Differential cytokine contributions of perivascular haematopoietic stem cell niches

Journal name:
Nature Cell Biology
Volume:
19,
Pages:
214–223
Year published:
DOI:
doi:10.1038/ncb3475
Received
Accepted
Published online

Abstract

Arterioles and sinusoids of the bone marrow (BM) are accompanied by stromal cells that express nerve/glial antigen 2 (NG2) and leptin receptor (LepR), and constitute specialized niches that regulate quiescence and proliferation of haematopoietic stem cells (HSCs). However, how niche cells differentially regulate HSC functions remains unknown. Here, we show that the effects of cytokines regulating HSC functions are dependent on the producing cell sources. Deletion of chemokine C-X-C motif ligand 12 (Cxcl12) or stem cell factor (Scf) from all perivascular cells marked by nestin-GFP dramatically depleted BM HSCs. Selective Cxcl12 deletion from arteriolar NG2+ cells, but not from sinusoidal LepR+ cells, caused HSC reductions and altered HSC localization in BM. By contrast, deletion of Scf in LepR+ cells, but not NG2+ cells, led to reductions in BM HSC numbers. These results uncover distinct contributions of cytokines derived from perivascular cells in separate vascular niches to HSC maintenance.

At a glance

Figures

  1. NG2-cre labels perivascular niche cells.
    Figure 1: NG2-cre labels perivascular niche cells.

    (a,b) Whole-mount images of sternums from NG2-cre/iTdTomato/Nes-GFP transgenic mice stained with anti-VE-cadherin antibody. The dashed lines delineate the borders between bone and BM. Representative image from 3 mice. Scale bars, 100μm in a, 20μm in b. NG2-cre-targeted cells overlap with both peri-arteriolar Nes-GFP+ cells (arrows) and peri-sinusoidal Nes-GFP+ cells (arrowheads). (c) Representative FACS plots showing the percentage of NG2-cre/iTdTomato-positive cells within CD45TER119CD31 Nes-GFP+ BM stromal cells isolated from NG2-cre/iTdTomato/Nes-GFP transgenic mice. n = 4 mice. (d) Whole-mount images of sternum from NG2-cre/iTdTomato transgenic mice stained with anti-LepR and anti-VE-cadherin antibodies. Scale bars, 20μm. All panels show the same area for different channels (NG2-cre, LepR, VE-cadherin and merged fluorescence images). (e) Representative FACS plots of CD45TER119CD31 BM stromal cells stained with anti-LepR antibody isolated from NG2-cre/iTdTomato mice. n = 3 mice. Data are represented as mean ± s.e.m. Statistics source data are available in Supplementary Table 1.

  2. NG2-cre-marked cells are the main source of Cxcl12 in the BM.
    Figure 2: NG2-cre-marked cells are the main source of Cxcl12 in the BM.

    (a) Whole-mount images of sternal BM from NG2-cre/iTdTomato/Cxcl12-GFP mice stained with anti-VE-cadherin antibody and DAPI. Representative images from 3 mice. All panels show the same area for different channels (NG2-cre, Cxcl12-GFP and merged fluorescence images). Scale bars, 20μm. (b) Representative FACS plots showing the percentage of Cxcl12-GFP+ cells within CD45TER119CD31 NG2-cre/iTdTomato+ cells. Data are represented as mean ± s.e.m. n = 5 mice. (c) Representative histogram showing the gating scheme for isolation of CD45TER119 CD31 cell subpopulations from Cxcl12-GFP mice used in d. (d) Gene expression analysis of Cxcl12 in sorted CD45TER119CD31 cells. n = 6 mice. Statistical analysis was done by one-way ANOVA. (e,f) FACS analyses of intracellular Cxcl12 protein in CXCL12-GFP mice. (e) Histograms showing intracellular Cxcl12 protein level of each cell subpopulation in CD45TER119 CD31 cells. (f) Quantification of intracellular Cxcl12 protein level. n = 4 mice from two independent experiments. Statistical analysis was done by one-way ANOVA MFI, median fluorescent intensity. (gi) Analyses of intracellular Cxcl12 protein in NG2-cre/iTdTomato/Cxcl12-GFP mice. (g) FACS plots showing gating scheme in CD45TER119 CD31 stromal cells of BM. TomatoGFP (circled one), Tomato+GFP (circled two), Tomato+GFPmid (circled three), Tomato+GFPhi (circled four). (h) Representative histograms showing intracellular Cxcl12 protein level in each population. (i) Quantification of intracellular Cxcl12 protein level. Summary of MFI of intracellular Cxcl12 relative to that of TdTomatoGFP cells. Statistical analysis was done by one-way ANOVA. n = 7 mice. Data are represented as ± s.e.m. (b,d,f,i). *P < 0.05, **P < 0.01, ****P < 0.0001. Statistics source data are available in Supplementary Table 1.

  3. Cxcl12 from distinct perivascular niche cells contributes differentially to HSC functions.
    Figure 3: Cxcl12 from distinct perivascular niche cells contributes differentially to HSC functions.

    (ac) Analyses of LepR-cre/Cxcl12fl/ mice. (a) Absolute numbers of HSCs in BM. n = 6 mice for each group. (b) FACS analyses of cell cycle of HSCs with Ki-67 and Hoechst 33342 staining. n = 5 mice per group. (c) HSC localization relative to arterioles. Error bars, n = 3 mice. The P value has been calculated using n = 129 HSCs for cre (−), 160 HSCs for cre (+), pooled from 3 mice per group. P = 0.9981. (dk) Analyses of NG2-cre/Cxcl12flox/ mice. (dCxcl12 mRNA expression relative to β-actin in CD45TER119CD31Nes-GFP+ cells from NG2-cre(−) Cxcl12f/ and NG2-cre(+) Cxcl12f/ mice. n = 4 mice for cre (−), n = 3 mice for cre (+), from two independent experiments. (e,f) BM cellularity (e) and absolute numbers of phenotypic CD150+CD48LineageSca-1+c-kit+ (LSK) HSCs (f) per femur. n = 10 mice. (g) Percentages of donor-derived cells after competitive reconstitution. n = 5 mice per group. (h) Quantification of cell cycle of HSCs with Ki-67 and Hoechst 33342 staining. n = 5 mice for cre (−), n = 7 mice for cre (+). (i) Representative images of whole-mount immunofluorescent staining of the sternal BM from 3 mice. Arrows indicate CD150+CD48CD41Lineage HSCs. Dashed lines depict the border between bone and BM. Scale bars, 100μm. (j) HSC localization relative to arterioles. Error bars, n = 3 mice for cre (−), n = 4 mice for cre (+). The P value has been calculated using n = 139 HSCs pooled from 3 mice for cre (−), 105 HSCs pooled from 4 mice for cre (+). P = 0.001. (k) Absolute numbers of HSCs in the spleen (left) and blood (right). n = 6 mice (cre(−)), 8 mice (cre(+)) for spleen. n = 5 mice (cre(−)), n = 7 mice (cre(+)) for blood. Data are represented as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data were analysed with two-tailed t-test (a,b,df,h,k) and two-sample Kolmogorov–Smirnov test (c,j).

  4. Cxcl12 deletion in NG2-creERTM-targeted cells alters HSC numbers and location in the BM.
    Figure 4: Cxcl12 deletion in NG2-creERTM-targeted cells alters HSC numbers and location in the BM.

    (a,b) Whole-mount images of sternum from NG2-creERTM /iTdTomato/Nes-GFP mice. Representative images from 3 mice. Scale bars, 100μm in a and 20μm in b. (c) Representative FACS plot of BM stromal cells isolated from NG2-creERTM/iTdTomato/Nes-GFP mice. (d) Quantitative real-time PCR of Cxcl12 and Scf in CD45TER119CD31+, CD45TER119CD31Nes-GFP+, and CD45TER119CD31 Nes-GFP+NG2-creERTMTdTomato+ cells after 8 weeks of tamoxifen administration. n = 4 mice. (e) Representative histogram showing intracellular Cxcl12 levels in NG2-creERTMTdTomato+ cells (left). MFI of intracellular Cxcl12 protein (right). n = 7 mice. (fk) Analyses of NG2-creERTM/Cxcl12fl/ mice. (f) Cellularity in the BM and spleen. BM; n = 10 mice for cre (−), n = 13 mice for cre (+), Spleen; n = 5 mice for cre (−), n = 8 mice for cre (+). (g) Numbers of CD150+ CD48LSK HSCs in BM. n = 10 mice for cre (−), n = 13 mice for cre (+). (h) Percentages of donor-derived cells after competitive reconstitution. n = 8 mice for cre (−), n = 13 mice for cre (+). (i) LSK cells in BM. n = 10 mice for cre (−), n = 13 mice for cre (+). (j) HSC numbers in the spleen (left) and LSK cells in blood (right). n = 5 mice for cre (−), n = 8 mice for cre (+). (k) HSC localization relative to arterioles. Error bars, n = 3 mice. The P value has been calculated using n = 254 HSCs for cre (−), 238 HSCs for cre (+) pooled from 3 mice per group. P < 0.0001. (l) Whole-mount sternal images from Myh11-creERT2/Nes-GFP/iTdTomato mice stained with anti-NG2 antibody. Representative images from 3 mice. Scale bars, 20μm. (m,n) Analyses of Myh11-creERT2/Cxcl12fl/ mice. (m) Numbers of CD150+CD48 LSK HSCs in BM. n = 5 mice for cre (−), n = 8 mice for cre (+). (n) HSC localization relative to arterioles. Error bars, n = 3 mice. The P value has been calculated using n = 220 HSCs pooled from 3 mice for cre (−), 239 HSCs pooled from 4 mice for cre (+). P = 0.0007. Data are represented as ± s.e.m. (dk,m,n). *P < 0.05, **P < 0.01, ***P < 0.001. Statistical significance was assessed using two-tailed t-test (ej,m), one-way ANOVA (d), and two-sample Kolmogorov–Smirnov test (k,n).

  5. NG2-cre-, but not NG2-creERTM-, targeted cells are the source of Scf in the BM.
    Figure 5: NG2-cre-, but not NG2-creERTM-, targeted cells are the source of Scf in the BM.

    (a) Whole-mount sternum from NG2-cre/iTdTomato/Scf-GFP mice, anti-VE-cadherin. Representative images from 3 mice. Scale bars, 20μm. (b) Representative FACS plot showing percentage of NG2-cre/iTdTomato+ cells within CD45TER119CD31Scf-GFP+ cells. n = 3 mice. (ce) Analyses of LepR-cre/Scffl/ mice. (c) Numbers of HSCs (left) in BM and LSK cells in spleen (right). n = 4 mice for cre (−), n = 3 mice for cre (+). (d) FACS analyses of HSC (CD150+CD48LSK) cell cycle with Ki-67 and Hoechst 33342 staining. n = 5 mice for cre (−), n = 6 mice for cre (+). (e) HSC localization relative to arterioles. Error bars, n = 3 mice. P value has been calculated using n = 272 HSCs for cre (−), 293 HSCs for cre (+) pooled from 3 mice per group. P = 0.3402. (fi) Analyses of NG2-cre/Scffl/ mice. (f) Numbers of total BM cells (left) and CD150+CD48LSK HSCs (right) in BM. n = 5 mice for cre (−), n = 7 mice for cre (+). (g) Percentages of donor-derived cells after competitive reconstitution. n = 5 mice for cre (−), n = 7 mice for cre (+). (h) FACS analyses of HSC cell cycle with Ki-67 and Hoechst 33342 staining. n = 6 mice for cre (−), n = 7 mice for cre (+). (i) HSC localization relative to arterioles. Error bars, n = 3 mice. P value has been calculated using n = 224 HSCs for cre (−), 274 HSCs for cre (+) pooled from 3 mice per group. P = 0.2872. (jl) Analyses of NG2-creERTM/Scffl/ mice. (j) Absolute numbers of total cells (left) and HSCs (right) per femur BM. n = 8 mice for cre (−), n = 6 mice for cre (+). (k) Percentages of donor-derived cells after competitive reconstitution. n = 9 mice for cre (−), n = 7 mice for cre (+). (l) HSC localization relative to arterioles. Error bars, n = 3 mice. P value has been calculated using n = 161 HSCs for cre (−), 152 HSCs for cre (+) pooled from 3 mice per group. P = 0.0868. Data are represented as ± s.e.m. (bl). *P < 0.05, **P < 0.01, ****P < 0.0001. Statistical significance was assessed using two-tailed t-test (c,d,fh,j,k) and two-sample Kolmogorov–Smirnov test (e,i,l). Statistics source data are available in Supplementary Table 1.

  6. Distinct contributions of vascular-associated cells in niche activity.
    Figure 6: Distinct contributions of vascular-associated cells in niche activity.

    (a) RNA-seq analysis of perivascular niche cells. The heat map of unsupervised hierarchical clustering of significant enriched genes for sorted CD45TER119CD31NG2-cre/TdTomato+, CD45TER119CD31LepR-cre/TdTomato+, CD45TER119CD31Myh11-creERT2/TdTomato+, and CD45TER119CD31+ cells was created with clustergrammer. 2 mice for each group. Enrichment analysis for each cluster as determined by hierarchical clustering of the rows was performed with Enrichr35. (b) Heat map expression levels of selected genes defined by previous studies for HSC niche cells4, pericytes34 and endothelial cells6. The values of log-transformed reads per kilobase per million mapped reads (RPKM) obtained from RNA-seq were visualized using GraphPad Prism7. Genes with absent expression were assigned the lowest value of the gene pool for visualization. 2 mice for each group. (c) Summary of phenotypes of niche-factor-deleted mice in perivascular stromal cells. PB HSPC, peripheral blood hematopoietic stem and progenitor cells.

  7. Characterization of NG2-cre derived BM stromal cells.
    Supplementary Fig. 1: Characterization of NG2-cre derived BM stromal cells.

    (ac) Fate mapping study of NG2-cre targeted cells. Immunofluorescence staining of osteocalcin (a), perilipin (b), and aggrecan (c) in femur bone marrow sections from NG2-cre/iTdTomato mice. Representative images from 3 mice. Scale bars, 200μm in low power field, 20μm in high power field. (d) Representative FACS plots showing the percentage of TdTomato positive cells within CD45TER119 VE-cadherin positive endothelial cells. n = 3 mice. (e) Representative FACS plots showing the percentage of NG2-cre/iTdTomato positive cells within CD45TER119 CD31+ endothelial cells. n = 4 mice. (f) Representative FACS plots showing the percentage of Nes-GFPdim and Nes-Gbright within CD45 TER119CD31 LepR-cre/TdTomato+ stromal cells (upper right), and of LepR-cre/TdTomato+ cells within CD45 TER119CD31 Nes-GFPbright cells (lower right). n = 4 mice. (g) Representative FACS plots showing the percentage of Nes-GFPdim and Nes-Gbright within CD45 TER119CD31 NG2-cre/TdTomato+ stromal cells (upper right), and of NG2-cre/TdTomato+ cells within CD45 TER119CD31 Nes-GFPbright cells (lower right). n = 3 mice. (h) Whole-mount images of the sternum from NG2-cre/iTdTomato mice stained with anti-NG2 antibody. Representative images from 3 mice. Scale bars, 20μm. All panels show the same area for different channels (NG2-cre, NG2 and merged fluorescence images with DAPI). Data are represented as mean ± s.e.m. Statistics Source Data are available in Supplementary Table 1.

  8. Cxcl12-GFP expression of LepR-cre marked stromal cells.
    Supplementary Fig. 2: Cxcl12-GFP expression of LepR-cre marked stromal cells.

    (a) Whole-mount sternum images from LepR-cre/iTdTomato/Cxcl12-GFP mice stained with anti-VE-cadherin antibody. All panels show the same area for different channels (LepR-cre, Cxcl12-GFP, VE-cadherin and merged fluorescence images). Representative images from 3 mice. Scale bars, 20μm. (b) Representative FACS plots showing the percentage of LepR-cre/iTdTomato positive cells within CD45TER119 CD31Cxcl12-GFP+ cells. (c) Representative histograms showing intracellular Cxcl12 protein level of each fraction in CD45 TER119CD31 cells from NG2-cre/Cxcl12fl/gfp mice. Representative histograms from 3 mice.

  9. Deletion of Cxcl12 from peri-sinusoidal niche cells.
    Supplementary Fig. 3: Deletion of Cxcl12 from peri-sinusoidal niche cells.

    (a) NG2-cre/Cxcl12flox/ mice enable Cxcl12 deletion from both arteriole associated Nes-GFP+, NG2+ cells and more broadly distributed Nes-GFP+, LepR+ stromal cells. (b) Cxcl12 mRNA expression relative to β-actin in CD45 TER119CD31 LepR+ cells from LepR-cre(−) Cxcl12fl/ and LepR-cre(+) Cxcl12fl/ mice. n = 4 mice for cre (−), n = 5 mice for cre (+). (c) Analyses of LepR-cre/Cxcl12fl/ mice. Absolute numbers of lineage Sca-1+ c-kit+ (LSK) cells in the blood (left) n = 6 mice, CFU-C in the blood (middle) n = 5 mice from 2 independent experiments, and HSCs in spleen (right) n = 6 mice. (d) Quantitative real-time PCR of Scf, Vcam-1, and Angiopoietin-1 (Angpt-1) relative to β-actin in sorted CD45 TER119CD31 Nes-GFP+ stromal cells from NG2-cre(−) Cxcl12f/ or NG2-cre(+) Cxcl12f/mice.n = 4 mice for cre (−), n = 3 mice for cre (+), from two independent experiments. (e) Absolute numbers of CD45TER119CD31Nes-GFP+ cells from NG2-cre(−) Cxcl12f/ and NG2-cre(+) Cxcl12f/mice.n = 4 mice for cre (−), n = 3 mice for cre (+), from two independent experiments. (fi) Analyses of NG2-cre/Cxcl12flox/mice. (f) The percentages of CD45.2 donor-derived cells in competitive reconstitution of bone marrow cells. The number of X-axis indicates the time (week) after transplantation. n = 5 mice per group. (g) Absolute numbers of common myeloid progenitor (CMP), granulocyte monocyte progenitor (GMP), and megakaryocyte erythroid progenitor (MEP) in the BM (left). Absolute numbers of common lymphoid progenitor (CLP) in the BM (right). n = 6 mice. (h) Representative FACS plots (CD150+CD48 LSK gated) of cell cycle of HSCs with Ki-67 and Hoechst 33342 staining. (i) Cellularity (left) and absolute number of phenotypic HSCs (right) in P0 newborn liver. n = 7 mice for cre (−), n = 5 mice for cre (+). Data are represented as mean ± s.e.m.

  10. Niche factor deletion from peri-arteriolar niche cells.
    Supplementary Fig. 4: Niche factor deletion from peri-arteriolar niche cells.

    (a) Representative histogram showing the percentage of Nes-GFPdim and Nes-GFPbright cells within CD45 TER119CD31 NG2-creERTM/TdTomato+ cells. n = 3 mice. (b) Representative FACS plots showing the gating strategy for sorting of LineageCD31 Nes-GFP+NG2-DsR+ PDGFRβ+ cells. Blue and red lines represent isotype control and anti- PDGFRβ antibody, respectively. (c) Gene expression analysis of Cxcl12 and Scf in sorted LineageCD31 Nes-GFPNG2-DsR cells, LineageCD31 Nes-GFP+ NG2-DsR, and LineageCD31 Nes-GFP+NG2-DsR+ PDGFRβ+ cells. n = 4 mice from two independent experiments. (d) Whole-mount images of sternum from NG2-creERTM/Cxcl12-GFP/iTdTomato mice stained with anti-VE-cadherin antibody and DAPI. All panels show the same area for different channels (Cxcl12-GFP, NG2-creERTM, VE-cadherin and merged fluorescence images with DAPI). Representative images from 3 mice. Scale bars, 20μm. (e) Whole-mount images of sternum from NG2-DsRed/Cxcl12-GFP/iTdTomato mice stained with anti-VE-cadherin antibody. Representative images from 2 mice. Scale bars, 20μm. (f) In NG2-creERTM/Cxcl12fl/ mice, Cxcl12 is deleted from NG2+ stromal cells associated arterioles but not broadly distributed Nes-GFP+, LepR+ stromal cells. (g) Gene expression analysis of Cspg4 (NG2) mRNA in sorted CD45 TER119CD31+, CD45TER119 CD31TdTomato, CD45TER119 CD31TdTomato+ stromal cells. NG2-creERTM/iTdTomato mice were analysed at 7–8 weeks after tamoxifen treatment. n = 3 mice from two independent experiments. (h,i) Analyses of NG2-creERTM/Cxcl12fl/ mice. (h) The percentages of CD45.2 donor-derived cells in competitive reconstitution of bone marrow cells. n = 8 mice for cre (−), n = 13 mice for cre (+). (i) Absolute numbers of lineage cells in the blood. n = 5 mice for cre (−), n = 8 mice for cre (+). (j) Whole-mount images of sternum from Myh11-creERT2/iTdTomato mice stained with anti-NG2 antibody and DAPI. Representative images from 3 mice. Scale bars, 100μm. (k) Representative FACS plots showing the percentage of Nes-GFP positive cells within CD45 TER119CD31 Myh11-creERT2/TdTomato positive cells. Representative data from 3 mice from 2 independent experiments. (l) Histogram showing intracellular Cxcl12 protein level of CD45 TER119CD31+ endothelial cells and CD45TER119 TdTomato+ cells from Myh11-creERT2/iTdTomato mice (left). Quantification of MFI of intracellular CXCL12 protein (right). n = 3 mice from two independent experiments. (m) Gene expression analysis of Myh11 mRNA in sorted CD45 TER119CD31+, CD45TER119 CD31TdTomato, and CD45TER119 CD31TdTomato+ stromal cells. Myh11-creERT2/iTdTomato mice were analysed at 7–8 weeks after tamoxifen treatment. n = 4 mice from two independent experiments. Data are represented as mean ± s.e.m. Statistical significance was assessed using two-tailed t-test (h,i,l) and one-way ANOVA (c,g,m). Statistics Source Data are available in Supplementary Table 1.

  11. Scf deletion from peri-vascular niche cells.
    Supplementary Fig. 5: Scf deletion from peri-vascular niche cells.

    (a) Whole-mount images of the sternum from NG2-creERTM/Scf-GFP/iTdTomato mice stained with anti-VE-cadherin antibodies. All images show the same area for different channels (Scf-GFP, NG2-creERTM, VE-cadherin and merged fluorescence images). Representative images from 3 mice. Scale bars, 20μm. (b) In LepR-cre/Scffl/ mice, Scf is deleted from broadly distributed Nes-GFP+, LepR+ stromal cells, but not from the arteriole-associated Nes-GFP+, NG2+ stromal cells. (cf) Analysis of NG2-cre/Scfflox/ mice. (c) The percentages of CD45.2 donor-derived cells in competitive reconstitution of bone marrow cells. n = 5 mice for cre (−), n = 7 mice for cre (+). (d) Absolute number of LSK cells in the blood (left) and HSCs in the spleen (right). n = 5 mice for cre (−), n = 7 mice for cre (+). (e) Differential leukocyte counts in the blood (left) and spleen (right). n = 5 mice for cre (−), n = 7 mice for cre (+). (f) Cellularity (left) and absolute number of phenotypic HSCs (right) in P0 newborn liver of NG2-cre/ Scffl/ mice. n = 8 mice for cre (−), n = 10 mice for cre (+). (g,h) Analysis of NG2-creERTM/Scfflox/ mice. (g) The percentages of CD45.2 donor-derived cells in competitive reconstitution of bone marrow cells. n = 9 mice for cre (−), n = 7 mice for cre (+). (h) Absolute numbers of HSCs in the spleen (left) and LSK cells in the blood (right). n = 8 mice for cre (−), n = 6 mice for cre (+). Data are represented as mean ± s.e.m. Statistical significance was assessed using two-tailed t-test (ch).

  12. Proposed model of HSC regulation by distinct perivascular niche cells via different cytokines.
    Supplementary Fig. 6: Proposed model of HSC regulation by distinct perivascular niche cells via different cytokines.

    Cxcl12, derived from NG2-expressing Nes-GFP+ stromal cells, closely associates with arterioles, regulating HSC maintenance. HSC distributions relative to arterioles are altered after Cxcl12 deletion in arteriole-associated stromal cells, suggesting that Cxcl12 derived from these niche cells may promote HSC tethering to the proper microenvironment for their maintenance. Non-myelinating Schwann cells regulating quiescence of HSCs are also tightly associated with arterioles, which suggest the possibility of coordination for HSC regulation in the peri-arteriolar niche. On the other hand, Cxcl12 derived from broadly distributed LepR-expressing, Nes-GFP+ stromal cells controls mobilization of HSCs to the circulation. Uniformly distributed LepR-expressing Nes-GFP+ stromal cells are the main source of Scf for HSC maintenance.

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Author information

  1. Present addresses: Department of Stem Cell Biology and Medicine/Cancer Stem Cell Research, Kyushu University, Fukuoka 812-8582, Japan (Y.K.); Department of Pathology, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais 31270-901, Brazil (A.B.).

    • Yuya Kunisaki &
    • Alexander Birbrair

Affiliations

  1. Gottesman Institute for Stem Cell and Regenerative Medicine Research, Albert Einstein College of Medicine, Bronx, New York 10461, USA

    • Noboru Asada,
    • Yuya Kunisaki,
    • Halley Pierce,
    • Alexander Birbrair &
    • Paul S. Frenette
  2. Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA

    • Noboru Asada,
    • Yuya Kunisaki,
    • Halley Pierce,
    • Alexander Birbrair &
    • Paul S. Frenette
  3. Department of Pharmacological Sciences, Mount Sinai Center for Bioinformatics, Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA

    • Zichen Wang,
    • Nicolas F. Fernandez &
    • Avi Maayan
  4. Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461, USA

    • Paul S. Frenette

Contributions

N.A. performed most of the experiments and analysed data; H.P. performed CFU-C experiments; Z.W., N.F.F. and A.M. analysed RNA-seq data; A.B. bred Myh11-creERT2 mice; P.S.F. initiated and directed the study. N.A., Y.K. and P.S.F. interpreted data and wrote the manuscript. All of the authors contributed to the design of experiments, discussed the results and commented on the manuscript.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Characterization of NG2-cre derived BM stromal cells. (1,122 KB)

    (ac) Fate mapping study of NG2-cre targeted cells. Immunofluorescence staining of osteocalcin (a), perilipin (b), and aggrecan (c) in femur bone marrow sections from NG2-cre/iTdTomato mice. Representative images from 3 mice. Scale bars, 200μm in low power field, 20μm in high power field. (d) Representative FACS plots showing the percentage of TdTomato positive cells within CD45TER119 VE-cadherin positive endothelial cells. n = 3 mice. (e) Representative FACS plots showing the percentage of NG2-cre/iTdTomato positive cells within CD45TER119 CD31+ endothelial cells. n = 4 mice. (f) Representative FACS plots showing the percentage of Nes-GFPdim and Nes-Gbright within CD45 TER119CD31 LepR-cre/TdTomato+ stromal cells (upper right), and of LepR-cre/TdTomato+ cells within CD45 TER119CD31 Nes-GFPbright cells (lower right). n = 4 mice. (g) Representative FACS plots showing the percentage of Nes-GFPdim and Nes-Gbright within CD45 TER119CD31 NG2-cre/TdTomato+ stromal cells (upper right), and of NG2-cre/TdTomato+ cells within CD45 TER119CD31 Nes-GFPbright cells (lower right). n = 3 mice. (h) Whole-mount images of the sternum from NG2-cre/iTdTomato mice stained with anti-NG2 antibody. Representative images from 3 mice. Scale bars, 20μm. All panels show the same area for different channels (NG2-cre, NG2 and merged fluorescence images with DAPI). Data are represented as mean ± s.e.m. Statistics Source Data are available in Supplementary Table 1.

  2. Supplementary Figure 2: Cxcl12-GFP expression of LepR-cre marked stromal cells. (819 KB)

    (a) Whole-mount sternum images from LepR-cre/iTdTomato/Cxcl12-GFP mice stained with anti-VE-cadherin antibody. All panels show the same area for different channels (LepR-cre, Cxcl12-GFP, VE-cadherin and merged fluorescence images). Representative images from 3 mice. Scale bars, 20μm. (b) Representative FACS plots showing the percentage of LepR-cre/iTdTomato positive cells within CD45TER119 CD31Cxcl12-GFP+ cells. (c) Representative histograms showing intracellular Cxcl12 protein level of each fraction in CD45 TER119CD31 cells from NG2-cre/Cxcl12fl/gfp mice. Representative histograms from 3 mice.

  3. Supplementary Figure 3: Deletion of Cxcl12 from peri-sinusoidal niche cells. (310 KB)

    (a) NG2-cre/Cxcl12flox/ mice enable Cxcl12 deletion from both arteriole associated Nes-GFP+, NG2+ cells and more broadly distributed Nes-GFP+, LepR+ stromal cells. (b) Cxcl12 mRNA expression relative to β-actin in CD45 TER119CD31 LepR+ cells from LepR-cre(−) Cxcl12fl/ and LepR-cre(+) Cxcl12fl/ mice. n = 4 mice for cre (−), n = 5 mice for cre (+). (c) Analyses of LepR-cre/Cxcl12fl/ mice. Absolute numbers of lineage Sca-1+ c-kit+ (LSK) cells in the blood (left) n = 6 mice, CFU-C in the blood (middle) n = 5 mice from 2 independent experiments, and HSCs in spleen (right) n = 6 mice. (d) Quantitative real-time PCR of Scf, Vcam-1, and Angiopoietin-1 (Angpt-1) relative to β-actin in sorted CD45 TER119CD31 Nes-GFP+ stromal cells from NG2-cre(−) Cxcl12f/ or NG2-cre(+) Cxcl12f/mice.n = 4 mice for cre (−), n = 3 mice for cre (+), from two independent experiments. (e) Absolute numbers of CD45TER119CD31Nes-GFP+ cells from NG2-cre(−) Cxcl12f/ and NG2-cre(+) Cxcl12f/mice.n = 4 mice for cre (−), n = 3 mice for cre (+), from two independent experiments. (fi) Analyses of NG2-cre/Cxcl12flox/mice. (f) The percentages of CD45.2 donor-derived cells in competitive reconstitution of bone marrow cells. The number of X-axis indicates the time (week) after transplantation. n = 5 mice per group. (g) Absolute numbers of common myeloid progenitor (CMP), granulocyte monocyte progenitor (GMP), and megakaryocyte erythroid progenitor (MEP) in the BM (left). Absolute numbers of common lymphoid progenitor (CLP) in the BM (right). n = 6 mice. (h) Representative FACS plots (CD150+CD48 LSK gated) of cell cycle of HSCs with Ki-67 and Hoechst 33342 staining. (i) Cellularity (left) and absolute number of phenotypic HSCs (right) in P0 newborn liver. n = 7 mice for cre (−), n = 5 mice for cre (+). Data are represented as mean ± s.e.m.

  4. Supplementary Figure 4: Niche factor deletion from peri-arteriolar niche cells. (703 KB)

    (a) Representative histogram showing the percentage of Nes-GFPdim and Nes-GFPbright cells within CD45 TER119CD31 NG2-creERTM/TdTomato+ cells. n = 3 mice. (b) Representative FACS plots showing the gating strategy for sorting of LineageCD31 Nes-GFP+NG2-DsR+ PDGFRβ+ cells. Blue and red lines represent isotype control and anti- PDGFRβ antibody, respectively. (c) Gene expression analysis of Cxcl12 and Scf in sorted LineageCD31 Nes-GFPNG2-DsR cells, LineageCD31 Nes-GFP+ NG2-DsR, and LineageCD31 Nes-GFP+NG2-DsR+ PDGFRβ+ cells. n = 4 mice from two independent experiments. (d) Whole-mount images of sternum from NG2-creERTM/Cxcl12-GFP/iTdTomato mice stained with anti-VE-cadherin antibody and DAPI. All panels show the same area for different channels (Cxcl12-GFP, NG2-creERTM, VE-cadherin and merged fluorescence images with DAPI). Representative images from 3 mice. Scale bars, 20μm. (e) Whole-mount images of sternum from NG2-DsRed/Cxcl12-GFP/iTdTomato mice stained with anti-VE-cadherin antibody. Representative images from 2 mice. Scale bars, 20μm. (f) In NG2-creERTM/Cxcl12fl/ mice, Cxcl12 is deleted from NG2+ stromal cells associated arterioles but not broadly distributed Nes-GFP+, LepR+ stromal cells. (g) Gene expression analysis of Cspg4 (NG2) mRNA in sorted CD45 TER119CD31+, CD45TER119 CD31TdTomato, CD45TER119 CD31TdTomato+ stromal cells. NG2-creERTM/iTdTomato mice were analysed at 7–8 weeks after tamoxifen treatment. n = 3 mice from two independent experiments. (h,i) Analyses of NG2-creERTM/Cxcl12fl/ mice. (h) The percentages of CD45.2 donor-derived cells in competitive reconstitution of bone marrow cells. n = 8 mice for cre (−), n = 13 mice for cre (+). (i) Absolute numbers of lineage cells in the blood. n = 5 mice for cre (−), n = 8 mice for cre (+). (j) Whole-mount images of sternum from Myh11-creERT2/iTdTomato mice stained with anti-NG2 antibody and DAPI. Representative images from 3 mice. Scale bars, 100μm. (k) Representative FACS plots showing the percentage of Nes-GFP positive cells within CD45 TER119CD31 Myh11-creERT2/TdTomato positive cells. Representative data from 3 mice from 2 independent experiments. (l) Histogram showing intracellular Cxcl12 protein level of CD45 TER119CD31+ endothelial cells and CD45TER119 TdTomato+ cells from Myh11-creERT2/iTdTomato mice (left). Quantification of MFI of intracellular CXCL12 protein (right). n = 3 mice from two independent experiments. (m) Gene expression analysis of Myh11 mRNA in sorted CD45 TER119CD31+, CD45TER119 CD31TdTomato, and CD45TER119 CD31TdTomato+ stromal cells. Myh11-creERT2/iTdTomato mice were analysed at 7–8 weeks after tamoxifen treatment. n = 4 mice from two independent experiments. Data are represented as mean ± s.e.m. Statistical significance was assessed using two-tailed t-test (h,i,l) and one-way ANOVA (c,g,m). Statistics Source Data are available in Supplementary Table 1.

  5. Supplementary Figure 5: Scf deletion from peri-vascular niche cells. (526 KB)

    (a) Whole-mount images of the sternum from NG2-creERTM/Scf-GFP/iTdTomato mice stained with anti-VE-cadherin antibodies. All images show the same area for different channels (Scf-GFP, NG2-creERTM, VE-cadherin and merged fluorescence images). Representative images from 3 mice. Scale bars, 20μm. (b) In LepR-cre/Scffl/ mice, Scf is deleted from broadly distributed Nes-GFP+, LepR+ stromal cells, but not from the arteriole-associated Nes-GFP+, NG2+ stromal cells. (cf) Analysis of NG2-cre/Scfflox/ mice. (c) The percentages of CD45.2 donor-derived cells in competitive reconstitution of bone marrow cells. n = 5 mice for cre (−), n = 7 mice for cre (+). (d) Absolute number of LSK cells in the blood (left) and HSCs in the spleen (right). n = 5 mice for cre (−), n = 7 mice for cre (+). (e) Differential leukocyte counts in the blood (left) and spleen (right). n = 5 mice for cre (−), n = 7 mice for cre (+). (f) Cellularity (left) and absolute number of phenotypic HSCs (right) in P0 newborn liver of NG2-cre/ Scffl/ mice. n = 8 mice for cre (−), n = 10 mice for cre (+). (g,h) Analysis of NG2-creERTM/Scfflox/ mice. (g) The percentages of CD45.2 donor-derived cells in competitive reconstitution of bone marrow cells. n = 9 mice for cre (−), n = 7 mice for cre (+). (h) Absolute numbers of HSCs in the spleen (left) and LSK cells in the blood (right). n = 8 mice for cre (−), n = 6 mice for cre (+). Data are represented as mean ± s.e.m. Statistical significance was assessed using two-tailed t-test (ch).

  6. Supplementary Figure 6: Proposed model of HSC regulation by distinct perivascular niche cells via different cytokines. (356 KB)

    Cxcl12, derived from NG2-expressing Nes-GFP+ stromal cells, closely associates with arterioles, regulating HSC maintenance. HSC distributions relative to arterioles are altered after Cxcl12 deletion in arteriole-associated stromal cells, suggesting that Cxcl12 derived from these niche cells may promote HSC tethering to the proper microenvironment for their maintenance. Non-myelinating Schwann cells regulating quiescence of HSCs are also tightly associated with arterioles, which suggest the possibility of coordination for HSC regulation in the peri-arteriolar niche. On the other hand, Cxcl12 derived from broadly distributed LepR-expressing, Nes-GFP+ stromal cells controls mobilization of HSCs to the circulation. Uniformly distributed LepR-expressing Nes-GFP+ stromal cells are the main source of Scf for HSC maintenance.

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