Haematopoietic stem cells (HSCs) reside in a perivascular niche but the specific location of this niche remains controversial1. HSCs are rare and few can be found in thin tissue sections2,3 or upon live imaging4, 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-catulinGFP), we discover that α-catulinGFP 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 Cxcl12high 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+Cxcl12high cells throughout the bone marrow.
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S.J.M. is a Howard Hughes Medical Institute investigator, the Mary McDermott Cook Chair in Pediatric Genetics, the director of the Hamon Laboratory for Stem Cells and Cancer, and a Cancer Prevention and Research Institute of Texas Scholar. J.G.P. is a National Science Foundation Graduate Research Fellow. M.M.M. was supported by a National Research Service Award from NIH. This work was supported by the NIH NHLBI (HL097760) and NIH Shared Instrumentation grant NIH S10RR029731. We thank K. Correll and M. Gross for mouse colony management; N. Loof and the Moody Foundation Flow Cytometry Facility; and A. Bugde of the University of Texas Southwestern Live Cell Imaging Facility; and Y. Liu from the Baylor College of Dentistry microCT facility. We also gratefully acknowledge D. Miranda and A. Tully from Bitplane, S. Terclavers from Zeiss, and L. Smith and H. Pudavar from Leica.
The authors declare no competing financial interests.
Extended data figures and tables
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 exon 1, from bacterial artificial chromosome clone RP24-146F11 by recombineering31. The retrieved genomic region was then modified to replace most of the exon 1 coding region and the exon 1 to intron 1 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 chimaeric mice. Upon confirmation of germline transmission by PCR, the α-catulinGFP-neo mice were crossed with FLPe mice32, 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, respectively) did not show any statistically significant differences among α-catulin+/+ and α-catulinGFP/GFP mice by microCT (microcomputed tomography) analysis (6 α-catulinGFP/GFP and 5 α-catulin+/+ controls at 10−12 weeks of age). HA, hydroxyapatite. All data represent mean ± s.d. The significance of differences between genotypes was assessed using Student’s t-tests; none were statistically significant.
Extended Data Figure 2 α-catulinGFP/GFP mice had normal haematopoiesis, 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), spleen cellularity (n = 6 mice for α-catulin+/+, n = 4 mice for α-catulinGFP/+ and n = 6 mice for α-catulinGFP/GFP genotype), and spleen mass (n = 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 microlitre of peripheral blood from 8−12 week old α-catulin+/+, α-catulinGFP/+, and α-catulinGFP/GFP mice (n = 9 mice per genotype). c, d, Frequencies of mature haematopoietic cells and progenitors in the bone marrow of 8−12 week old α-catulin+/+ and α-catulinGFP/GFP mice (pre-pro-B cells were B220+sIgM−CD43+CD24−; pro-B cells were B220+sIgM−CD43+CD24+; pre-B cells were B220+sIgM−CD43-; common lymphoid progenitors (CLP) were Lin−c-kitlowSca-1lowCD127+CD135+; common myeloid progenitors (CMP) were Lin−c-kit+Sca-1−CD34+CD16/32−; granulocyte macrophage progenitors (GMP) were Lin−c-kit+Sca-1−CD34+CD16/32+; and megakaryocyte erythroid progenitors (MEP) were Lin−c-kit+Sca-1−CD34−CD16/32− (n = 3 mice per genotype). e, Bone marrow CD150+CD48−LSK HSC frequency, bone marrow CD150−CD48−LSK MPP frequency (n = 12 mice per genotype in 12 independent experiments), and spleen HSC frequency (n = 3 mice per 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, granulocyte macrophage colonies; GEMM, granulocyte erythroid macrophage megakaryocyte colonies; Mk, megakaryocyte colonies; n = 5 mice per 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 3,000,000 WBM cells from primary recipient mice shown in h 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 α-catulin−GFP expression among haematopoietic 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+c-kit+ cells from Fig. 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+c-kit+ cells had high forward scatter, lacked reconstituting potential, and were gated out when isolating HSCs by flow cytometry and when identifying HSCs during imaging (see Extended Data Fig. 5 for further explanation). d, Lin−c-kitlowSca-1lowCD127+CD135+ common lymphoid progenitors (CLPs), Lin−c-kit+Sca-1−CD34+CD16/32− common myeloid progenitors (CMPs), Lin−c-kit+Sca-1−CD34+CD16/32+ granulocyte-macrophage progenitors (GMPs), and Lin−c-kit+Sca-1−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 per genotype in 2 independent experiments).
Extended Data Figure 4 α-catulin−GFP+c-kit+ bone marrow cells are highly enriched for HSC activity and are quiescent.
a, Competitive reconstitution assays in which 1 donor α-catulin−GFP+c-kit+ bone marrow cell was transplanted along with 300,000 recipient bone marrow cells into irradiated recipient mice. Each line represents 1 of the 9 mice (out of 34 transplanted; see Table 1) that were long-term multilineage reconstituted by donor myeloid, B, and T cells. b, Three million WBM cells from primary recipient mice 1−4 from a (indicated by an asterisk) were transplanted into secondary recipient mice (7 secondary recipients from primary recipient-1; 4 secondary recipients from primary recipient-2; 3 secondary recipients from primary recipient-3; 3 secondary recipients from primary recipient-4 for an overall 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+c-kit+ 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+c-kit+ 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+c-kit+ 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 two to five independent experiments. Statistical significance was assessed using Student’s t-tests (*P < 0.05; **P < 0.01).
Extended Data Figure 5 All HSC activity resides among α-catulin−GFP+c-kit+ cells with low forward and side scatter.
a, Most α-catulin−GFP+c-kit+ cells (63 ± 7.2%) had low forward and side scatter, but a distinct minority population (36 ± 7.2%) had higher forward and side scatter that was not typical of HSCs. b, We sorted the low scatter and the high scatter α-catulin−GFP+c-kit+ cell populations gated in a and measured their diameters (three 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+c-kit+ cells identified by confocal microscopy in bone marrow plugs from the tibia diaphysis (6 bones analysed 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 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 centre of the marrow to the endosteum (a) or to equal radial deciles from the centre 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 centre to the endosteum as cylinder volume increases (f). g, When we plotted our HSC localization data by volumetric deciles (as in Fig. 2a), HSC were enriched towards the central marrow. h, When we plotted our HSC localization data by radial deciles, the number of HSCs per cylinder increased towards the endosteum as cylinder volume increased, similar to random spots. All data represent mean ± s.d.
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 2D. 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 2D optical plane for presentation. j, For comparison purposes, a single 2 μm thick optical slice from the tibia in i is shown. 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 different morphology. Statistical significance was assessed using Student’s t-tests (*P < 0.05; **P < 0.01; ***P < 0.001). All data represent mean ± s.d.
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 centre of the marrow.
a, Four-colour 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 a. c, Ki-67 staining was largely or exclusively nuclear, co-localizing with DAPI. d–g, Low 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 fibres that run longitudinally along the central bone marrow, where innervated arterioles are located24. α-catulin–GFP+c-kit+ cells were identified and annotated with blue spheres using the Imaris spot function in e and g. Note, the blue spheres are larger than the actual HSCs because at their actual size, HSCs would be extremely difficult to see at this magnification. As the HSCs are represented as large blue spheres, they appear denser than they actually are. For clarity, other haematopoietic cells and endothelial cells are not shown in 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 d–g show a 505 μm thick specimen. This causes α-catulin–GFP+ cells and c-kit+ cells to appear more frequent than they actually were because all of the cells from the thick specimen were collapsed into a single 2D optical plane for presentation. 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 2D image. For this reason, α-catulin–GFP+c-kit+ cells cannot be reliably identified in low magnification 2D projected images. In all cases, cells that we identified as α-catulin–GFP+c-kit+ were manually examined at high magnification in 3D to confirm double labelling of single cells, as shown in h. Few HSCs were closely associated with nerve fibres in these images when they were examined at high magnification and in 3D.
Extended Data Figure 9 Bone marrow blood vessel types can be distinguished based on vessel diameter, continuity of basal lamina, morphology, and position; and no difference in the distribution of HSCs in the bone marrow of male and female mice was detected.
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, and then colour-coded using published criteria17,30,46,47. 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 (TZ) 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–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–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 1,345 α-catulin–GFP+c-kit+ cells from 3 female tibias and 1,632 α-catulin–GFP+c-kit+ cells from 3 male tibias. The statistical significance of differences was assessed using Kolmogorov–Smirnov tests in d–g and Student’s t-tests in h–k; none of the differences were statistically significant.
Extended Data Figure 10 Expression of NG2–CreER was not detected in Scf- or Cxcl12-expressing cells; and conditional deletion of Scf or Cxcl12 using NG2–CreER did not affect HSC frequency or haematopoiesis.
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 tdTomato+ periartieriolar smooth muscle cells (arrow) as well as glia associated with nerve fibres (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 tdTomato expression (reflecting recombination by NG2–CreER) or Lepr antibody staining (mean ± s.d. from 4 mice in 3 independent experiments). Scf–GFP+ stromal cells were uniformly positive for Lepr expression but negative for NG2–CreER recombination. c–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 per genotype in 5 independent experiments with 4/5 recipient mice per donor in each experiment). g, A 20 μm optical section from the diaphysis of a 130-μm-thick cleared half tibia from an 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–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 per genotype in 4 independent experiments with 4/5 recipient mice per donor in each experiment).
A segment of tibia was fixed, stained with antibodies against c-kit (red), α-Catulin-GFP (white), and laminin (green), then cleared and imaged. Bone was imaged as second harmonic generation (gray). Endothelial cells were α-Catulin-GFP+ and HSCs were α-Catulin-GFP+ c-kit+ . To show the spatial relationship between HSCs and bone, blood vessels and other hematopoietic cells were masked, then laminin staining was unmasked to show the relationship between HSCs and blood vessels. (MP4 28230 kb)
A bone marrow plug from the tibia of a Cxcl12DsRed/+; α- CatulinGFP/+ mouse was stained with antibodies against GFP (green) and c-kit (white). Hematopoietic progenitors are c-kit+ (white), HSCs are α-Catulin-GFP+ c-kit+ (green and white), and Cxcl12high stromal cells are DsRed+ (red). To make it possible to see examples of interactions between HSCs and Cxcl12high stromal cells, all channels 20-30µm beyond the spot of interest were occasionally masked. (MP4 28191 kb)
A bone marrow plug from the tibia of a α-CatulinGFP/+ mouse was stained with antibodies against GFP (green), c-kit (red), and laminin (white). Hematopoietic progenitors are c-kit+ (red), HSCs are α-Catulin-GFP+ c-kit+ (green and red), and blood vessels are marked by laminin (white). Hematopoietic cells other than HSCs are masked throughout most of the video to make it possible to see HSCs throughout the marrow. Note that HSCs tend to localize in the central marrow around sinusoids that are dimly stained for laminin. The α-Catulin-GFP signal from c-kit negative endothelial cells was also masked for clarity. (MP4 28633 kb)
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Acar, M., Kocherlakota, K., Murphy, M. et al. Deep imaging of bone marrow shows non-dividing stem cells are mainly perisinusoidal. Nature 526, 126–130 (2015). https://doi.org/10.1038/nature15250
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