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. 2017 Mar;19(3):214-223.
doi: 10.1038/ncb3475. Epub 2017 Feb 20.

Differential Cytokine Contributions of Perivascular Haematopoietic Stem Cell Niches

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Free PMC article

Differential Cytokine Contributions of Perivascular Haematopoietic Stem Cell Niches

Noboru Asada et al. Nat Cell Biol. .
Free PMC article

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.

Conflict of interest statement

Competing financial interests

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. NG2-cre labels peri-vascular niche cells
(a,b) Whole-mount images of sternums from NG2-cre/ iTdTomato/ Nes-GFP transgenic mice stained with anti-VE-cadherin antibody. Dashed lines delineate the borders between bone and bone marrow. Representative image from 3 mice. Scale bars, 100 μm in (a), 20 μm 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 CD45 TER119 CD31 Nes-GFP+ bone marrow 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 CD45 TER119 CD31 bone marrow stromal cells stained with anti-LepR antibody isolated from NG2-cre/ iTdTomato mice. n=3 mice. Data are represented as mean ± SEM. Statistics Source Data are available in Supplementary Table1.
Figure 2
Figure 2. NG2-cre-marked cells are main source of Cxcl12 in the bone marrow
(a) Whole-mount images of sternal bone marrow 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, 20 μm. (b) Representative FACS plots showing the percentage of Cxcl12-GFP+ cells within CD45 TER119 CD31 NG2-cre/ iTdTomato+ cells. Data are represented as mean ± SEM. n=5 mice. (c) Representative histogram showing the gating scheme for isolation of CD45 TER119 CD31 cell subpopulations from Cxcl12-GFP mice used in (d). (d) Gene expression analysis of Cxcl12 in sorted CD45 TER119 CD31 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 CD45 TER119 CD31 cells. (f) Quantification of intracellular Cxcl12 protein level. n=4 mice from two independent experiments. Statistical analysis was done by one-way ANOVA. (g–i) Analyses of intracellular Cxcl12 protein in NG2-cre/ iTdTomato/ Cxcl12-GFP mice. (g) FACS plots showing gating scheme in CD45 TER119 CD31 stromal cells of bone marrow. TomatoGFP (➀), Tomato+GFP (➁), Tomato+GFPmid (➂), Tomato+GFPhi (➃). (h) Representative histograms showing intracellular Cxcl12 protein level in each population. (i) Quantification of intracellular Cxcl12 protein level. Summary of median fluorescent intensity (MFI) of intracellular Cxcl12 relative to that of TdTomato GFP cells. Statistical analysis was done by one-way ANOVA. n=7 mice. Data are represented as ± S.E.M. (b, d, f, i). Statistics Source Data are available in Supplementary Table1.
Figure 3
Figure 3. Cxcl12 from distinct peri-vascular niche cells contributes differentially to HSC functions
(a–c) 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. (d–k) Analyses of NG2-cre/ Cxcl12flox/−. mice (d) Cxcl12 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) Bone marrow cellularity (e) and absolute numbers of phenotypic CD150+CD48LineageSca-1+c-kit+ (LSK) HSCs (f) per one 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 bone marrow from 3 mice. Arrows indicate CD150+CD48CD41Lineage HSCs. Dashed lines depict the border between bone and bone marrow. Scale bars, 100 μH. (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. Data were analysed with two-tailed t-test (a, b, d–f, h, k) and Two-sample Kolmogorov-Smirnov test (c, j).
Figure 4
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, 100 μm in (a) and 20 μm in (b). (c) Representative FACS plot of bone marrow 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 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. (f–k) Analyses of NG2-creERTM/Cxcl12fl/− mice. (f) Cellularity in the bone marrow 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+ CD48 LSK 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, 20 μm. (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. (d–k, m, n). Statistical significance was assessed using two-tailed t-test (e–j, m), one-way ANOVA (d), and Two-sample Kolmogorov-Smirnov test (k, n).
Figure 5
Figure 5. NG2-cre, but not NG2-creERTM, targeted cells are the source of Scf in the bone marrow
(a) Whole-mount sternum from NG2-cre/ iTdTomato/ Scf-GFP mice, anti-VE-cadherin. Representative images from 3 mice. Scale bars, 20 μm. (b) Representative FACS plot showing percentage of NG2-cre/ iTdTomato+ cells within CD45TER119CD31Scf-GFP+ cells. n=3 mice. (c–e) 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. (f–i) 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. (j–l) Analyses of NG2-creERTM/ Scffl/− mice. (j) Absolute numbers of total cells (left) and HSCs (right) per one 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. (b–l). Statistical significance was assessed using two-tailed t-test (c, d, f–h, j, k) and Two-sample Kolmogorov-Smirnov test (e, i. l). Statistics Source Data are available in Supplementary Table1.
Figure 6
Figure 6. Distinct contributions of vascular-associated cells in niche activity
(a) RNA-seq analysis of peri-vascular niche cells. Heatmap 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 Enrichr. (b) Heatmap expression levels of selected genes defined by previous studies for HSC niche cell, pericyte, and endothelial cells. 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 peri-vascular stromal cells.

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