative analysis. Mean ± s.e.m., n = 9 repeats in triplicates from three independent experiments. Two-tailed Student’s t-test; ***P < 0.005.

Extended Data Figure 3 Expression pattern of molecules involved in cellular trafficking by distinct types of blood vessels.

ah, Expression levels (MFI) of indicated surface or intracellular molecules by distinct types of BMEC as measured by flow cytometry analysis. Mean ± s.e.m., n = 8 Sca-1-EGFP and wild-type mice from two independent experiments). Two-tailed Student’s t-test; **P < 0.01, ***P < 0.005.

Extended Data Figure 4 Femural and calvarial comparison and monitoring of calvarial trafficking.

a, Evans blue dye (EBD) absorbance following extraction from the femurs or calvarias, was measured using spectrophotometric analysis at 620 nm and 740 nm and normalized to total protein content per femur (Bradford). Mean ± s.e.m., n = 6 mice from two independent experiments. be, Mean ± s.e.m., n = 8 mice from two independent experiments. Two-tailed Student’s t-test; **P < 0.01, ***P < 0.005. b, Total BMEC frequency as determined by flow cytometry analysis. c, Sca-1+ aBMEC frequency as determined by flow cytometry analysis. d, e, VE-cadherin and ZO-1 expression (MFI) on distinct types of BMECs as determined by flow cytometry analysis. f, A representative plot showing the flow speed of an HSPC passing through a network of nestin-GFP+/− blood vessels as a function of time. Note that the cell temporarily stops within a sinus at ~0.4 s and slowly roles until it adheres again at ~0.7 s. g, Snapshot images from 0, 0.03, 0.10, 0.53, and 6.3 s taken from Supplementary Video 5. Nestin-GFP (green), HSPC (red), blood vessels (grey), and bone (blue) are displayed. The cell is overlaid on the pre-acquired nestin-GFP, blood vessels, and bone images. Yellow arrows indicate for the location of the trafficking HSPC. Scale bars indicate 100 μm.

Extended Data Figure 5 Properties of distinct types of bone marrow blood vessels under HSPC mobilization conditions and the role of the endothelial CXCL12–CXCR4 axis.

ae, C57BL/6 or nestin-GFP mice received a single injection of AMD3100 (5 mg per kg) and were analysed 5 min (for pCXCR4) or 30 min later. Mean ± s.e.m., n = 7 mice from three independent experiments. Two-way ANOVA with Bonferroni’s multiple comparison post-hoc test; *P < 0.05, **P < 0.01, ***P < 0.005. a, Evans blue dye (EBD) absorbance following EBD (30 mg per kg) injection together with AMD3100. bd, Flow cytometry quantitative analysis and representative histogram plots of VE-cadherin, membrane-bound CXCL12, and membranal SCF MFIs. e, Intracellular CXCR4 phosphorylation (pCXCR4) levels (MFI) in distinct types of BMECs as measured by flow cytometry analysis and representative histogram plots. f, C57BL/6 mice received two injections (30 min interval) of 50 μg 12G5 CXCR4 neutralizing antibodies or IgG control followed by EBD injection. EBD absorbance following extraction from the femur was measured using spectrophotometric analysis at 620 nm and 740 nm. Mean ± s.e.m., n = 6 mice from two independent experiments. Two-tailed Student’s t-test, **P < 0.01. g, Endothelial cell (EC)-specific inducible deletion of Cxcr4 (EndoΔCxcr4) or Fgfr1/2 (EndoΔFgfr1/2) in mice. Mice harbouring loxP sites flanking Cxcr4 or Fgfr1 and Fgfr2 genes were crossed with a mouse line with endothelial-cell-specific VE-cadherin promoter-driven CreERT2 (VE-cadherin (Cdh5, PAC)-CreERT2). Specificity of VE-cadherin (Cdh5, PAC)-CreERT2 was validated in reporter mice carrying enhanced YFP protein following floxed stop codon (EndoYFP). Cxcr4 or Fgfr1/2 deletion or YFP expression in endothelial cells was induced by tamoxifen injection. Mouse analysis was performed 4 weeks after tamoxifen-induced Cre activity. Mice carrying only the Cxcr4lox/lox or Fgfr1/2lox/lox mutations or VE-cadherin (Cdh5, PAC)-CreERT2 transgene served as controls. hj, Mean ± s.e.m., n = 12 mice from four independent experiments. Two-tailed Student’s t-test; ***P < 0.005. h, Representative flow cytometry histogram and dot plots confirming BMEC specific induction of Cre activity by exclusive expression of YFP in ~70% of BMEC. i, Frequency of YFP expression and representative histogram plot, among BMEC sub-populations, was determined by flow cytometry quantitative analysis 4 weeks after tamoxifen induction of Cre activity. Note higher Cre activity, indicated by higher YFP signal, in aBMECs. Black line indicates for a positive signal region. j, Fluorescent representative images of YFP expression by distinct BMBVs (sinusoids and arteries). km, Tamoxifen-treated wild-type and EndoΔCxcr4 mice were allowed to recover for 4 weeks before studies. Mean ± s.e.m., n = 9 mice from three independent experiments. Two-tailed Student’s t-test; *P < 0.05, **P < 0.01, ***P < 0.005. k, EBD absorbance in wild-type and EndoΔCxcr4 mice. l, Flow cytometry quantitative analysis of VE-cadherin MFI on BMEC from wild-type and EndoΔCxcr4 mice. m, Flow cytometry quantitative analysis of blood LSK HSPCs and CD34LSK HSPCs of wild-type or EndoΔCxcr4 mice.

Extended Data Figure 6 FGF-2 administration remodels the bone marrow vasculature and the stromal compartment while retaining HSPCs in the bone marrow.

a, b, Bone marrow cells were incubated for 2 h with (25% blood plasma) or without (control) peripheral blood plasma. Mean ± s.e.m., n = 9 repeats from three independent experiments. Two-tailed Student’s t-test; ***P < 0.005. a, Frequencies of cycling Ki67+ SLAM LSK HSPC. b, Frequencies of apoptotic annexinV+ SLAM LSK HSPC. af, C57BL/6 or nestin-GFP mice were treated with FGF-2 (200 μg per kg) for 7 days. Mean ± s.e.m., n = 9 mice from three independent experiments. Two-tailed Student’s t-test; *P < 0.05, ***P < 0.005, **P < 0.01. c, d, Quantitative analysis of VE-cadherin and ZO-1 MFIs on BMECs. e, EBD absorbance. f, g, Flow cytometry quantitative analysis of BMEC frequencies expressing Sca-1, nestin and intracellular Ki67 cell cycling markers. h, Diameters of distinct types of bone marrow blood vessels in the metaphysis region as determined by ImageJ software analysis of high-resolution confocal images. i, Fluorescent representative images of LDL (red) uptake by sinusoidal BMEC and other bone marrow cells following diffusion into the parenchymal marrow. Note lower LDL uptake and diffusion following FGF-2 treatment. Scale bar indicates 20 μm. j, Representative confocal images of Sca-1+ (green) arterial blood vessels in the metaphysis region. Note higher abundance of arterial blood vessels following FGF-2 treatment. Scale bar indicates 200 μm. k, For the homing assay, bone marrow cells from c-Kit-EGFP labelled mice were lineage depleted, and transplanted to the indicated recipient mice. Four hours after transplantation, bones from recipient mice were recovered, flushed and crushed, and the numbers of homed Linc-Kit-EGFP+Sca-1+CD34 HSPCs were determined per femur by flow cytometry quantitative analysis. lq, Mice were treated with FGF-2 (200 μg per kg) for 7 days. Mean ± s.e.m., unless indicated otherwise n = 12 mice from three independent experiments. Two-tailed Student’s t-test; **P < 0.01, ***P < 0.005. l, HSPC homing per femur. Mean ± s.e.m., n = 8 mice from three independent experiments. m, Numbers of LSK HSPCs in the blood. n, Levels of chimaerism indicating LTR-HSC contribution from blood transplant. Mean ± s.e.m., n = 20 mice from two independent experiments. o, Frequencies and representative density plots of bone marrow αSMA+ pericytes as determined by flow cytometry analysis. p, q, Expression levels (MFI) and representative histograms of glucose uptake by HSPCs and MSPCs, respectively, were determined by flow cytometry analysis.

Extended Data Figure 7 Genetic breaching of the endothelial barrier remodels the bone marrow vasculature and the stromal compartment while enhancing HSPC egress in a ROS dependent manner.

a, EBD absorbance. Mean ± s.e.m., n = 6 mice from three independent experiments. Two-tailed Student’s t-test; **P < 0.01. b, c, Quantitative analysis of VE-cadherin and ZO-1 MFIs on BMEC. Mean ± s.e.m., n = 6 mice from three independent experiments. Two-tailed Student’s t-test; *P < 0.05. dh, Flow cytometry quantitative analysis of BMEC frequencies, surface and intracellular molecules expression (MFI), in wild-type or EndoΔFgfr1/2 mice. Mean ± s.e.m., n = 9 mice from three independent experiments. Two-tailed Student’s t-test; **P < 0.01, ***P < 0.005. i, Diameters of distinct types of bone marrow blood vessels in the metaphysis region as determined by ImageJ software analysis of high-resolution confocal images. j, Representative confocal representative images of Sca-1+ (green) arterial blood vessels in the metaphysis region. Note lower abundance of arterial blood vessels in EndoΔFgfr1/2 mice. Scale bar indicates 200 μm. k, l, Mean ± s.e.m., n = 16 bone marrow sections were analysed from n = 4 mice. Two-way ANOVA with Bonferroni’s multiple comparison post-hoc test; **P < 0.01, ***P < 0.005). k, Frequency of ROShigh cells scored among total bone marrow cells found in proximity (<20 μm) to different bone marrow blood vessels, in wild-type or EndoΔFgfr1/2 mice. l, Frequency of ROShigh cells scored among total bone marrow cells found in proximity (<20 μm) to different bone marrow blood vessels, in C57BL/6 mice treated with neutralizing rat anti-VE-cadherin antibodies or rat IgG control antibodies (50 μg per mouse per day) for 2 days. mo, Flow cytometry quantitative analysis of HSPC glucose uptake (m) (MFI), frequency of cycling HSPC (n) and apoptotic HSPC (o). Mean ± s.e.m., n = 9 mice from three independent experiments. Two-tailed Student’s t-test; **P < 0.01, ***P < 0.005. p, q, Frequencies of donor-derived lymphoid B220+ or myeloid CD11b+ cells in the PB of recipient mice, as were determined 24 weeks after transplantation by flow cytometry. Mean ± s.e.m., n = 18 donor mice from two independent experiments, for 3 recipient mice per donor. Two-tailed Student’s t-test; ***P < 0.005. rt, Wild-type or EndoΔFgfr1/2 mice were treated with NAC (130 mg per kg) or PBS for 7 days. Mean ± s.e.m., n = 9 mice from three independent experiments. Two-tailed Student’s t-test; **P < 0.01, ***P < 0.005. r, Number of circulating peripheral blood HSPC as determined by quantitative flow cytometry analysis. s, Number of bone marrow SLAM LSK HSPC as determined by quantitative flow cytometry analysis. t, Levels of chimaerism, indicating LTR-HSC contribution, were determined 24 weeks after transplantation by flow cytometry ratio analysis (CD45.2/(CD45.2 + CD45.1)). Mean ± s.e.m., n = 24 donor mice from two independent experiments, for 3 recipient mice per donor.

Extended Data Figure 8 Endothelial barrier manipulation affects stromal properties, development and the levels of bone remodelling hormones.

ad, Mean ± s.e.m., n = 9 mice from three independent experiments. Two-tailed Student’s t-test; **P < 0.01, ***P < 0.005. a, Glucose uptake by bone marrow MSPC as determined by quantitative flow cytometry (MFI) analysis. b, Frequencies of bone marrow αSMA+ pericytes as determined by flow cytometry analysis. c, Average number of scored (ImageJ) CFU-F per well and representative images. d, Average determined (ImageJ) percentage of mineralized area per well and representative images. ei, C57BL/6 mice were treated with neutralizing rat anti-VE-cadherin antibodies or rat IgG control antibodies (50 μg per mouse per day) for 5 days. Mean ± s.e.m., n = 9 mice from three independent experiments. Two-tailed Student’s t-test and one-way ANOVA with Bonferroni’s multiple comparison post-hoc test; **P < 0.01, ***P < 0.005. e, Frequency of bone marrow MSPC as determined by flow cytometry quantitative analysis. f, Glucose uptake by bone marrow MSPC as determined by quantitative flow cytometry (MFI) analysis. g, Frequencies of bone marrow αSMA+ pericytes as determined by flow cytometry analysis. h, Average number of scored (ImageJ) CFU-F per well and representative images. i, Average determined (ImageJ) percentage of mineralized area per well and representative images. jo, Bone marrow supernatants from wild-type or EndoΔFgfr1/2, PBS or FGF-2 (200 μg per kg) treated for 7 days, and IgG or rat anti-VE-cadherin (50 μg per mouse per day) for 5 days, were isolated and bone marrow concentrations of calcitonin and PTH hormones were determined using an ELISA assay. Mean ± s.e.m., n = 9 mice per group from three independent experiments. Two-tailed Student’s t-test; ***P < 0.005.

Extended Data Figure 9 Pharmacological breaching of the endothelial barrier remodels the bone marrow vasculature and the stromal compartment while enhancing HSPCs egress in a ROS-dependent manner.

a–j, C57BL/6 mice were treated with neutralizing rat anti-VE-cadherin antibodies or rat IgG control antibodies (50 μg per mouse per day) for 5 days. Mean ± s.e.m., unless otherwise indicated, n = 9 mice from three independent experiments. Two-tailed Student’s t-test; *P < 0.05, **P < 0.01, ***P < 0.005. a, EBD absorbance. b, HSPCs homing per femur. c, Quantitative analysis of blood LSK HSPC and chimaerism levels indicating LTR-HSC contribution. Mean ± s.e.m., n = 10 mice from two independent experiments. d, Chimaerism levels indicating LTR-HSC contribution. Mean ± s.e.m., n = 18 donor mice from two independent experiments, for 3 recipient mice per donor. e, f, Quantitative analysis and representative histogram plots of HSPCs and PαS MSPCs ROS MFI. g, Representative images of ROShigh (red) cells in proximity to blood vessels. Scale bar indicates 20 μm. hk, C57BL/6 mice were treated with neutralizing rat anti-VE-cadherin antibodies or rat IgG control antibodies (50 μg per mouse per day) for 2 days. Where indicated, mice were also treated with NAC (130 mg per kg) or PBS for 2 days. Mean ± s.e.m., n = 9 mice from three independent experiments. Two-tailed Student’s t-test and one-way ANOVA with Bonferroni’s multiple comparison post-hoc test; *P < 0.05, ***P < 0.005. h, White blood cell (WBC) numbers in the blood circulation were determined using a haematocytometer and Turk lysis of erythrocytes. i, Flow cytometry quantitative analysis of CD34- LSK HSPC in the blood circulation. jk, Bone marrow MNC or bone marrow lineage depleted cells from treated mice were seeded on a 5 μm pore transwell and allowed to migrate for 2 h towards CXCL12 (125 ng ml−1). Following migration, the frequency of migrated bone marrow MNC or CD34/LSK HSPC was determined by flow cytometry quantitative analysis. Note preferential HSPC enhanced migration after VE-cadherin neutralization. lp, C57BL/6 mice were treated with neutralizing rat anti-VE-cadherin antibodies or rat IgG control antibodies (50 μg per mouse per day) for 5 days. Mean ± s.e.m., n = 9 mice from three independent experiments. Two-tailed Student’s t-test; **P < 0.01, ***P < 0.005. l, Glucose uptake levels (MFI) by HSPC as determined by flow cytometry quantitative analysis. mn, Frequencies of Ki67+ cycling and AnnexinV+ SLAM LSK HSPC as determined by flow cytometry quantitative analysis. o, Frequency of Sca-1+ aBMEC out of the total as determined by flow cytometry quantitative analysis. p, Diameters of distinct types of bone marrow blood vessels in the metaphysis region as determined by ImageJ software analysis of high-resolution confocal images. q, Confocal representative images of Sca-1+ (green) arterial blood vessels in the metaphysis region. Note lower abundance of arterial blood vessels following 5 days of anti-VE-cadherin treatment. Scale bar indicates 200 μm. rt, C57BL/6 mice were treated with NAC (130 mg per kg) or PBS for 7 days. Mean ± s.e.m., n = 8 mice from two independent experiments. Two-tailed Student’s t-test; ***P < 0.005. r, Flow cytometry quantitative analysis of CD34 LSK HSPC in the blood circulation. s, EBD absorbance following extraction from the femur was measured using spectrophotometric analysis at 620 nm and 740 nm. t, VE-cadherin expression levels (MFI) on distinct types of BMEC as determined by flow cytometry quantitative analysis (arterial and sinusoidal respectively).

Extended Data Figure 10 Illustration of proposed bone marrow blood vessels model and regulation of haematopoiesis.

Bone marrow vasculature is composed of two main types of blood vessels which are arterial blood vessels and sinusoids. Blood enters the bone marrow via the arteries, branching to smaller arterioles, which in proximity to endosteal areas, further branch into small-diameter endosteal arterioles. These endosteal arterioles reconnect to downstream sinusoids which drain the blood into the central sinus and out of the bone marrow. Arterial BMEC have elongated nuclear morphology, express Sca-1 and nestin markers, and display high barrier integrity properties. In addition, arterial blood vessels display the highest blood flow speed and shear rate. Arterial BMEC maintain a microenvironment that promotes low ROS state of HSCs in its surrounding. The second layer of cells associated with arteries is composed of αSMA+ pericytes, while endosteal arterioles are associated with HSC-supportive MSPCs. The association of MSPCs and ROSlow HSCs with endosteal capillaries suggests the existence of an osteo-vascular niche where the residing HSCs are influenced by both endosteal and vascular elements simultaneously. Innervating Schwann cell nerve fibres, shown to maintain HSC dormancy, were found to be associated with arteries and endosteal arterioles. More permeable fenestrated sinusoids induce higher ROS state in their surroundings, and have slower internal blood flow, all of which makes them the ultimate candidate to serve as the site for bone marrow cellular trafficking. Megakaryocytes found in sinusoidal sites support and maintain HSPC in a ROS low state. Live real-time imaging indicates that all leukocyte trafficking occurs exclusively via sinusoids. Furthermore, experimental systems manipulating endothelial barrier integrity provide evidence that more fenestrated endothelial state promotes trafficking at the expense of stem cell maintenance. Yet, conditions enhancing endothelial integrity, reducing cellular trafficking promote bone marrow stem cell expansion and maintenance. Peripheral blood plasma, which can penetrate into the bone marrow more easily via fenestrated blood vessels, enhances HSPC migratory capacity but hampers their long-term repopulation capacity and survival. Thus, the state of the endothelial blood–bone-marrow barrier in distinct blood vessels and under steady state or ‘stress’ conditions may have a strong regulatory impact on tissue residing stem cells.

Supplementary information

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. (PDF 76 kb)

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. (MP4 4324 kb)

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. (MP4 3145 kb)

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. (MP4 67409 kb)

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. (MP4 65017 kb)

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). (MP4 1325 kb)

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. (MP4 22500 kb)

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. (MP4 3109 kb)

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. (MP4 462 kb)

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. (MP4 422 kb)

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. (MP4 777 kb)

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Itkin, T., Gur-Cohen, S., Spencer, J. et al. Distinct bone marrow blood vessels differentially regulate haematopoiesis. Nature 532, 323–328 (2016). https://doi.org/10.1038/nature17624

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