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. 2015 Oct 1;526(7571):126-30.
doi: 10.1038/nature15250. Epub 2015 Sep 23.

Deep imaging of bone marrow shows non-dividing stem cells are mainly perisinusoidal

Melih Acar  1 , Kiranmai S Kocherlakota  2   1 , Malea M Murphy  1 , James G Peyer  1 , Hideyuki Oguro  1 , Christopher N Inra  1 , Christabel Jaiyeola  1 , Zhiyu Zhao  1 , Katherine Luby-Phelps  3 , Sean J Morrison  2   1
Affiliations

Deep imaging of bone marrow shows non-dividing stem cells are mainly perisinusoidal

Melih Acar et al. Nature. .

Abstract

Haematopoietic stem cells (HSCs) reside in a perivascular niche but the specific location of this niche remains controversial. HSCs are rare and few can be found in thin tissue sections or upon live imaging, making it difficult to comprehensively localize dividing and non-dividing HSCs. Here, using a green fluorescent protein (GFP) knock-in for the gene Ctnnal1 in mice (hereafter denoted as α-catulin(GFP)), we discover that α-catulin(GFP) is expressed by only 0.02% of bone marrow haematopoietic cells, including almost all HSCs. We find that approximately 30% of α-catulin-GFP(+)c-kit(+) cells give long-term multilineage reconstitution of irradiated mice, indicating that α-catulin-GFP(+)c-kit(+) cells are comparable in HSC purity to cells obtained using the best markers currently available. We optically cleared the bone marrow to perform deep confocal imaging, allowing us to image thousands of α-catulin-GFP(+)c-kit(+) cells and to digitally reconstruct large segments of bone marrow. The distribution of α-catulin-GFP(+)c-kit(+) cells indicated that HSCs were more common in central marrow than near bone surfaces, and in the diaphysis relative to the metaphysis. Nearly all HSCs contacted leptin receptor positive (Lepr(+)) and Cxcl12(high) niche cells, and approximately 85% of HSCs were within 10 μm of a sinusoidal blood vessel. Most HSCs, both dividing (Ki-67(+)) and non-dividing (Ki-67(-)), were distant from arterioles, transition zone vessels, and bone surfaces. Dividing and non-dividing HSCs thus reside mainly in perisinusoidal niches with Lepr(+)Cxcl12(high) cells throughout the bone marrow.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Extended data figure 1
Extended data figure 1. Generation of α-catulinGFP mice
a, The targeting strategy to generate the α-catulinGFP allele is shown. The targeting vector was generated by retrieving a genomic fragment of the α-catulin gene, including exon1, from BAC clone RP24-146F11 by recombineering. The retrieved genomic region was then modified to replace most of the exon1 coding region and the exon1-intron1 junction with an EGFP-bGH-pA-FRT-neo-FRT cassette in frame with the first ATG of α-catulin. The final targeting vector was then linearized and electroporated into C57BL-derived Bruce4 ES cells. b, New NsiI and SpeI sites introduced with the EGFP-bGH-pA-FRT-neo-FRT cassette were used to screen correctly targeted ES cell clones by Southern blotting for 5′ and 3′ probes. Correctly targeted ES cells were used to generate chimeric mice. Upon confirmation of germ-line transmission by PCR, the α-catulinGFP−neo mice were crossed with Flpe mice, to remove the neomycin resistance cassette. c, PCR genotyping of α-catulin+ (WT) and α-catulinGFP alleles from α-catulin+/+, α-catulinGFP/+ and α-catulinGFP/GFP mice. d, α-catulin+/+ and α-catulinGFP/GFP mice did not show any difference in size or body mass (n=9 α-catulin+/+ and 8 α-catulinGFP/GFP male mice, n=7 α-catulin+/+ and 7 α-catulinGFP/GFP female mice, all were 8–10 weeks old). e, α-catulinGFP/+ and α-catulinGFP/GFP mice were born at mendelian frequencies, survived into adulthood in normal numbers, and were apparently developmentally normal. The statistics reflect mice genotypes at 8–10 weeks of age. f, Cortical and trabecular femur bone (CB and TB) did not show any statistically significant differences among α-catulin+/+ and α-catulinGFP/GFP mice by microCT (micro Computed Tomography) analysis (6 α-catulinGFP/GFP and 5 α-catulin+/+ controls at 10–12 weeks of age). HA refers to Hydroxyapatite. All data represent mean±s.d. The significance of differences between genotypes was assessed using Student’s t-tests. None were statistical significant.
Extended data figure 2
Extended data figure 2. α-catulinGFP/GFP mice had normal hematopoiesis, normal HSC frequency, and normal HSC function
a, Hindlimb bone marrow cellularity (n=9 mice for α-catulin+/+,n=4 mice for α-catulinGFP/+ and n=9 mice for α-catulinGFP/GFP genotype) and spleen cellularity (n=6 mice for α-catulin+/+,n=4 mice for α-catulinGFP/+ and n=6 mice for α-catulinGFP/GFP genotype), spleen mass (7 mice for α-catulin+/+,n=4 mice for α-catulinGFP/+ and n=7 mice for α-catulinGFP/GFP genotype). b, White blood cell (WBC), red blood cell (RBC) and platelet (PLT) counts per microliter of peripheral blood from 8–12 week old α-catulin+/+, α-catulinGFP/+, and α-catulinGFP/GFP mice (n=9 mice/genotype). c,d, Frequencies of mature hematopoietic cells and progenitors in the bone marrow of 8–12 week old α-catulin+/+ and α-catulinGFP/GFP mice (Pre-ProB cells were B220+sIgM−CD43+CD24−; ProB cells were B220+sIgM−CD43+CD24+; Pre-B cells were B220+sIgM−CD43−; common lymphoid progenitors (CLPs) were Lin−c-kitlowSca1lowCD127+CD135+; common myeloid progenitors (CMPs) were Lin−c-kit+Sca1−CD34+CD16/32−; granulocyte-macrophage progenitors (GMPs) were Lin−c-kit+Sca1−CD34+CD16/32+; and megakaryocyte-erythroid progenitors (MEPs) were Lin−c-kit+Sca1−CD34−CD16/32− (n=3 mice/genotype). e, Bone marrow CD150+CD48−LSK HSC frequency, bone marrow CD150−CD48−LSK MPP frequency (n=12 mice/genotype in 12 independent experiments), and spleen HSC frequency (n=3 mice/genotype in 3 experiments). f, Percentage of HSCs and whole bone marrow cells that incorporated a 3 day pulse of BrdU in vivo (n=6 α-catulin+/+, 9 α-catulinGFP/+, and 7 α-catulinGFP/GFP 8–12 week old mice in 3 independent experiments). g, Colony formation by HSCs in methylcellulose cultures (GM means granulocyte-macrophage colonies, GEMM means granulocyte-erythroid-macrophage-megakaryocyte colonies, Mk means megakaryocyte colonies; (n=5 mice/genotype in 5 independent experiments). h, Reconstitution of irradiated mice by 300,000 donor bone marrow cells from 8–12 week old α-catulin+/+, α-catulinGFP/+, or α-catulinGFP/GFP mice competed against 300,000 recipient bone marrow cells (n=4 donor mice and 16 recipient mice for α-catulin+/+, n=3 donor mice and 9 recipient mice for α-catulinGFP/+, and n=4 donor mice and 18 recipients for α-catulinGFP/GFP in 3 independent experiments). i, Serial transplantation of 3 million WBM cells from primary recipient mice shown in panel d into irradiated secondary recipient mice (n=4 primary α-catulin+/+ recipients were transplanted into 17 secondary recipients and n=6 primary α-catulinGFP/GFP recipients were transplanted into 20 secondary recipients). All data represent mean±s.d. The statistical significance of differences between genotypes was assessed using Student’s t-tests or ANOVAs. None were significant.
Extended data figure 3
Extended data figure 3. α-catulin-GFP expression among hematopoietic cells is highly restricted to HSCs
a, The frequency of α-catulin-GFP+ bone marrow cells in negative control α-catulin+/+ (WT) mice and α-catulinGFP/+ mice (n=14 mice per genotype in 11 independent experiments). In all cases in this figure percentages refer to the frequency of each population as a percentage of WBM cells. b, α-catulin-GFP+ckit+ cells from Figure 1b are shown (blue dots) along with all other bone marrow cells in the same sample (red dots). c, CD150+CD48−LSK HSCs express α-catulin-GFP but CD150−CD48−LSK MPPs do not (n=17 mice in 12 independent experiments). A minority of the α-catulin-GFP+ckit+ cells had high forward scatter, lacked reconstituting potential, and were gated out when flow cytometrically isolating HSCs and when identifying HSCs during imaging (see Extended data figure 5 for further explanation). d, Lin−c-kitlowSca1lowCD127+CD135+ common lymphoid progenitors (CLPs), Lin−c-kit+Sca1−CD34+CD16/32− cells common myeloid progenitors (CMPs), Lin−c-kit+Sca1−CD34+CD16/32+ granulocyte-macrophage progenitors (GMPs), and Lin−c-kit+Sca1−CD34−CD16/32− megakaryocyte-erythroid progenitors (MEPs) did not express α-catulin-GFP. α-catulinGFP/+ and control cell populations had similar levels of background GFP signals that accounted for fewer than 1% of the cells in each population (n=9 mice/genotype in 2 independent experiments).
Extended data figure 4
Extended data figure 4. α-catulin-GFP+ckit+ bone marrow cells are highly enriched for HSC activity and are quiescent
a, Competitive reconstitution assays in which one donor α-catulin-GFP+ckit+GFP+ bone marrow cell was transplanted along with 300,000 recipient bone marrow cells into irradiated recipient mice. Each line represents one of the nine mice (out of 34 transplanted; see Table 1) that were long-term multilineage reconstituted by donor cells. b, Three million WBM cells from primary recipient mice 1–4 from panel a (indicated by an asterisk) were transplanted into secondary recipient mice (7 secondary recipients/primary recipient-1, 4 secondary recipients/primary recipient-2, 3 secondary recipients/primary recipient-3, 3 secondary recipients/primary recipient-4 for a total of 17 secondary recipients). Each line shows the average (±s.d.) levels of donor cell reconstitution in secondary recipient mice from each primary donor. c, DNA content of WBM cells, α-catulin-GFP+ckit+ HSCs, and CD150+CD48−LSK HSCs. While 11.5% of WBM cells had greater than 2N DNA content (in S/G2/M phases of the cell cycle), only around 1% of α-catulin-GFP+ckit+ HSCs or CD150+CD48−LSK HSCs had greater than 2N DNA content. d, BrdU incorporation into WBM cells, c-kit+ cells, α-catulin-GFP− CD150+CD48−LSK cells, α-catulin-GFP+CD150+CD48−LSK HSCs, and α-catulin-GFP+ckit+ HSCs after 3 days of continuous BrdU administration (BrdU treated). Untreated negative control mice are also shown. e, Percentage of BrdU+ cells in each cell population. In each panel, the number of mice used for analysis (without being pooled) is indicated. All data reflect mean±s.d. from 2 to 5 independent experiments. Statistical significance was assessed using Students t-tests (*, P<0.05; **, P<0.01).
Extended data figure 5
Extended data figure 5. All HSC activity resides among α-catulin-GFP+ckit+ cells with low forward and side scatter
a, Most α-catulin-GFP+ckit+ cells (63±7.2%) had low forward and side scatter but a distinct minority population (36±7.2%) had higher forward and side scatters that were not typical of HSCs. b, We sorted the low scatter and the high scatter α-catulin-GFP+ckit+ cell populations gated in panel a and measured their diameters (3 independent experiments). c, Competitive reconstitution assays in irradiated mice revealed that all HSC activity resided in the low scatter cell fraction. For each recipient mouse, the indicated donor cells (based on the number of cells from each population contained within 300,000 bone marrow cells) were transplanted into irradiated mice along with 300,000 recipient bone marrow cells (mean±s.d. from 2 independent experiments with 20 total recipient mice in the GFP(−) group, 14 total recipient mice in the c-kit(+)GFP(+) FSC&SSC low group, 11 total recipient mice in the c-kit(+)GFP(+) FSC&SSC high group and 9 total recipient mice in the c-kit(−)GFP(+) group). d, The size distribution of all α-catulin-GFP+ckit+ cells identified by confocal microscopy in bone marrow plugs from the tibia diaphysis (6 bones analyzed in 6 independent experiments). In keeping with the flow cytometry data, the largest 40% of imaged cells were not considered HSCs, excluding all cells with diameter larger than 7 μm.
Extended data figure 6
Extended data figure 6. HSCs are enriched in the central marrow and depleted near the endosteum in the diaphysis
a,b, The distribution of HSCs from the central marrow to the endosteum can be determined by drawing concentric cylinders that correspond to equal volumetric deciles from the center of the marrow to the endosteum (a) or to equal radial deciles from the center to the endosteum (b). c,d, Each volumetric decile (as in a) contains 10% of the marrow volume (c). However, cylinders based on radial deciles (as in b), contain successively larger volumes of marrow as they approach the endosteum because the circumference of the cylinders becomes larger (d). e,f, The distribution of random spots among volumetric deciles (a) is nearly equal because each cylinder contains an equal marrow volume (e). However, the number of random spots per cylinder based on radial deciles (b) increases from the center to the endosteum as cylinder volume increases (f). g, When we plotted our HSC localization data by volumetric deciles (as in Figure 2a), HSC were enriched toward the central marrow. h, When we plotted our HSC localization data by radial deciles, the number of HSCs per cylinder increased toward the endosteum as cylinder volume increased, similar to random spots.
Extended data figure 7
Extended data figure 7. HSC density is higher in the diaphysis as compared to the metaphysis
a, Schematic of a femur showing the separation of epiphysis/metaphysis from diaphysis. We divided metaphysis from diaphysis at the point where the central sinus branched (see red line in panels a,f and i). This is also the point at which the density of trabecular bone declines moving into the diaphysis. b, A bisected femur before and after clearing. c, The frequency of CD150+CD48−LSK cells and α-catulin-GFP+c-kit+ cells by flow cytometry in the epiphysis/metaphysis versus diaphysis of femurs (n=9 mice in 2 independent experiments). Note that bone marrow cells were extracted from crushed bones. d, The distance (μm) from α-catulin-GFP+c-kit+ cells to the nearest bone surface in the femur diaphysis based on deep imaging (n=368 cells in 3 bisected femurs). e, The distance (μm) from α-catulin-GFP+c-kit+ cells to the nearest bone surface in the femur diaphysis based on analysis of thin (7 μm) sections (n=45 cells). f, Schematic of a tibia showing the separation of epiphysis/metaphysis from diaphysis (red line). g, The frequency of CD150+CD48−LSK cells and α-catulin-GFP+c-kit+ cells by flow cytometry in the epiphysis/metaphysis versus diaphysis of tibias (n=9 mice in 2 independent experiments). h, The frequency of α-catulin-GFP+c-kit+ cells in the tibia epiphysis/metaphysis versus diaphysis based on deep confocal imaging (n=3 bisected tibias in 3 independent experiments). i, Deep imaging of a bisected tibia showing the separation of metaphysis and diaphysis (red line) where the central sinus branches. Note that these tibias were digitally reconstructed from two different imaging sessions, above and below the diagonal white line. This image shows a 349 μm thick specimen collapsed into 2 dimensions. Note that this causes α-catulin-GFP+ cells and c-kit+ cells to appear much more frequent than they actually were because all of the cells from the thick specimen were collapsed into a single 2 dimensional optical plane for presentation. j, For comparison purposes, a single 2 μm thick optical slice from the tibia in panel i. k, High magnification images of single α-catulin-GFP+c-kit+ cells from the same tibia. Note that α-Catulin-GFP is also expressed by sinusoidal endothelial cells but these cells are easily distinguished from HSCs because the endothelial cells lack c-kit expression and have a very different morphology. Statistical significance was assessed using Students t-tests (*, P<0.05; ** P<0.01; ***, P<0.001).
Extended data figure 8
Extended data figure 8. c-kit and α-Catulin-GFP staining do not reflect autofluorescence or background staining and GFAP+ non-myelinating Schwann cells tend to localize in the center of the marrow
a, 4-color confocal analysis of a bone marrow plug from a tibia diaphysis stained with primary and secondary antibodies against Ki-67, α-catulin-GFP, c-kit, and laminin. A 2 μm optical section is shown from a thick specimen to illustrate typical staining. b, Negative control in which a bone marrow plug from a tibia diaphysis was stained with isotype control and secondary antibodies then imaged under the same conditions as shown in panel a. c, Ki-67 staining was largely or exclusively nuclear, co-localizing with DAPI. Low (d–g) magnification images of bone marrow plugs from tibia diaphysis stained with antibodies against α-catulin-GFP, c-kit, and GFAP. GFAP+ non-myelinating Schwann cells are associated with nerve fibers that run longitudinally along the central bone marrow, where innervated arterioles are located. α-catulin-GFP+c-kit+ cells were identified and annotated with blue spheres using the Imaris spot function in panels e and g. Note that the blue spheres are larger than the actual HSCs because at their actual size, HSCs would be extremely difficult to see at this magnification. Since the HSCs are represented as large blue spheres, they appear more dense than they actually are. For clarity, other hematopoietic cells and endothelial cells are not shown in panels e and g. h, A higher magnification image showing two α-catulin-GFP+c-kit+ cells (arrows) and their localization relative to GFAP positive glia (white) and α-catulin-GFP+ endothelial cells (green). The images in panels d–g show a 505 μm thick specimen. This causes α-catulin-GFP+ cells and c-kit+ cells to appear much more frequent than they actually were because all of the cells from the thick specimen were collapsed into a single 2 dimensional optical plane for presentation. Note as well that because these were thick specimens, there were cases in which an α-catulin-GFP+ cell and a c-kit+ cell were present in different optical planes such that they appeared to be a single α-catulin-GFP+c-kit+ cell when collapsed into a single 2 dimensional image. For this reason, α-catulin-GFP+c-kit+ cells cannot be reliably identified in low magnification 2 dimensional projected images. In all cases, cells that we identified as α-catulin-GFP+c-kit+ were manually examined at high magnification in 3 dimensions to confirm double labeling of single cells, as shown in panel h. Note as well that few HSCs were closely associated with nerve fibers in these images when they were examined at high magnification and in three dimensions.
Extended data figure 9
Extended data figure 9. Bone marrow blood vessel types can be distinguished based upon vessel diameter, continuity of basal lamina, morphology, and position, and we could not detect any difference in the distribution of HSCs in the bone marrow of male and female mice
a,b, Schematic (a) and properties (b) of blood vessels in the bone marrow. Blood enters the marrow through arterioles that branch as they become smaller in diameter and approach the endosteum, where they connect to smaller diameter transition zone capillaries near the bone surface. These transition zone capillaries connect to the large diameter sinusoids that feed blood into the central sinuous through which it leaves the bone marrow in venous circulation. c, Each type of blood vessel was distinguished based on vessel diameter, continuity of basal lamina, morphology, and position then color coded using published criteria,,,. To create distinct digital surfaces associated with each type of blood vessel we first designated all laminin-stained blood vessels in the outer 20% of the marrow volume (adjacent to the endosteum) as transition zone vessels (blue). Arterioles were identified and manually traced in the remaining 80% of marrow volume based on high intensity laminin staining, continuous basal lamina, and morphology. Remaining blood vessels with low intensity laminin staining, fenestrated basal lamina, large diameter, and sinusoidal morphology were designated sinusoids. The longitudinal images (top) show bone marrow plugs that were 550 μm thick and the cross-sectional images (bottom) were 49 μm thick. d, The distribution of α-catulin-GFP+c-kit+ cells in concentric cylinders corresponding to equal volumetric deciles from central marrow to endosteal marrow (near the bone surface) in bone marrow plugs from the tibia diaphysis of male and female mice. e,f,g, The distance from α-catulin-GFP+c-kit+ cells in male or female mice to the nearest arteriole (e), sinusoid (f), or transition zone vessel (g) in tibia based on deep imaging. h,i,j, The percentage of α-catulin-GFP+c-kit+ cells within 10 μm of arterioles (h), sinusoids (i) and transition zone vessels (j) in the tibias of male versus female mice. k, The percentage of α-catulin-GFP+c-kit+ cells closest to arterioles, sinusoids, or transition zone vessels in the tibias of male versus female mice. These data show mean±s.d. for a total of 1345 α-catulin-GFP+c-kit+ cells from 3 female tibias and 1632 α-catulin-GFP+c-kit+ cells from 3 male tibias. The statistical significance of differences were assessed using Kolmogorov–Smirnov tests in panels d–g and Student��s t tests in panels h–k. None of the differences were statistically significant.
Extended data figure 10
Extended data figure 10. We were not able to detect any expression of NG2-CreER in Scf or Cxcl12 expressing cells and conditional deletion of Scf or Cxcl12 using NG2-CreER did not affect HSC frequency or hematopoiesis
a, A 20 μm optical section from a 390 μm thick cleared bone marrow plug from the tibia diaphysis of an NG2-CreER; RosatdTomato/+; ScfGFP/+ mouse (image is representative of bones from 4 mice). The image shows rare Tomato+ periartieriolar smooth muscle cells (arrow) as well as glia associated with nerve fibers (arrowhead); however, we were unable to detect Scf expression by any of these cells. b, Representative flow cytometry plots showing the percentage of Scf-GFP+ stromal cells that were positive for Tomato expression (reflecting recombination by NG2-CreER) or LepR antibody staining (mean±SD from 4 mice in 3 independent experiments). Scf-GFP+ stromal cells were uniformly positive for LepR expression but negative for NG2-CreER recombination. c,d,e,f, Conditional deletion of Scf in NG2-CreER; ScfGFP/fl mice had no effect on bone marrow cellularity (c), HSC frequency (d), CMP, GMP, or MEP frequency (e) or bone marrow reconstituting capacity upon transplantation into irradiated mice (f) (n=5 mice/genotype in 5 independent experiments with 4–5 recipient mice/donor in each experiment). g, A 20 μm optical section from the diaphysis of a 130 μm thick cleared half tibia from a NG2-CreER; RosaYFP/+; Cxcl12dsRed/+ mouse. The image shows rare YFP+ periartieriolar smooth muscle cells; however, we were unable to detect Cxcl12 expression by these cells. h, Representative flow cytometry plots showing the percentage of Cxcl12-DsRed+ stromal cells that were positive for YFP expression (reflecting recombination by NG2-CreER) or LepR antibody staining. Cxcl12-DsRed+ stromal cells were uniformly positive for LepR expression but negative for NG2-CreER. i,j,k,l, Conditional deletion of Cxcl12 in NG2-CreER; Cxcl12−/fl mice had no effect on bone marrow cellularity (i), HSC frequency (j), CMP, GMP, or MEP frequency (k) or bone marrow reconstituting capacity upon transplantation into irradiated mice (l) (n=4 mice/genotype in 4 independent experiments with 4–5 recipient mice/donor in each experiment).
Figure 1
Figure 1. Deep imaging of α-catulin-GFP+ HSCs in digitally reconstructed bone marrow
a, Only 0.021±0.006% of α-catulinGFP/+ bone marrow cells were GFP+ (n=14 mice in 11 independent experiments). b, Nearly all α-catulin-GFP+c-kit+ bone marrow cells were CD150+CD48− (n=9 mice in 3 independent experiments; Extended data figure 3b shows ungated cells from this analysis and Extended data figure 5 shows light scatter properties). c–d, A half tibia before (c) and after clearing (d). e–l, Deep imaging of α-catulin-GFP+c-kit+ HSCs in the epiphysis and metaphysis of a half tibia (360 μm thick) showing digital bone surfaces (second harmonic generation, white), as well as blood vessels (laminin, blue), hematopoietic progenitors (c-kit+, red), and α-catulin-GFP+ cells (green). Endothelial cells express α-catulin-GFP but were easily distinguished from α-catulin-GFP+c-kit+ HSCs based on c-kit expression and morphology. In two dimensional projected images of thick specimens, α-catulin-GFP+ cells and c-kit+ cells can appear much more frequent than they actually are because all of the cells from the thick specimens are collapsed into a single optical plane. f) Same as (e), digitally masked to reveal only HSCs and bone. α-catulin-GFP+c-kit+ HSCs are represented by yellow spheres to make them visible at this magnification. g–i) A higher magnification view of the boxed region from panel f. Panel h digitally masks all hematopoietic cells other than HSCs (yellow spheres). Panel i digitally masks blood vessels and hematopoietic cells other than HSCs. j–l) A higher magnification view of the boxed area from panel i (arrows point to α-catulin-GFP+c-kit+ cells). Images are representative of three independent experiments. Supplementary video 1 shows a three dimensional digital reconstruction of bone and bone marrow. The positions of HSCs and other structures can appear to change in thick specimens when magnification is changed due to the rendering perspective for 3 dimensional display of volume data.
Figure 2
Figure 2. HSCs localize adjacent to Cxcl12high and LepR+ niche cells but distant from bone surfaces
a, The distribution of α-catulin-GFP+c-kit+ HSCs and random spots in concentric cylinders corresponding to equal volumetric deciles from central marrow to endosteal marrow (near the bone surface) from the tibia diaphysis (2977 HSCs in 6 bone marrow plugs (390–550 μm thick) in 6 independent experiments). See Extended data figure 6 for further explanation. Relative to random spots, HSCs were significantly enriched in central marrow. b,c, Distance from HSCs or random spots to the nearest bone surface in the diaphysis (b) or metaphysis (c). d,e, Percentages of all HSCs and random spots within 10μm of a bone surface in the diaphysis (d) or metaphysis (e; b–e show 817 HSCs in the diaphysis and 218 HSCs in the metaphysis of 3 bisected tibias that were 360–400 μm thick). f, Distance to the nearest GFAP+ Schwann cell (n=608 HSCs in bone marrow plugs (430 to 500 μm thick) from the diaphysis of 3 tibias). g, Percentages of all HSCs and random spots within 10μm of a GFAP+ Schwann cell. h, The distance to the nearest Cxcl12high stromal cell (n=596 HSCs in bone marrow plugs (235–450 μm thick) from the diaphysis of 4 tibias). i, Distance to the nearest LepR+ stromal cell in LepR-Cre; tdTomato bone marrow (n=384 HSCs in bone marrow plugs (500 μm thick) from the diaphysis of 3 tibias). j,k, An α-catulin-GFP+c-kit+ cell in apparent contact with a Cxcl12-expressing (j) or a LepR-expressing (k) cell. All data reflect mean±s.d. from at least three independent experiments. The statistical significance of differences in panels a,b,c,f,h,i was assessed by Kolmogorov–Smirnov analysis. The statistical significance of differences in panels d, e, g, h, and i were assessed using Student’s t tests (*, P<0.05).
Figure 3
Figure 3. HSCs localize adjacent to sinusoids but distant from arterioles and transition zone vessels in tibias
a–c, Distance to the nearest arteriole (a), sinusoid (b), or transition zone vessel (c). d–f, Percentages of all HSCs and random spots within 10μm of an arteriole (d), sinusoid (e), or transition zone vessel (f). g, Percentages of HSCs and random spots that were closest to arterioles, sinusoids, or transition zone vessels. h–j, Representative images of α-catulin-GFP+c-kit+ HSCs (arrows) that localized immediately adjacent to an arteriole (h), a sinusoid (i), or a transition zone vessel (j). All data reflect mean±s.d. from bone marrow plugs (390–550 μm thick) from the diaphysis of 6 tibias. A total of 2977 HSCs were analyzed in 6 independent experiments. In panels a–c the statistical significance was assessed by Kolmogorov–Smirnov analysis. Statistical significance in panels d–g was assessed by Student’s t-tests (*, P<0.05; **, P<0.01; ***, P<0.001).
Figure 4
Figure 4. Dividing and non-dividing HSCs are most closely associated with sinusoids
a,b, Representative images of a Ki-67−α-catulin-GFP+c-kit+ non-dividing HSC (a, arrow) and a Ki-67+α-catulin-GFP+c-kit+ dividing HSC (b, arrow). c, 15±2.0% of HSCs were Ki-67+. All data reflect mean±s.d. from bone marrow plugs (410–440 μm thick) from the diaphysis of 5 tibias. A total of 2132 HSCs were analyzed in 5 independent experiments. d–f, Distance to the nearest arteriole (d), sinusoid (e), or transition zone vessel (f). g–i, The percentages of all Ki-67− non-dividing HSCs, Ki-67+ dividing HSCs, or random spots within 10μm of an arteriole (g), a sinusoid (h), or a transition zone vessel (i). j, Most Ki-67− non-dividing HSCs and Ki-67+ dividing HSCs were most closely associated with sinusoids. k, The distributions of Ki-67− non-dividing HSCs, Ki-67+ dividing HSCs, and random spots in concentric cylinders corresponding to equal volumetric deciles from central marrow to endosteal (outside) marrow. Non-dividing HSCs were significantly enriched in central marrow while dividing HSCs were significantly enriched toward the endosteum (d–k reflect mean±s.d. from bone marrow plugs from the diaphysis of 5 tibias. A total of 1840 Ki-67− HSCs and 292 Ki-67+ HSCs were analyzed in 5 independent experiments). In panels d–f and k the statistical significance of distribution differences was assessed by Kolmogorov–Smirnov analysis. In panels g–j, statistical significance was assessed by Student’s t-tests (*, P<0.05).
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