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Bone marrow adipocytes promote the regeneration of stem cells and haematopoiesis by secreting SCF

Abstract

Endothelial cells and leptin receptor+ (LepR+) stromal cells are critical sources of haematopoietic stem cell (HSC) niche factors, including stem cell factor (SCF), in bone marrow. After irradiation or chemotherapy, these cells are depleted while adipocytes become abundant. We discovered that bone marrow adipocytes synthesize SCF. They arise from Adipoq-Cre/ER+ progenitors, which represent 5% of LepR+ cells, and proliferate after irradiation. Scf deletion using Adipoq-Cre/ER inhibited haematopoietic regeneration after irradiation or 5-fluorouracil treatment, depleting HSCs and reducing mouse survival. Scf from LepR+ cells, but not endothelial, haematopoietic or osteoblastic cells, also promoted regeneration. In non-irradiated mice, Scf deletion using Adipoq-Cre/ER did not affect HSC frequency in long bones, which have few adipocytes, but depleted HSCs in tail vertebrae, which have abundant adipocytes. A-ZIP/F1 ‘fatless’ mice exhibited delayed haematopoietic regeneration in long bones but not in tail vertebrae, where adipocytes inhibited vascularization. Adipocytes are a niche component that promotes haematopoietic regeneration.

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Figure 1: Irradiation disrupted sinusoids and depleted HSCs, endothelial cells, and LepR+ stromal cells while dramatically increasing adipocytes in the bone marrow.
Figure 2: Scf was highly expressed by LepR+ stromal cells and adipocytes in the bone marrow before and after irradiation.
Figure 3: Scf from LepR+ stromal cells, but not endothelial cells, is necessary for haematopoietic regeneration and mouse survival after irradiation.
Figure 4: Adipoq-Cre/ER recombines in most adipocytes and in a subset of LepR+ stromal cells in the bone marrow.
Figure 5: Adipoq-Cre/ER+ bone marrow stromal cells form adipocytes, but rarely osteoblasts, in vivo.
Figure 6: Scf from adipocytes is required for the regeneration of HSCs and haematopoiesis and mouse survival after irradiation.
Figure 7: Scf from adipocytes is required for normal HSC frequency and haematopoiesis in the caudal vertebrae of normal young adult mice.
Figure 8: A-ZIP/F1 ‘fatless’ mice exhibit delayed haematopoietic regeneration in the femur but accelerated regeneration of haematopoiesis and vasculature in caudal vertebrae.

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Acknowledgements

S.J.M. is a Howard Hughes Medical Institute Investigator, the Mary McDermott Cook Chair in Pediatric Genetics, the Kathryn and Gene Bishop Distinguished Chair in Pediatric Research, the director of the Hamon Laboratory for Stem Cells and Cancer, and a Cancer Prevention and Research Institute of Texas Scholar. B.O.Z. was supported by a fellowship from the Leukemia and Lymphoma Society and the Thousand Talents Plan-Youth in China. This work was funded by the National Institute on Aging (R37 AG024945) and the Cancer Prevention and Research Institute of Texas. We thank E. Jeffery for advice on the manuscript, N. Loof and the Moody Foundation Flow Cytometry Facility, and K. Correll for mouse colony management.

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Authors and Affiliations

Authors

Contributions

B.O.Z. performed most of the experiments. H.Y. and R.Y. helped in some imaging and transplantation experiments, respectively. Z.Z. performed statistical analyses. J.J.R. provided human bone marrow specimens. O.N. participated in the interpretation of results from A-ZIP/F1 mice. B.O.Z. and S.J.M. designed the experiments, interpreted the results, and wrote the manuscript.

Corresponding authors

Correspondence to Bo O. Zhou or Sean J. Morrison.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Irradiation depletes bone marrow hematopoiesis.

One million mechanically dissociated bone marrow cells from wild-type mice were transplanted into irradiated wild-type mice. The statistical significance of differences among treatments was assessed using repeated measures one-way ANOVAs with the Geisser-Greenhouse method for sphericity correction and Tukey’s multiple comparisons tests (ae). indicates statistical significance relative to non-irradiated controls (con) while #indicates statistical significance between 2 and 4 weeks after irradiation (P < 0.05, or ## P < 0.01, P < 0.001). All data represent mean ± s.d. from two femurs and two tibias in n = 5 mice/genotype/condition from 5 independent experiments. (ae) Numbers of B (a) and T (b) cells in the bone marrow as well as WBC (c), RBC (d), and platelet (e) counts in non-irradiated control (Con) mice as well as mice at 2 and 4 weeks after irradiation and bone marrow transplantation. (f) Confocal imaging showed that perilipin+ adipocytes were uniformly Tomato+ in Leprcre; R26tdTomao mice 2 weeks after irradiation and bone marrow transplantation (representative image from 3 independent experiments).

Supplementary Figure 2 Scf was expressed by adipocytes but not by hematopoietic cells or osteoblasts in the bone marrow of non-irradiated or irradiated mice.

(a) Virtually all Scf-GFPhigh cells were Tomato+ in the bone marrow of irradiated and non-irradiated Leprcre; R26tdTomao; ScfGFP mice (representative plots from 3 independent experiments). (b) Scf-GFP was not expressed by CD45+ or Ter119+ hematopoietic cells in the bone marrow of non-irradiated or irradiated ScfGFP mice (representative plots from 3 independent experiments). (c) Tomato+ osteoblasts did not express Scf-GFP in non-irradiated or irradiated Col1a12.3-cre; R26tdTomato; ScfGFP mice (representative plots from 3 independent experiments). (d,e) Scf-GFP was not detectably expressed by Tomato+ osteoblasts or osteocytes in femur sections from non-irradiated or irradiated Col1a12.3-cre; R26tdTomato; ScfGFP mice (representative images from 3 independent experiments). (f,g) Scf-GFP and Tomato were expressed by perilipin+ adipocytes in the bone marrow of normal Leprcre; R26tdTomao; ScfGFP mice (f) as well as 2 weeks after irradiation and wild-type bone marrow transplantation (g; representative images from 3 independent experiments).

Supplementary Figure 3 Scf is expressed by bone marrow adipocytes.

(a) Confocal imaging showed five consecutive optical images (each 1.5 μm) demonstrating the expression of Scf-GFP in perilipin+ adipocytes from non-irradiated mice (representative images from 3 independent experiments). (bf) Quantitative RT-PCR analysis of leptin (b), Ob-Rb (c), Adipoq (d), perilipin (e) and FABP4 (f) transcript levels (normalized to β-Actin) in LepR+ stromal cells (LepR+), bone marrow adipocytes (Adip-BM) and intraperitoneal adipocytes (Adip-IP) relative to unfractionated whole bone marrow cells (WBM). The transcript levels in WBM were normalized to 1. All data represent mean ± s.d. from n = 3 mice in 3 independent experiments. One-way ANOVAs with Tukey’s multiple comparisons tests were used to assess statistical significance (, P < 0.05; , P < 0.01; , P < 0.001). (g) Abundant adipocytes in human tibia bone marrow sections from an 8 year-old donor (representative image from 3 independent experiments).

Supplementary Figure 4 Scf from osteoblasts and hematopoietic cells is dispensable for the regeneration of HSCs and hematopoiesis after irradiation.

(af) White blood cell (a) red blood cell (b), and platelet counts (c), as well as bone marrow cellularity (d) and numbers of LSK cells (e) and HSCs (f) from paired Col1a12.3-cre; ScfGFP/fl mice and ScfGFP/fl controls that were non-irradiated (Con) or analyzed at 2 or 4 weeks after irradiation and bone marrow transplantation (n = 5 mice/genotype/condition from 3 independent experiments). HSCs could not be detected at 2 weeks after irradiation. Two-way ANOVAs with Sidak’s multiple comparisons tests (ae) or two-tailed Student’s t-tests with Holm-Sidak’s multiple comparisons test (f) were used to assess the significance of differences between Col1a12.3-cre; ScfGFP/fl mice and ScfGFP/fl controls. (g) Competitive reconstitution assay in which 106 donor bone marrow cells from the indicated primary recipient mice were transplanted 4 weeks after irradiation along with recipient-type competitor cells into irradiated secondary recipient mice (n = 12 recipient mice/genotype from 3 independent experiments). The statistical significance of differences was assessed using two-way repeated measures ANOVAs with Sidak’s multiple comparisons tests. (hm) White blood cell (h) red blood cell (i), and platelet counts (j), as well as bone marrow cellularity (k) and numbers of LSK cells (l) and HSCs (m) from paired Vav1-cre; ScfGFP/fl mice and ScfGFP/fl controls that were non-irradiated (Con) or analyzed at 2 or 4 weeks after irradiation and bone marrow transplantation (n = 5 mice/genotype/condition from 3 independent experiments). Two-way ANOVAs with Sidak’s multiple comparisons tests (hl) or two-tailed Student’s t-tests with Holm-Sidak’s multiple comparisons test (m) were used to assess the significance of differences between Vav1-cre; ScfGFP/fland ScfGFP/fl mice. (n) Competitive reconstitution assay in which 106 donor bone marrow cells from the indicated primary recipient mice were transplanted 4 weeks after irradiation along with recipient-type competitor cells into irradiated secondary recipient mice (n = 12 recipient mice/genotype from 3 independent experiments). The statistical significance of differences was assessed using two-way repeated measures ANOVAs with Sidak’s multiple comparisons tests. All data in this figure represent mean ± s.d.

Supplementary Figure 5 Percentage of LepR+ cells in Tomato+ bone marrow cells from Leprcre; R26tdTomato mice dropped after irradiation.

(ac) Confocal imaging of thin femur sections from Leprcre; R26tdTomato mice co-stained with anti-LepR and anti-perilipin antibodies. (Representative results from 3 independent experiments). (d) Quantification of the percentage of LepR+ cells in Tomato+ bone marrow cells from Leprcre; R26tdTomato mice dropped after irradiation. Data represent mean ± s.d. from n = 3 mice in 3 independent experiments. A one-way ANOVA with Tukey’s multiple comparisons test was used to assess statistical significance (P < 0.05,  or ###P < 0.001). (e) Western blot of SCF protein levels in the bone marrow of Adipoq-cre/ER; ScfGFP/fl mice, Leprcre; ScfGFP/fl mice, and ScfGFP/fl controls, either non-irradiated (Con) or 2 weeks after irradiation and transplantation of wild-type bone marrow cells (n = 3 mice/genotype from 3 independent experiments).

Supplementary Figure 6 Adipoq-Cre/ER+ CFU-F had increased adipogenic activity as compared to Adipoq-Cre/ER CFU-F in culture.

(a,b) Representative images of CFU-F colonies derived from Tomato (a) or Tomato+ bone marrow stromal cells (b) from Adipoq-cre/ER; R26tdTomato mice. The colonies were stained with anti-perilipin antibody after culturing in DMEM plus 20% fetal bovine serum for one week. The average number of perilipin+ adipocytes that spontaneously differentiated per CFU-F colony was quantified in Fig. 4j. (c,d) Most bone marrow adipocytes that formed after irradiation were Tomato+ in Adipoq-cre/ER; R26tdTomato mice that had been administered tamoxifen 2 weeks before irradiation (n = 4 mice/time point from 3 independent experiments). (e) Whole-mount imaging of a thick femur section 12 days after 5-FU treatment (representative image from n = 3 mice from 3 independent experiments).

Supplementary Figure 7 We did not detect any effect of Scf deletion on the morphology or frequency of megakaryocytes, endothelial cells, or blood vessels in the bone marrow.

(a,b) Few CD41+ megakaryocyte lineage cells expressed c-kit in mechanically dissociated bone marrow cells (a) or in femur sections (b; results are representative of n = 3 mice analyzed in 3 independent experiments). (c,d) Few endothelial cells expressed c-kit in enzymatically dissociated bone marrow cells (c) or in blood vessels identified in bone marrow sections by laminin staining (d; n = 3 mice from 3 independent experiments). (eh) The morphologies and frequencies of megakaryocytes, endothelial cells, and blood vessels in the bone marrow did not significantly differ between Adipoq-cre/ER; ScfGFP/fl mice (mut) and ScfGFP/fl controls (con) at 4 weeks after irradiation and bone marrow transplantation. Two-tailed Student’s t-tests were used to assess the significance of differences between genotypes but the differences were not statistically significant (n = 5 mice per genotype analyzed in 3 independent experiments). (ik) Diphtheria toxin ablated Tomato+ cells in the bone marrow of Adipoq-cre/ER; R26iDTR; R26tdTomato mice at 3 days after treatment but adipocytes regenerated from unrecombined Tomato negative progenitors within 14 days after treatment (n = 5 mice per genotype analyzed in 3 independent experiments). All data in this figure represent mean ± s.d.

Supplementary Figure 8

Unprocessed scans of western-blots from Supplementary Fig. 5e.

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Zhou, B., Yu, H., Yue, R. et al. Bone marrow adipocytes promote the regeneration of stem cells and haematopoiesis by secreting SCF. Nat Cell Biol 19, 891–903 (2017). https://doi.org/10.1038/ncb3570

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