Bone marrow adipose tissue-derived stem cell factor mediates metabolic regulation of hematopoiesis

Abstract

Hematopoiesis is dynamically regulated by metabolic cues in homeostatic and stressed conditions; however, the cellular and molecular mechanisms mediating the metabolic sensing and regulation remain largely obscure. Bone marrow adipose tissue remodels in various metabolic conditions and has been recently proposed as a niche for hematopoietic stem cells after irradiation. Here, we investigated the role of marrow adipose tissue-derived hematopoietic cytokine stem cell factor in unperturbed hematopoiesis by selectively ablating the Kitl gene from adipocytes and bone marrow stroma cells using Adipoq-Cre and Osx1-Cre, respectively. We found that both Adipoq-Kitl knockout (KO) and Osx1-Kitl KO mice diminished hematopoietic stem and progenitor cells and myeloid progenitors in the bone marrow and developed macrocytic anemia at the steady-state. The composition and differentiation of hematopoietic progenitor cells in the bone marrow dynamically responded to metabolic challenges including high fat diet, β3-adrenergic activation, thermoneutrality, and aging. However, such responses, particularly within the myeloid compartment, were largely impaired in Adipoq-Kitl KO mice. Our data demonstrate that marrow adipose tissue provides stem cell factor essentially for hematopoiesis both at the steady state and upon metabolic stresses.

Figure 1.

Role of adipose stem cell factor (SCF) in brown fat function. (A and B) Stromal vascular faction (SVF) cells from iWAT of Kitl^fl/fl and Adipoq-Cre⁺;Kitl^fl/fl mice were differentiated into adipocytes in vitro and analyzed by Oil O Red staining (A) and western blotting (B). Densitometry of UCP1 shown in Online Supplementary Figure S1C. (C and D) Expression of proteins involved in adipogenesis and thermogenesis in BAT (C) and iWAT (D) from 7-week old Kitl^fl/fl and Adipoq-Cre⁺;Kitl^fl/fl male mice. Densitometry of UCP1 shown in Online Supplementary Figure S1D and E. (E and F) Kitl^fl/fl and Adipoq-Cre⁺;Kitl^fl/fl mice (n=6) were treated with CL 316,243 for seven days. Ucp1 mRNA levels (E) and UCP1 protein levels (F) in BAT and iWAT were determined. Densitometry of UCP1 shown in Online Supplementary Figure S1J and K. Data are presented as mean±Standard Deviation.

Figure 2.

Adipocyte-derived stem cell factor (SCF) is a niche factor for hematopoietic stem and progenitor cells. (A) Co-staining of Perilipin and EGFP in the cMAT and rMAT of Kitl^EGFP mice. Scale bar=50 μm. (B and C) Levels of total Kitl mRNA (B) and SCF protein (C) in the flushed bone marrow (BM) from tibia of Kitl^fl/fl (n = 5) and Adipoq-Cre⁺;Kitl^fl/fl (n=6) 13-week old male mice. (D) BM cellularity in the femur of 13-week old Kitl^fl/fl (n = 7) and Adipoq-Cre⁺;Kitl^fl/fl (n=8) male mice. (E) Representative flow cytometry plots showing LSK and MP cells among the lineage⁻ CD127⁻ population in 13-week old Kitl^fl/fl and Adipoq-Cre⁺;Kitl^fl/fl male mice. Average frequencies are shown as inserts. (F) Quantification of absolute numbers of LSK and MP cells in the femur of 13-week old Kitl^fl/fl (n=7) and Adipoq-Cre⁺;Kitl^fl/fl (n=8) male mice and phenotypic LT-HSC in the femur of 8-month old Kitl^fl/fl and Adipoq-Cre⁺;Kitl^fl/fl (n=4) male mice. (G) Representative flow cytometry plots showing CMP, MEP, and GMP cells among the MP population in 13-week old Kitl^fl/fl and Adipoq-Cre⁺;Kitl^fl/fl male mice. Average frequencies are shown as inserts. (H) Absolute numbers of CMP, MEP, and GMP cells in the femur of 13-week old Kitl^fl/fl (n=7) and Adipoq-Cre⁺;Kitl^fl/fl (n=8) male mice. (I) The ratio of marrow MEP to GMP in 13-week old Kitl^fl/fl (n=7) and Adipoq-Cre⁺;Kitl^fl/fl (n=7) male mice. (J) The absolute number of CLP in 13-week old Kitl^fl/fl (n=7) and Adipoq-Cre⁺;Kitl^fl/fl (n=7) male mice. (K) The ratio of marrow CMP to CLP in 13-week old Kitl^fl/fl (n=7) and Adipoq-Cre⁺;Kitl^fl/fl (n=7) male mice. (L) Colony formation assay of 2×10⁴ BM cells from Kitl^fl/fl and Adipoq-Cre⁺;Kitl^fl/fl (n=4) mice. (M) Spleen weight of Kitl^fl/fl and Adipoq-Cre⁺;Kitl^fl/fl (n=4) mice. (N) Colony formation assay of 2×10⁵ splenic cells from Kitl^fl/fl and Adipoq-Cre⁺;Kitl^fl/fl (n=4) mice. Data are presented as mean±Standard Deviation. *P<0.05; **P<0.01; ***P<0.001 by unpaired student t-test (B-K) or one-way ANOVA (N).

Figure 3.

Impaired hematopoiesis when stem cell factor (SCF) is ablated in Osx1⁺ cells. (A) Bone marrow cellularity in the femur of 8-month old control (n=4), Osx1-Cre⁺;Kitl^fl/+ (n=5), and Osx1-Cre⁺;Kitl^fl/fl (n=4) male mice. (B) Frequencies of LSK, phenotypic LT-HSC, MP, CMP, GMP, MEP, and CLP populations in the bone marrow (BM) of 8-month old control (n=4), Osx1-Cre⁺;Kitl^fl/+ (n=5), and Osx1-Cre⁺;Kitl^fl/fl (n=4) male mice, determined by flow cytometry. (C and D) Ratios of MEP to GMP (C) and CMP to CLP (D) in 8-month old control and Osx1-Cre⁺;Kitl^fl/fl (n=4) male mice. (E) Complete blood count of 10-week old control (n=8, including 6 Kitl^fl/fl and 2 Osx1-Cre⁺) and Osx1-Cre⁺;Kitl^fl/fl (n=7) male mice. (F) Colony formation assay of 2×10⁴ BM cells from Kitl^fl/fl and Osx1-Cre⁺;Kitl^fl/fl (n=4) mice. (G) Spleen weight of Kitl^fl/fl and Osx1-Cre⁺;Kitl^fl/fl (n=4) mice. (H) Colony formation assay of 2×10⁵ splenic cells from Kitl^fl/fl and Osx1-Cre⁺;Kitl^fl/fl (n=4) mice. Data are presented as mean±Standard Deviation. ^*P<0.05; ^**P<0.01; ^***P<0.001 by one-way ANOVA followed with Tukey’s multiple comparison (A, B, and H) or unpaired Student t-test (C, E, and G).

Figure 4.

High-fat diet (HFD)-stressed hematopoiesis in control and Adipoq-Cre⁺;Kitl^fl/fl male mice. (A and B) Kitl^fl/fl and Adipoq-Cre⁺;Kitl^fl/fl male mice at the age of eight weeks were fed with normal chow (NC) or HFD (n=3-4) for another eight weeks. (A) Representative images of femur sections. (B) Quantification of adipocyte numbers in the BM from the growth plate to 4.5 mm away distally. (C-I) 8-week old Kitl^fl/fl (n=6 for each diet) and Adipoq-Cre⁺;Kitl^fl/fl (n=8 for NC and n=6 for HFD) male mice were fed with NC or HFD for eight weeks. BM cellularity (C), LSK number (D), GMP number (E), CLP number (F), CMP number (G), MEP number (H), and the MEP/GMP ratio (I) were determined by flow cytometry. (J-N) Complete blood count of NC-and HFD-fed Kitl^fl/fl (n=16 and 13, respectively) and Adipoq-Cre⁺;Kitl^fl/fl (n=12 and 6, respectively) male mice showing granulocyte number (J), monocyte number (K), lymphocyte number (L), megakaryocyte-erythrocyte (MkE) to granulo-cyte-monocyte (GrMo) ratio (M), and the ratio of lymphocyte to all myeloid cells including MkE and GrMo (N). Data are presented as mean±Standard Deviation. ^*P<0.05; ^**P<0.01; ^***P<0.001 by two-way ANOVA followed by multiple comparison using Tukey’s correction (B) or Sidak correction (C-N).

Figure 5.

Sympathetic nervous system (SNS)-activated hematopoiesis in control and Adipoq-Cre⁺;Kitl^fl/fl mice. (A and B) 12-week old Kitl^fl/fl and Adipoq-Cre⁺;Kitl^fl/fl male mice were treated with the saline vehicle or CL 316,243 for one week. (A) Representative images of femur sections. (B) Quantification of adipocyte numbers in the bone marrow (BM) from the growth plate to 4.5 mm away distally. (C) BM cellularity of 12-week old Kitl^fl/fl (n=7 for vehicle and n=6 for CL) and Adipoq-Cre⁺;Kitl^fl/fl (n=8 for vehicle and n=6 for CL) male mice were treated with the saline vehicle or CL 316,243 for one week. (D and E) Relative Kitl mRNA levels in the BM (D) or iWAT (E) of wild-type mice treated with vehicle or CL 316,243 (n=6) for one week. (F-M) 12-week old Kitl^fl/fl (n=7 for vehicle and n=6 for CL) and Adipoq-Cre⁺;Kitl^fl/fl (n=8 for vehicle and n=6 for CL) male mice were treated with the saline vehicle or CL 316,243 for one week. LSK number (F), MP number (G), CMP number (H), MEP number (I), GMP number (J), CLP number (K), the MEP/GMP ratio (L), and the CLP/CMP ratio (M) were determined by flow cytometry. (N and O) CL 316,243 treatment-induced changes in MEP/GMP ratio (N) and CLP/CMP ratio (O) in Kitl^fl/fl (n=6) and Adipoq-Cre⁺;Kitl^fl/fl (n=6) male mice. Data are presented as mean±Standard Deviation. *P<0.05; **P<0.01; ***P<0.001 by two-way ANOVA followed by multiple comparison using Sidak correction (F-M) or unpaired, two-tailed Student t-test (D, N, and O).

Figure 6.

The effect of aging on hematopoietic stem and progenitor cells (HSPC) in Kitl^fl/fl and Adipoq-Cre⁺;Kitl^fl/fl mice. (A) Body weight of Kitl^fl/fl and Adipoq-Cre⁺;Kitl^fl/fl (n=6) male mice at three [n=9 and 10 for control and knock-out (KO), respectively] and ten (n=6 and 5 for control and KO, respectively) months of age. (B-J) Flow cytometric analyses of bone marrow from Kitl^fl/fl and Adipoq-Cre⁺;Kitl^fl/fl (n=6) male mice at three (n=9 and 10 for control and KO, respectively) and ten (n=6 and 5 for control and KO, respectively) months of age, showing total cellularity (B), LSK number (C), MP number (D), CMP number (E), MEP number (F), GMP number (G), CLP number (H), MEP/GMP ratio (I), and CLP/CMP ratio (J). Data are presented as mean±Standard Deviation. Two-way ANOVA followed by multiple comparison using Tukey’s correction was performed. (Top) P-values for interaction and between groups. ^*P<0.05; ^**P<0.01; ^***P<0.001 between 3M and 10M mice in indicated genotypes.