Distinct bone marrow blood vessels differentially regulate haematopoiesis

doi:10.1038/nature17624

. 2016 Apr 21;532(7599):323-8.

doi: 10.1038/nature17624. Epub 2016 Apr 13.

Distinct bone marrow blood vessels differentially regulate haematopoiesis

Tomer Itkin¹, Shiri Gur-Cohen¹, Joel A Spencer^{2

3}, Amir Schajnovitz^{4

5

6}, Saravana K Ramasamy⁷, Anjali P Kusumbe⁷, Guy Ledergor^{1

8}, Yookyung Jung^{2

3}, Idan Milo¹, Michael G Poulos⁹, Alexander Kalinkovich¹, Aya Ludin¹, Orit Kollet¹, Guy Shakhar¹, Jason M Butler⁹, Shahin Rafii⁹, Ralf H Adams⁷, David T Scadden^{4

5

6}, Charles P Lin^{2

3}, Tsvee Lapidot¹

Affiliations

¹ Department of Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel.
² Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA.
³ Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA.
⁴ Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
⁵ Harvard Stem Cell Institute, Cambridge, Massachusetts 02114, USA.
⁶ Center for Regenerative Medicine and Cancer Center, Massachusetts General Hospital, Boston, Massachusetts 02114, USA.
⁷ Max Planck Institute for Molecular Biomedicine, Department of Tissue Morphogenesis and Faculty of Medicine, University of Münster, D-48149 Münster, Germany.
⁸ Internal Medicine Department, Tel-Aviv Sourasky Medical Center, Tel-Aviv 64239, Israel.
⁹ Department of Genetic Medicine, Weill Cornell Medical College, New York, New York 10065, USA.

PMID: 27074509
PMCID: PMC6450701
DOI: 10.1038/nature17624

Distinct bone marrow blood vessels differentially regulate haematopoiesis

Tomer Itkin et al. Nature. 2016.

. 2016 Apr 21;532(7599):323-8.

doi: 10.1038/nature17624. Epub 2016 Apr 13.

Authors

Affiliations

¹ Department of Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel.
² Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA.
³ Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA.
⁴ Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
⁵ Harvard Stem Cell Institute, Cambridge, Massachusetts 02114, USA.
⁶ Center for Regenerative Medicine and Cancer Center, Massachusetts General Hospital, Boston, Massachusetts 02114, USA.
⁷ Max Planck Institute for Molecular Biomedicine, Department of Tissue Morphogenesis and Faculty of Medicine, University of Münster, D-48149 Münster, Germany.
⁸ Internal Medicine Department, Tel-Aviv Sourasky Medical Center, Tel-Aviv 64239, Israel.
⁹ Department of Genetic Medicine, Weill Cornell Medical College, New York, New York 10065, USA.

PMID: 27074509
PMCID: PMC6450701
DOI: 10.1038/nature17624

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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Conflict of interest statement

The authors do not declare competing financial interests.

Figures

**Extended Data figure 1:**
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). P-values, two-tailed student’s t-test (***P<0.005). b, Representative confocal image showing CD31(red)/Sca-1⁺ (green) arterial BVs on proximity to endosteal regions in the metaphysis and representative confocal image of endosteal regions in the metaphysis showing Sca-1⁺ (green) arterial BVs, αSMA⁺ (blue) pericytes, and OPN (red) for endosteal borders. Scales indicate 200 μm. c, Frequencies of Sca-1⁺ arterial BVs distribution among zones representing growing distances from the endosteum in the calvaria and femur. d, Average diameters of distinct types of BVs in the clavarial and femoral marrow. **e-f**, Representative images of arterial BV (green, left) and of sinusoidal BVs (green, right) explaining how frequency of ROS^High cells around these BVs was scored. The gray-masked areas surrounding the BVs indicate the region of distance <20 μm from the BVs. 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 ROS^High (red) cells in the region of interest. ImageJ cell counter plugin was used to analyze and score the number of total cells and ROS^High cells in the region of interest. Yellow numbers in the center of the BVs indicate how many ROS^High cells are scored out of total cells. Scale bar indicates 20 μm. g, Frequency of ROS^high cells scored among total BM cells found in proximity (<20 μm) to different BM BVs. (Mean ± s.e.m., n=24 BM sections were analyzed from n=6 mice). P-values, two-tailed student’s t-test (***P<0.005). **h-k**, White arrow heads indicate for SLAM HSPC h, Representative confocal images with ROS probe (red) of ROS^low/-, CD150⁺ (pink)/CD48⁻ (blue) SLAM HSPC, found away (>20 μm) from Sca-1⁺ (green) endosteal BVs, neighboring a megakaryocyte. Yellow dashed line indicates sinusoidal borders. Scale bar indicates 20 μm. i, Representative confocal images of ROS^high (red), CD150⁺ (pink)/CD48⁻ (blue) SLAM-HSPC, found away (>20 μm) from Sca-1⁺ (green) endosteal BVs, surrounded by mature hematopoietic cells. Yellow dashed line indicates for sinusoidal borders. Scale bar indicates 20 μm. j, Representative confocal images of cells with ROS^High (red) levels among CD150⁺ (pink)/CD48⁻ (blue) SLAM-HSPC neighboring (<20 μm) Sca-1⁺ (green) endosteal arteriole. Scale bar indicates 20 μm. k, Representative tile scan confocal images of BM merged Z-stalk showing **(I)** CD31⁺ BVs (blue) and their neighboring CD150⁺ (green) CD48/Lin (red) negative SLAM HSPC. **(II)** Cells nuclei are visualized (green) together with CD48/Lin (red) and CD31⁺ BVs (blue). Scale bars indicate 30 μm.

**Extended Data figure 2:**
a, Representative fluorescence images of Sca-1⁺ (green) BVs and their neighboring 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) BVs and nestin-GFP labeling (green) BVs and MSPCs (white arrows). Scale bar indicates 20 μm. c, Representative fluorescence images of nestin⁺ (green) BVs and VE-cadherin (red) staining, showing that nestin⁺ BV structures are co-stained with VE-cadherin while neighboring sinusoids are VE-cadherin⁺/nestin⁻. Scale bar indicates 20 μm. d, Representative fluorescence images of nestin⁺ (green) BVs and their neighboring 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) BVs and BVs 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-1⁻/nestin⁻ sBMECs and CD45⁻/CD31⁺/Sca-1⁺/nestin⁺ aBMECs, CD45⁻/CD31⁻/Sca-1^+/−/nestin⁺ MSPCs, and CD45⁺/CD31⁻/Sca-1^+/−/nestin⁺ hematopoietic 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)/CD31⁺ (white) arterial blood vessels branching into smaller endosteal nestin⁺/CD31⁺ arterioles which are not associated with αSMA⁺ pericytes. Endosteal nestin⁺ BVs are surrounded by nestin⁺ MSPCs. Scale bars indicate 50 μm. **j-k**, Representative confocal images of diaphysial area (a) and metaphysial area (b) showing GFAP (red, Schwann cell marker) fibers associated with Sca-1⁺ (green) arterial BV (a) or with Sca-1⁺ endosteal arterioles (b). Scale bar indicates 50 μm for (a) and 100 μm for (b). l, BM cells were incubated with 20 ng/ml TGFβ1 or vehicle for 2 h. ROS MFI levels in BM SLAM HSPCs were determined by flow cytometry quantitative analysis. (Mean ± s.e.m., n=9 repeats in triplicates from three independent experiments). P-values, two-tailed student’s t-test (***P<0.005).

**Extended Data figure 3:**
**a-h**, 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 mice from two independent experiments). P-values, two-tailed student’s t-test (**P<0.01, ***P<0.005).

**Extended Data figure 4:**
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).b-e, (Mean ± s.e.m., n=8 mice from two independent experiments). P-values, 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^+/− BVs 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 (gray), 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:**
**a-e**, C57BL/6 or nestin-GFP mice received a single injection of AMD3100 (5 mg/kg) and were analyzed 5 min (for pCXCR4) or 30 min later. (Mean ± s.e.m., n=7 mice from three independent experiments). P-values, 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/kg) injection together with AMD3100. **b-d**, 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). P-values, two-tailed student’s t-test (**P<0.01). g, Endothelial cell (EC)-specific inducible deletion of CXCR4 (eΔCXCR4) or FGFR1/2 (eΔFGFR1/2) in mice. Mice harboring 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 (eYFP). CXCR4 or FGFR1/2 deletion or YFP expression in endothelial cells was induced by tamoxifen injection. Mice analysis was performed 4 weeks post tamoxifen-induced Cre activity. Mice carrying only the CXCR4^lox/lox or FGFR1/2^lox/lox mutations or VE-cadherin(Cdh5, PAC)-CreERT2 transgene served as controls. **h-j**, (Mean ± s.e.m., n=12 mice from four independent experiments). P-values, 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 post 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). **k-m**, Tamoxifen treated WT and eΔCXCR4 mice were allowed to recover for 4 weeks before studies. (Mean ± s.e.m., n=9 mice from three independent experiments). P-values, two-tailed student’s t-test (*P<0.05, ***P<0.005, **P<0.01). k, EBD absorbance in WT and eΔCXCR4 mice. l, Flow cytometry quantitative analysis of VE-cadherin MFI on BMEC from WT and eΔCXCR4 mice. m, Flow cytometry quantitative analysis of blood LSK HSPCs and CD34⁻LSK HSPCs of WT or eΔCXCR4 mice.

**Extended Data figure 6:**
**a-b**, BM cells were incubated for 2h with (25% P) or without (control) PB plasma. (Mean ± s.e.m., n=9 repeats from three independent experiments). P-values, 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. **a-f**, C57BL/6 or nestin-GFP mice were treated with FGF-2 (200 μg/kg) for 7 days. (Mean ± s.e.m., n=9 mice from three independent experiments). P-values, 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 marker. h, Diameters of distinct types of BM BVs in the metaphysis region as determined by ImageJ software analysis of high-resolution confocal images. i, Fluorescent representative images of LDL (red) up-taken by sinusoidal BMEC and other BM cells following diffusion into the parenchymal marrow. Note lower LDL uptake and diffusion following FGF-2 treatment. Scale bar indicates 20 μm. j, Confocal representative images of Sca-1⁺ (green) arterial BVs in the metaphysis region. Note higher abundance of arterial BVs following FGF-2 treatment. Scale bar indicates 200 μm. k, For performance of homing assay, BM cells from c-Kit-EGFP labeled mice were lineage depleted, and transplanted to indicated recipient mice. Four hours post-transplantation bones from recipient mice were recovered, flushed and crushed, and the numbers of homed Lin⁻/c-Kit-EGFP⁺/Sca-1⁺/CD34⁻ HSPCs were determined per femur by flow cytometry quantitative analysis. **l-q**, Mice were treated with FGF-2 (200 μg/kg) for 7 days. (Mean ± s.e.m., unless indicated otherwise n=12 mice from three independent experiments). P-values, 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 chimerism 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 BM 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:**
a, EBD absorbance. (Mean ± s.e.m., n=6 mice from three independent experiments). P-values, 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). P-values, two-tailed student’s t-test (*P<0.05). **d-h**, Flow cytometry quantitative analysis of BMEC frequencies, surface and intracellular molecules expression (MFI), in WT or eΔFGFR1/2 mice. (Mean ± s.e.m., n=9 mice from three independent experiments). P-values, two-tailed student’s t-test (**P<0.01, ***P<0.005). i, Diameters of distinct types of BM BVs in the metaphysis region as determined by ImageJ software analysis of high-resolution confocal images. j, Confocal representative images of Sca-1⁺ (green) arterial BVs in the metaphysis region. Note lower abundance of arterial BVs in eΔFGFR1/2 mice. Scale bar indicates 200 μm. **k-l**, (Mean ± s.e.m., n=16 BM sections were analyzed from n=4 mice). P-values, two-way ANOVA with Bonferroni’s multiple comparison post-hoc test (**P<0.01, ***P<0.005). k, Frequency of ROS^high cells scored among total BM cells found in proximity (<20 μm) to different BM BVs, in WT or eΔFGFR1/2 mice. l, Frequency of ROS^high cells scored among total BM cells found in proximity (<20 μm) to different BM BVs, in C57BL/6 mice treated with neutralizing Rat anti VE-cadherin antibodies or Rat IgG control antibodies (50 μg/mouse/day) for 2 days. **m-o**, Flow cytometry quantitative analysis of (a) HSPC glucose uptake (MFI), frequency of (b) cycling HSPC and (c) apoptotic HSPC. (Mean ± s.e.m., n=9 mice from three independent experiments). P-values, 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 post transplantation by flow cytometry. (Mean ± s.e.m., n=18 donor mice from two independent experiments, for 3 recipient mice per donor). P-values, two-tailed student’s t-test (***P<0.005). **r-t**, WT or eΔFGFR1/2 mice were treated with NAC (130 mg/kg) or PBS for 7 days. (Mean ± s.e.m., n=9 mice from three independent experiments). P-values, two-tailed student’s t-test (**P<0.01, ***P<0.005). r, Number of circulating PB HSPC as determined by quantitative flow cytometry analysis. s, Number of BM SLAM LSK HSPC as determined by quantitative flow cytometry analysis. t, Levels of chimerism, indicating LTR-HSC contribution, were determined 24 weeks post 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:**
**a-d**, (Mean ± s.e.m., n=9 mice from three independent experiments). P-values, two-tailed student’s t-test (**P<0.01, ***P<0.005). a, Glucose uptake by BM MSPC as determined by quantitative flow cytometry (MFI) analysis. b, Frequencies of BM 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. **e-i**, C57BL/6 mice were treated with neutralizing Rat anti VE-cadherin antibodies or Rat IgG control antibodies (50 μg/mouse/day) for 5 days. (Mean ± s.e.m., n=9 mice from three independent experiments). P-values, 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 BM MSPC as determined by flow cytometry quantitative analysis. f, Glucose uptake by BM MSPC as determined by quantitative flow cytometry (MFI) analysis. g, Frequencies of BM 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. **j-o**, BM supernatants from WT or eΔFGFR1/2, PBS or FGF-2 (200 μg/kg) treated for 7 days, and IgG or Rat anti VE-cadherin (50 μg/mouse/day) for 5 days, were isolated and BM concentrations of calcitonin and PTH hormones were determined using ELISA assay. (Mean ± s.e.m., n=9 mice per group from three independent experiments). P-values, two-tailed student’s t-test (***P<0.005).

**Extended Data figure 9:**
**a-j**, C57BL/6 mice were treated with neutralizing Rat anti VE-cadherin antibodies or Rat IgG control antibodies (50 μg/mouse/day) for 5 days. (Mean ± s.e.m., unless otherwise indicated n=9 mice from three independent experiments). P-values, 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 chimerism levels indicating LTR-HSC contribution. (Mean ± s.e.m., n=10 mice from two independent experiments). d, Chimerism 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 ROS^High (red) cells in proximity to BVs. Scale bar indicates 20 μm. **h-k**, C57BL/6 mice were treated with neutralizing Rat anti VE-cadherin antibodies or Rat IgG control antibodies (50 μg/mouse/day) for 2 days. Where indicated mice were also treated with NAC (130 mg/kg) or PBS for 2 days. (Mean ± s.e.m., n=9 mice from three independent experiments). P-values, 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 hematocytometer and Turk lysis of erythrocytes. i, Flow cytometry quantitative analysis of CD34⁻ LSK HSPC in the blood circulation. **j-k**, BM MNC or BM lineage depleted cells from treated mice were seeded on a 5 μm pore transwell and allowed to migrate for 2 hours towards CXCL12 (125 ng/mL). Following migration the frequency of migrated BM MNC or CD34⁻/LSK HSPC was determined by flow cytometry quantitative analysis. Note preferential HSPC enhanced migration post VE-cadherin neutralization. **l-p**, C57BL/6 mice were treated with neutralizing Rat anti VE-cadherin antibodies or Rat IgG control antibodies (50 μg/mouse/day) for 5 days. (Mean ± s.e.m., n=9 mice from three independent experiments). P-values, 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. **m-n**, Frequencies of Ki67⁺ cycling and AnnexinV⁺ SLAM LSK HSPC as determined by flow cytometry quantitative analysis. o, Frequency of Sca-1⁺ aBMEC out of total as determined by flow cytometry quantitative analysis. p, Diameters of distinct types of BM BVs in the metaphysis region as determined by ImageJ software analysis of high-resolution confocal images. q, Confocal representative images of Sca-1⁺ (green) arterial BVs in the metaphysis region. Note lower abundance of arterial BVs following 5 days anti VE-cadherin treatment. Scale bar indicates 200 μm. **r-t**, C57BL/6 mice were treated with NAC (130 mg/kg) or PBS for 7 days. (Mean ± s.e.m., n=8 mice from two independent experiments). P-values, 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 proposed BM BVs model and regulation of hematopoiesis.**
BM vasculature is composed of two main types of blood vessels which are arterial BVs, and sinusoids. Blood enters the BM 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 BM. Arterial BMEC have elongated nuclear morphology, express Sca-1 and nestin markers, and display high barrier integrity properties. In addition arterial BVs 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 ROS^low 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. Also, innervating Schwann cell nerve fibers, 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 which makes them the ultimate candidate to serve as the site for BM 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 expanse of stem cell maintenance. Yet, conditions enhancing endothelial integrity, reducing cellular trafficking promote BM stem cell expansion and maintenance. PB plasma, which can penetrate into the BM more easily via fenestrated BVs, 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 BVs and under steady state or “stress” conditions may have a strong regulatory impact on tissue residing stem cells.

**Figure 1:. Sca-1 and nestin distinguish less permeable arterial BM BVs, which sustain ROS^low HSC.**
a, Representative flow cytometry density and histogram plots for BMECs. (Mean ± s.e.m., n=6 mice from three independent experiments). b, Representative fluorescence images of a small diameter blood vessel from the metaphysial area expressing Sca-1-EGFP (green), junctional VE-cadherin (red) and elongated nuclei (Hoechst, blue). Scale bar indicates 20 μm. c, VE-cadherin and ZO-1 flow cytometry representative histogram plots for mean fluorescent expression (MFI) by BMECs. (n=9 mice from three independent experiments). d, Representative confocal tile scan of Sca-1-EGFP (Green) femur. Scale bar indicates 300 μm. e, Representative confocal images of endosteal regions in the metaphysis and diaphysis showing Sca-1⁺ (green)/CD31⁺ (red) arterial BVs and αSMA⁺ (red) pericytes. Scale bars in panels indicate 50 μm except for lower right panel where scale bar indicates 100 μm. f, Representative fluorescence images of ROS^high (red) expressing cells in the proximate microenvironment of Sca-1⁺ (green) or VE-cadherin⁺ (green) arterioles (I, II respectively) and VE-cadherin⁺ (green) sinusoids (III). Scale bar indicates 20 μm.

**Figure 2:. Leaky sinusoids are the exclusive site for cellular trafficking.**
**a-g**, (Mean ± s.e.m., n=53 BVs were analyzed for permeability measurements and n=62 BVs were analyzed for blood flow/shear rates, from three independent experiments each). P-values, two-tailed student’s t-test (***P<0.005). a, Standard deviation heat map of Rhodamine-Dextran leakage. Color scale shows pixel intensity over 30 seconds of data acquisition. Regions of interest are bordered with white line. b, Time traces of fluorescence signal. c, Average vascular permeability. d, A plot of permeability measurements as a function of vessel diameter. **e-f**, Average blood flow speed and shear rate. g, Representative maximum intensity images demonstrating (I) homing of adoptively transferred lineage depleted HPC (hematopoietic progenitor cell, red) and BM MNC (blue) within the BM of a nestin-GFP (green) mouse, (II) transmigrating BM MNC (circled, blue) and adherent Lin⁻ HPC (circled, red)(Scale bars indicate 50 μm), (III) upper and lower planes of the Z-stack taken from supplementary video 9 representing the relative proximity of a nestin⁺ BV to a sinusoidal trafficking site (Scale bar indicates 25 μm, n=309 trafficking events were monitored and analyzed).

**Figure 3:. Plasma penetration through leaky endothelium dictates HSPC trafficking and development.**
a, HSPCs ROS MFI quantitative analysis and representative histogram plot. (Mean ± s.e.m., n=6 mice from three independent experiments). P-values, two-tailed student’s t-test (***P<0.005). **b-e**, BM cells were incubated for 2h with (25% P) or without (control) PB plasma. (Mean ± s.e.m., n=9 repeats in triplicates from three independent experiments). P-values, two-tailed student’s t-test (**P<0.01, ***P<0.005). b, HSPCs ROS MFI quantitative analysis and representative histogram plot. c, HSPC migration frequency. d, Chimerism levels indicating LTR-HSC contribution. (Mean ± s.e.m., n=27 donor from three independent experiments, with at least 3 recipient mice per donor). e, Average number of CFU-GM. f, Numbers of LSK HSPCs in the blood. (Mean ± s.e.m., n=9 mice from three independent experiments). P-values, two-way ANOVA with Bonferroni’s multiple comparison post-hoc test (**P<0.01). g, HSPC homing per femur. (Mean ± s.e.m., n=5 mice from 2 independent experiments). P-values, two-way ANOVA with Bonferroni’s multiple comparison post-hoc test (*P<0.05, **P<0.01).

**Figure 4:. Reducing endothelial barrier integrity hampers stem cell maintenance.**
**a-h**, (Mean ± s.e.m., n=9 mice from three independent experiments). P-values, two-tailed student’s t-test (**P<0.01, ***P<0.005). **a-b**, Quantitative analysis and representative plots of HSPC populations. c, Chimerism levels indicating LTR-HSC contribution. (Mean ± s.e.m., n=18 donor mice from two independent experiments, with 3 recipient mice per donor). d, Repopulating units frequency in the BM indicating HSC. (n=60 donor mice from two independent experiments, for 3 recipient mice per dilution per donor). **e-f**, Quantitative analysis and representative density plots of MSPCs. **g-h**, Quantitative analysis and representative histogram plots of HSPCs and PαS MSPCs ROS MFIs. i, Representative images of ROS^High (red) cells in proximity to BVs. Scale bar indicates 20 μm.

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Dissecting the bone marrow HSC niches.
Zhao M, Li L. Zhao M, et al. Cell Res. 2016 Sep;26(9):975-6. doi: 10.1038/cr.2016.71. Epub 2016 Jun 14. Cell Res. 2016. PMID: 27297237 Free PMC article.

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References

1. Rafii S, Butler JM & Ding BS Angiocrine functions of organ-specific endothelial cells. Nature 529, 316–325 (2016). - PMC - PubMed
1. Lapidot T, Dar A & Kollet O How do stem cells find their way home? Blood 106, 1901–1910 (2005). - PubMed
1. Morrison SJ & Scadden DT The bone marrow niche for haematopoietic stem cells. Nature 505, 327–334 (2014). - PMC - PubMed
1. Kusumbe AP, Ramasamy SK & Adams RH Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone. Nature 507, 323–328 (2014). - PMC - PubMed
1. Kiel MJ, Yilmaz OH, Iwashita T, Terhorst C & Morrison SJ SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 121, 1109–1121 (2005). - PubMed

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[1] Rafii S, Butler JM & Ding BS Angiocrine functions of organ-specific endothelial cells. Nature 529, 316–325 (2016). - PMC - PubMed

[2] Rafii S, Butler JM & Ding BS Angiocrine functions of organ-specific endothelial cells. Nature 529, 316–325 (2016). - PMC - PubMed

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

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

[5] Morrison SJ & Scadden DT The bone marrow niche for haematopoietic stem cells. Nature 505, 327–334 (2014). - PMC - PubMed

[6] Morrison SJ & Scadden DT The bone marrow niche for haematopoietic stem cells. Nature 505, 327–334 (2014). - PMC - PubMed

[7] Kusumbe AP, Ramasamy SK & Adams RH Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone. Nature 507, 323–328 (2014). - PMC - PubMed

[8] Kusumbe AP, Ramasamy SK & Adams RH Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone. Nature 507, 323–328 (2014). - PMC - PubMed

[9] Kiel MJ, Yilmaz OH, Iwashita T, Terhorst C & Morrison SJ SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 121, 1109–1121 (2005). - PubMed

[10] Kiel MJ, Yilmaz OH, Iwashita T, Terhorst C & Morrison SJ SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 121, 1109–1121 (2005). - PubMed

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Distinct bone marrow blood vessels differentially regulate haematopoiesis

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Distinct bone marrow blood vessels differentially regulate haematopoiesis

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