Cell cycle quiescence is a critical feature contributing to haematopoietic stem cell (HSC) maintenance. Although various candidate stromal cells have been identified as potential HSC niches, the spatial localization of quiescent HSCs in the bone marrow remains unclear. Here, using a novel approach that combines whole-mount confocal immunofluorescence imaging techniques and computational modelling to analyse significant three-dimensional associations in the mouse bone marrow among vascular structures, stromal cells and HSCs, we show that quiescent HSCs associate specifically with small arterioles that are preferentially found in endosteal bone marrow. These arterioles are ensheathed exclusively by rare NG2 (also known as CSPG4)+ pericytes, distinct from sinusoid-associated leptin receptor (LEPR)+ cells. Pharmacological or genetic activation of the HSC cell cycle alters the distribution of HSCs from NG2+ periarteriolar niches to LEPR+ perisinusoidal niches. Conditional depletion of NG2+ cells induces HSC cycling and reduces functional long-term repopulating HSCs in the bone marrow. These results thus indicate that arteriolar niches are indispensable for maintaining HSC quiescence.
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We thank P. P. Pandolfi for providing Pml−/− mice and D. Rowe, J. Butler and S. Rafii for providing Col2.3–GFP mice. We are grateful to L. Tesfa and O. Uche for technical assistance with sorting. S.P. is supported by a New York Stem Cell Foundation-Druckenmiller Fellowship and C.S. by the German Research Foundation (DFG) (Emmy-Noether-Program). J.A. was supported by Training Grant T32 063754. This work was enabled by the National Institutes of Health (R01 grants DK056638, HL069438, HL097700) to P.S.F.
The authors declare no competing financial interests.
Extended data figures and tables
Extended Data Figure 1 Whole-mount immunofluorescence imaging techniques of the sternal and long-bone bone marrow using in vivo immunofluorescence staining of vessels.
a, Whole-mount immunofluorescence techniques of the sternal bone marrow. Tissue preparation and representative images of the vasculature and haematopoietic cells. b, Analysis of the localization of sinusoids, arterioles and HSCs in the femoral bone marrow of transverse-shaved whole-mount immunofluorescence images. The central vein was identified and localization of sinusoids, arterioles and HSCs were plotted on the axis between central vein and bone as a ratio from 0 to 1. c, Representative FACS plots of bone marrow CD45− Ter119− stromal cells of three independent experiments. Anti-Sca-1 antibody administered intravenously stains a fraction of CD31+ endothelial cells whereas CD31− cells are not stained. d, e, Haematopoietic (d) or Nes–GFPbright mesenchymal (e) progenitor cells are not stained by intravenously injected anti-Sca-1 antibody. f, Average distances between individual sinusoidal vessels in the femoral bone marrow. n = 6 mice.
a, FACS plots of bone marrow endothelial cells. Bone marrow endothelial cells are identified as a VEGFR2+ CD31+ population. Representative data of 3 mice. ∼90% of bone marrow endothelial cells are VEGFR2+ VEGFR3+ Sca-1lo (sinusoidal) and ∼10% are VEGFR2+ VEGFR3− Sca-1hi (arteriolar). b, Whole-mount images of femoral bone marrow from TIE2–GFP mice stained with anti-VEGFR3, anti-Sca-1, anti-VE-cadherin and anti-PECAM1 antibodies. Scale bar, 25 μm. c, Whole-mount images of the sternal bone marrow stained with Alexa Fluor 633 and Dil-Ac-LDL in vivo. Alexa Fluor 633+ arteriolar vessels do not take up Dil-Ac-LDL. Scale bar, 50 μm. d, e, Whole-mount images of sternal (d) and transverse-shaved femoral (e) bone marrow from Nes–GFP mice stained with Alexa Fluor 633 in vivo (d, e) and anti-PECAM1, anti-VE-cadherin antibodies (e). Alexa Fluor 633 specifically stains vessels accompanied by Nes–GFPbright cells (arterioles). Scale bar, 50 μm. f, Intravital imaging of the mouse calvarial bone marrow stained with intravenously injected Rhodamine 6G and Alexa Fluor 633. Sinusoidal vessels identified by Rhodamine 6G are not stained with Alexa Fluor633. Scale bars, 100 μm.
Extended Data Figure 3 Three-dimensional analysis of sinusoids, arterioles and HSCs by the whole-mount immunofluorescence imaging technique of the bone marrow.
a, Illustrative example of transverse-shaved femoral bone marrow. Arrowheads denote HSCs. Scale bar, 100 μm. b, c, Strategy to identify phenotypic CD150+ CD41− CD48− Lineage− HSCs. Megakaryocytes are distinguished by their size and CD41 expression. b, Two representative areas highlighted in dashed squares in Fig. 1f are shown in high magnification. Arrowheads denote HSCs, arrows show CD150+ Lin/CD48/CD41+ cells. Scale bar, 50 μm. c, Three-dimensionally reconstructed images. Grid, 50 μm. d, Estimated HSC number per sternal segment measured by FACS and whole-mount image analysis. e, f, Distances of HSCs to Nes–GFPbright cells, Nes–GFPdim(n = 98 HSCs from 5 mice), arterioles or sinusoids (n = 119 HSCs from 5 mice) shown in absolute numbers (e) and absolute numbers of adjacent HSCs to those structures (f) per sternal segment (75-μm thickness). Similar distribution patterns were obtained when plotting distances of HSCs from Nes–GFPperi cells or arterioles (two-sample Kolmogorov–Smirnov test; P = 0.97), and from Nes–GFPdim cells or sinusoids (two-sample Kolmogorov–Smirnov test; P = 0.45).
Extended Data Figure 4 A null modelling of the spatial relationship between HSCs and arteriolar or sinusoidal vessels.
a, Computational simulation of randomly distributed HSCs on images of whole-mount prepared sterna. To establish the null-model, binary spatial maps of the sinusoids, arterioles (marked by Nes–GFPbright cells, details in Fig. 2a, b and Extended Data Figs 2d, e and 5a–h) were defined from the images of whole-mount prepared sterna. To simulate a null model in which HSCs are not preferentially localized in the marrow, we randomly placed 20 HSCs (to reflect the mean HSCs/sternum observed in situ) on the unoccupied regions of the spatial maps and measured the Euclidean distance of HSCs to the nearest vascular structure. The means of 1,000 simulations defined a distribution of mean distances one would observe for non-preferentially localized HSCs in relation to the respective structures. If the in situ distance measurements were not statistically significantly different from those obtained by a random placement of HSCs on the same structures in silica, this would indicate a non-preferential spatial HSC distribution. The cumulative probability P(X ≤ μ) of observing the in situ mean X was calculated based on the normal distribution, N(μ,σ2 ), with mean (μ) ± s.d. (σ) obtained from our simulation on a map of each bone marrow structure. b, c, Illustrative examples depicting measurements of randomly placed HSCs (b), and distributions of actual (white) and randomly placed (red) HSCs (c) on the image in Fig. 1f.
a, Quantification of GFP fluorescence intensity of Nes–GFP+ cells by whole-mount imaging of the femoral bone marrow. Two distinct populations, Nes–GFPbright and Nes–GFPdim, can be distinguished based on fluorescence intensity. n = 423 cells from 4 mice. b, The absolute numbers of Nes–GFPbright (Nesperi) and Nes–GFPdim (Nesretic) cells in the femoral bone marrow analysed by FACS. n = 3 mice. c–f, Whole-mount images of longitudinally shaved femoral bone marrow from Nes–GFP mice stained with anti-PECAM1, anti-VE-cadherin and anti-Sca-1 antibodies in vivo. Low-power overview (c) and enlarged images (d–f). Scale bars, 100 μm (c), 50 μm (d), 25 μm (e, f). g, Whole-mount images of sternum from Nes–GFP mice stained with anti-PECAM1 and anti-VE-cadherin, showing morphological differences of arterioles (arrows) and sinusoids (arrowheads). Scale bar, 25 μm. h, Whole-mount images of transverse-shaved femoral bone marrow from Nes–GFP mice, stained with anti-PECAM1 and anti-VE-cadherin in vivo. Scale bar, 10 μm. i, Whole-mount images of the Nes–GFP mouse sternum stained with anti-VE-cadherin and anti-tyrosine hydroxylase (TH) antibodies. Scale bar, 50 μm. j, Quantification of CFU-F content of Nes− CD31−, Nesretic and Nesperi stromal cells. n = 4 mice per group. k, Quantitative PCR analysis of HSC niche-related genes within Nesperi, Nesretic and Nes–GFP− CD31− stromal cells. n = 8, 9, 10, 9, 6 independent experiments from left to right. *P < 0.05, **P < 0.01, ***P < 0.001.
a, FACS plots of HSC cell cycle analysed by staining with anti-Ki-67 antibody and Hoechst 33342. Representative data of 5 mice. b, c, Percentages of cells adjacent to Nesperi cells in total or EdU+ HSCs in the long-bone bone marrow (b; n = 244, 52 HSCs from 3 mice) and in Ki-67− or Ki-67+ HSCs in the sternal bone marrow (c; n = 64, 116 HSCs from 7 mice). d, Localization of Ki-67− and Ki-67+ HSCs relative to arterioles in the sternal bone marrow shown as number per sternum segment (75 μm thickness). n = 64, 116 HSCs from 7 mice. e, Distributions of distances of HSCs from arterioles, bone or Col2.3–GFP+ osteoblasts. n = 109, 109, 121 HSCs from 5, 5, 3 mice, respectively. f, Probability distribution of the mean distances from 1,000 simulations of 20 randomly positioned HSCs from Col2.3–GFP+ cells (Fig. 4e). A dashed line depicts the actual mean distance observed in situ. A solid line and dotted lines show the mean ± 2 s.d. (95% confidential interval) calculated by the simulation. The observed mean distance of HSCs was not significantly different from that predicted by the simulation (P = 0.33). g, Localization of total, Ki-67− and Ki-67+ HSCs relative to Col2.3–GFP+ osteoblasts shown in absolute numbers per sternum segment (75 μm thickness). n = 121, 80, 41 HSCs from 3 mice. h, i, Localization of HSCs relative to Nesperi cells after 5-FU treatment shown in number per sternum segment (75-μm thickness). Distribution of HSCs (h), kinetics of the number of HSCs located within 20 μm from Nesperi cells compared to total HSCs (h, inset) and adjacent cells (i). n = 98, 39, 70, 55, 112 HSCs from 5, 9, 7, 4, 4 mice per control, day 3, day 7, day 14 and day 21 groups, respectively.
a, FACS analysis for HSC (CD150+ CD48− Sca-1+ c-kit+ Lineage− gated) cell cycle by using Ki-67 and Hoechst 33342 staining after Poly(I:C) injection. n = 4, 6 mice for control and treatment groups, respectively. b, HSC localization relative to Nesperi cells after Poly(I:C) treatment. n = 106, 123 HSCs from 9, 4 mice for control and treatment groups, respectively. Two-sample Kolmogorov–Smirnov test; P = 0.007. c, Altered distances of HSCs from arterioles in Pml−/− sternal bone marrow. n = 118, 146 HSCs from 5, 4 mice for wild-type control and Pml−/− groups, respectively. Wild type/Pml−/−: 32.9 ± 5.4%/11.5 ± 4.2% in 0–20-μm proximity, 25.4 ± 3.4%/51.1 ± 6.8% > 80-μm distance. Two-sample Kolmogorov–Smirnov test; P = 1.9 × 10−6. d, e, Quantification of distances of Ki-67− quiescent (d; n = 68, 73 HSCs from 5, 4 mice per wild-type control and Pml−/− groups, respectively) and Ki-67+ non-quiescent (e; n = 53, 73 HSCs from 5, 4 mice per wild-type control and Pml−/− groups, respectively). HSCs from arteriolar niches in Pml−/− bone marrow. Two-sample Kolmogorov–Smirnov test; P = 9.6 × 10−8 (d), P = 0.48 (e). f, Localization of HSCs relative to Nesperi cells in the sternal bone marrow after G-CSF treatment. n = 98, 73, 122, 94 HSCs from 6, 3, 4, 5 mice for control, day 1, day 2 and day 4 groups, respectively. Two-sample Kolmogorov–Smirnov test; day 1, P = 0.026; day 2, P = 0.012; day 4, P = 0.23 compared to control. g, Absolute numbers of HSCs that located within 20-μm distance from Nesperi cells in the sternal bone marrow after G-CSF treatment. n = 6, 3, 4, 5 mice for control, day 1, day 2 and day 4 groups, respectively. *P < 0.05, **P < 0.01.
Extended Data Figure 8 Peri-sinusoidal Nesretic cells, not periarteriolar Nesperi, overlap with LEPR+ cells.
a–c, Analyses of the bone marrow from Lepr-cre/loxp-tdTomato/Nes–GFP transgenic mice. Whole-mount immunofluorescence images of sternum (a, b) and FACS analysis of the femoral bone marrow (c). Representative data of 3 mice. Scale bars, 50 μm.
a, b, Immunofluorescence images of the bone marrow from wild-type mice stained with anti-NG2 antibody in a sectioned femur (a) and a whole-mount sternum (b). c–f, Whole-mount immunofluorescence (c–e) and FACS (f) analyses of the bone marrow from Nes–GFP mice stained with anti-α-smooth muscle actin (αSMA) (c, d) or anti-NG2 (e, f) antibodies. g–i, Analysis of the bone marrow from NG2-creERTM/loxp-tdTomato/Nes–GFP transgenic mice. Whole-mount images of sternal bone marrow (g, i) and recombination efficiency for tdTomato protein expression on Nesperi cells analysed by whole-mount imaging after tamoxifen treatment (h). Arrowheads denote Nesretic cells, arrows show Nesperi cells. n = 6 mice. Scale bars, 50 μm.
Extended Data Figure 10 Periarteriolar NG2+ cells are essential for HSC maintenance in the bone marrow and spleen.
Functional analyses of NG2+ cells using NG2-creERTM/iDTR mice. a–c, Quantification of Nesperi cells (a) and vascular volume (b, c), accessed by whole-mount imaging of the sternal bone marrow from NG2-creERTM/iDTR/Nes–GFP mice. Scale bars, 100 μm. n = 3 mice per group. d, e, Cxcl12, Kitl and Angpt1 gene expressions assessed by quantitative PCR in sorted Sca-1hi arteriolar (d) and Sca-1lo sinusoidal (e) endothelial (CD45− Ter119− CD31+) cells after NG2+ cell depletion. n = 4 mice per group. f, HSC localization relative to sinusoids in the sternal bone marrow. n = 69, 71 HSCs from 3, 4 mice per control and NG2-creERTM/iDTR groups, respectively. Two-sample Kolmogorov–Smirnov test, P = 0.29. g, Quantification of bone marrow cellularity, frequency and number of phenotypic CD150+ CD48− Sca-1+ c-kit+ Lineage− HSCs in spleen. n = 6 mice per group. h, i, Quantification of long-term reconstituting HSCs by LTC-IC assays. n = 3 mice per group. j, Numbers of total leukocytes and phenotypic CD150+ CD48− Sca-1+ c-kit+ Lineage− HSCs in blood. n = 3 mice per group. *P < 0.05, **P < 0.01.
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Kunisaki, Y., Bruns, I., Scheiermann, C. et al. Arteriolar niches maintain haematopoietic stem cell quiescence. Nature 502, 637–643 (2013). https://doi.org/10.1038/nature12612
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