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Differential cytokine contributions of perivascular haematopoietic stem cell niches

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.

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Figure 1: NG2-cre labels perivascular niche cells.
Figure 2: NG2-cre-marked cells are the main source of Cxcl12 in the BM.
Figure 3: Cxcl12 from distinct perivascular niche cells contributes differentially to HSC functions.
Figure 4: Cxcl12 deletion in NG2-creERTM-targeted cells alters HSC numbers and location in the BM.
Figure 5: NG2-cre-, but not NG2-creERTM-, targeted cells are the source of Scf in the BM.
Figure 6: Distinct contributions of vascular-associated cells in niche activity.

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Acknowledgements

We thank C. Prophete, P. Ciero and C. Cruz for technical assistance and L. Tesfa, Y. Wang and D. Sun for help with cell sorting. We thank T. Nagasawa and S. Morrison for providing reagents. This work was supported by R01 grants from the National Institutes of Health (NIH) (DK056638, HL116340, HL097819 to P.S.F.), New York Stem Cell Foundation and NIH’s Common Fund (U54HL127624, U54CA189201 to A.M.). We are also grateful to the New York State Department of Health (NYSTEM Program) for shared facility (C029154) and research support (N13G-262) and the Leukaemia and Lymphoma Society’s Translational Research Program. Y.K. is supported by JSPS Grant-in Aid for Scientific Research (B)(15H04859) and the Takeda Science Foundation. N.A. is supported by JSPS Postdoctoral Fellowships for Research Abroad.

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Authors and Affiliations

Authors

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.

Corresponding author

Correspondence to Paul S. Frenette.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 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, 200 μm in low power field, 20 μm 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, 20 μm. 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.

Supplementary Figure 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, 20 μm. (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.

Supplementary Figure 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.

Supplementary Figure 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, 20 μm. (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, 20 μm. (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, 100 μm. (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.

Supplementary Figure 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, 20 μm. (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).

Supplementary Figure 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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Asada, N., Kunisaki, Y., Pierce, H. et al. Differential cytokine contributions of perivascular haematopoietic stem cell niches. Nat Cell Biol 19, 214–223 (2017). https://doi.org/10.1038/ncb3475

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