The mitochondrial respiratory chain is essential for haematopoietic stem cell function

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Abstract

Adult and fetal haematopoietic stem cells (HSCs) display a glycolytic phenotype, which is required for maintenance of stemness; however, whether mitochondrial respiration is required to maintain HSC function is not known. Here we report that loss of the mitochondrial complex III subunit Rieske iron-sulfur protein (RISP) in fetal mouse HSCs allows them to proliferate but impairs their differentiation, resulting in anaemia and prenatal death. RISP-null fetal HSCs displayed impaired respiration resulting in a decreased NAD+/NADH ratio. RISP-null fetal HSCs and progenitors exhibited an increase in both DNA and histone methylation associated with increases in 2-hydroxyglutarate (2HG), a metabolite known to inhibit DNA and histone demethylases. RISP inactivation in adult HSCs also impaired respiration resulting in loss of quiescence concomitant with severe pancytopenia and lethality. Thus, respiration is dispensable for adult or fetal HSC proliferation, but essential for fetal HSC differentiation and maintenance of adult HSC quiescence.

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Figure 1: Loss of RISP in haematopoietic cells impairs mitochondrial oxidative metabolism and results in severe fetal anaemia.
Figure 2: RISP is essential for fetal HSC differentiation.
Figure 3: Ablation of RISP depletes fetal myeloid progenitors.
Figure 4: RISP deficiency impairs fetal HSC competitive repopulation capacity.
Figure 5: Loss of RISP in fetal HSCs leads to deregulation of gene expression, DNA and histone hypermethylation, and histone hypoacetylation.
Figure 6: RISP deficiency decreases NAD+/NADH ratio and increases 2HG.
Figure 7: Loss of RISP in adult haematopoietic stem cells impairs mitochondrial oxidative metabolism and leads to a severe pancytopenia phenotype.
Figure 8: RISP deficiency in adult HSCs leads to impaired differentiation, loss of quiescence, increased apoptosis and increased DNA damage.

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Acknowledgements

This work was supported by the NIH (R35CA197532) to N.S.C., NIH (T32 GM008061) to L.P.D., NIH (T32 T32HL076139) to S.E.W. J.X. is supported by the NIH/NIDDK grants K01DK093543 and R01DK111430 and the Cancer Prevention and Research Institute of Texas (CPRIT) New Investigator award (RR140025). We thank Robert H. Lurie Cancer Center Flow Cytometry facility supported by NCI CCSG P30 CA060553 for their invaluable assistance. Proteomics services were performed by the Northwestern Proteomics Core Facility, generously supported by NCI CCSG P30 CA060553 awarded to the Robert H. Lurie Comprehensive Cancer Center and the National Resource for Translational and Developmental Proteomics supported by P41 GM108569.

Author information

E.A., S.E.W. and L.P.D. carried out most of the experiments in the paper. B.J.T. and S.M. provided technical expertise and carried out some of the initial experiments. X.L., Y.Z. and Z.S. performed RNA sequence analysis. M.S. and K.M.M. conducted and analysed metabolomics. P.T.S. generated the RISP-KO mice. E.A., S.E.W., J.X., L.P.D., J.D.C. and N.S.C. provided intellectual input and wrote the paper.

Correspondence to Navdeep S. Chandel.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Loss of TFAM in hematopoietic cells results in perinatal death due to severe fetal anemia.

(a) Genotypes from crosses between Tfamfl/fl (TFAM WT) and Tfamfl/fl Vav-iCre (TFAM KO) mice. (b) Protein Expression of TFAM in WT, Heterozygous (HET) and KO lineage negative (Lin) FL cells analyzed by Western blot. Experiment performed 3 times and figure displayed is one representative experiment. Image of the whole blot is displayed in Supplementary Fig. 8. (c) Pictures at E15.5 and E18.5 days of TFAM WT and KO fetuses. Images are representative of 10 animals of each genotype isolated on at least three separate days. (d) Total cell number per FL at E15.5 days in TFAM WT and KO fetuses (WT n = 17; HET n = 10; KO n = 10). (e) Peripheral blood cell parameters at E18.5 days fetuses, including RBC (red blood cells), HB (hemoglobin), HCT (hematocrit), WBC (white blood cells) and PLT (platelets) in TFAM WT and KO fetuses. (WT n = 11; HET n = 4; KO n = 8). All data in this figure represent mean ± s.e.m. P ≤ 0.05; P ≤ 0.01. All P values calculated using Student’s t-test.

Supplementary Figure 2 Fetal HSC and progenitors gating strategy.

(a) Within the lineage negative population: LSKs were defined as Sca-1high, CD48low. Within the LSK population: HSCs (hematopoietic stem cells) were defined as Mac-1high, CD150high, and MPPs (Multipotent progenitors) were defined as Mac-1low, CD150low. (b) Within the lineage negative population: MPs (myeloid progenitors) were defined as c-kithigh, Sca-1low. Within the MP population: MEPs (Megakaryocyte-erythroid progenitors) were defined as CD34low, FcγRIIIlow, CMPs (common myeloid progenitors) were defined as CD34high, FcγRIIIlow, and GMPs (granulocyte-monocyte progenitors) were defined as CD34high, FcγRIIIhigh. (c) Fetal liver hematopoiesis populations described in manuscript and gating definitions.

Supplementary Figure 3 Defective hematopoiesis due to loss of RISP is independent of ROS.

(a) MitoSOX mean fluorescence intensity (MFI) of RISP-WT FL and RISP-KO FL MPs. Data are shown as relative to WT (WT n = 9; KO n = 8). (b) TMRE MFI of RISP-WT FL and RISP-KO FL MPs. Results are CCCP corrected. Data are shown as relative to WT (WT n = 9; KO n = 7). (c) Cell frequency and cell number of erythroid populations defined by staining against surface markers CD71 and Ter119 in RISP-WT FL and RISP-KO FL mice treated with NAC (1 mg ml−1) in vivo (WT n = 8; KO n = 5). (d) Quantification of CFU-GM, BFU-E and CFU-GEMM colonies from Methocult cultures of 105 RISP WT or KO FL cells treated with NAC (+) in vivo (1 mg ml−1) and in vitro (500 μM) (WT n = 8; KO n = 5). (e) Cell frequency and cell number of erythroid populations defined by staining against surface markers CD71 and Ter119 in RISP-WT FL and RISP-KO FL mice treated with BSO (2 mM) in vivo (WT n = 7; KO n = 4). (f) Quantification of CFU-GM, BFU-E and CFU-GEMM colonies from Methocult cultures of 105 FL cells from RISP WT or KO fetuses treated with BSO in vivo (2 mM) and in vitro (500 μM) (WT n = 7; KO n = 4). (g) Quantitative RT-PCR analysis of p16INK4a in RISP WT and KO FL Lin cells, and WT FL Lin cells treated with different doses of BSO for two days. Results are expressed as relative fold change of WT untreated cells. Experiment was performed twice and three experimental replicates were done per experiment. (WT n = 3; KO n = 3; Different BSO doses on WT cells n = 3). (h) Cell frequency and cell number of erythroid populations defined by staining against surface markers CD71 and Ter119 in RISP-WT FL and RISP-KO FL mice treated with BSO (20 mM) in vivo (WT n = 6; KO n = 4). All experiments were done with FL cells from E15.5 day fetuses. All data in this figure represent mean ± s.e.m. P ≤ 0.05; P ≤ 0.01. All P values calculated using Student’s t-test.

Supplementary Figure 4 Ablation of RISP in the HSCs impaired long-term repopulation activity and competitive repopulation capacity.

(A) Design of the competitive repopulation assay. (B) The contribution of CD45.2 FL donor cells to reconstitute the peripheral blood of previously irradiated CD45.1 recipient mice in competitive transplantation assays. (C) The contribution of FL donor cells to peripheral multilineage reconstitution including B-cell lineage (B220+), T-cell lineage (CD3+) and myeloid lineage (Mac-1+, Gr-1+). Data represent the average contribution for n = 6 recipient mice per genotype in Supplementary Fig. 4b, c. Data presented in Supplementary Fig. 4b, c are mean ± s.e.m. P ≤ 0.05; P ≤ 0.01. All p values calculated using Student’s t-test. (d) The total contribution of CD45.2 fetal liver (FL) donor cells to the peripheral blood of irradiated CD45.1 recipient mice in a competitive transplantation assay following engraftment and subsequent Poly (I:C) treatment. Data represent the percentage of CD45.2 donor cells relative to the initial chimerism. (e) The contribution of CD45.2 FL donor cells to specific lineages in the peripheral blood of irradiated CD45.1 recipient mice in a competitive transplantation assay following engraftment and subsequent Poly (I:C) treatment. B-cell lineage (B220+), T-cell lineage (CD3+) and myeloid lineage (Mac-1+, Gr-1+). Data represent the percentage of CD45.2 donor cells relative to the initial chimerism. Data represent the average contribution for Rispfl/fl (RISP-WT FL; n = 9 mice) and Rispfl/fl Mx1-Cre (RISP-KO FL; n = 6 mice) in Supplementary Fig. 4D and S4E. Data in Supplementary Fig. 4D and S4E are mean ± s.e.m. P ≤ 0.05; P ≤ 0.01. All P values calculated using Student’s t-test.

Supplementary Figure 5 Metabolite levels in RISP deficient fetal liver lineage negative cells.

Relative abundance of glycolysis (a), TCA cycle (b), urea cycle metabolites (c), redox (d), nucleotides (e) and amino acids (f) extracted from RISP-WT FL and-KO FL Lin FL cells. Peak areas of each metabolite were normalized to cell number. Metabolites were measured from WT n = 7 mice; KO n = 5 mice. Raw metabolite data is available in Supplementary Table 3. Data in this figure represent mean ± s.e.m. P ≤ 0.05; P ≤ 0.01. All P values calculated using Student’s t-test.

Supplementary Figure 6 RISP deficiency impairs adult HSC competitive repopulation capacity.

(a) The contribution of CD45.2 BM donor cells to specific lineages in the peripheral blood of irradiated CD45.1 recipient mice in a competitive transplantation assay following engraftment and subsequent Poly (I:C) treatment. B-cell lineage (B220+), T-cell lineage (CD3+) and myeloid lineage (Mac-1+, Gr-1+). Data represent the percentage of CD45.2 donor cells relative to the initial chimerism. (b) The contribution of CD45.2 BM donor cells to specific lineages (LSK, ST-HSC, LT-HSC, MPP, MP, CMP, GMP, and MEP) in the bone marrow of irradiated CD45.1 recipient mice in a competitive transplantation assay after engraftment and subsequent Poly (I:C) treatment. Bone marrow was assayed four months post-Poly (I:C) treatment. Data represent the percentage of CD45.2 donor cells relative to the initial chimerism. Data represent the average contribution for Rispfl/fl (RISP-WT BM; n = 9 mice) and Rispfl/fl Mx1-Cre (RISP-KO BM; n = 4 mice) in Supplementary Fig. 7a, b. Data are mean ± s.e.m. P ≤ 0.05; P ≤ 0.01. All P values calculated using Student’s t-test. Supplementary Fig. 7c, d represent the 2nd independent experiment. (c) The total contribution of CD45.2 BM donor cells to the peripheral blood of irradiated CD45.1 recipient mice in a competitive transplantation assay following engraftment and subsequent Poly (I:C) treatment. Data represent the percentage of CD45.2 donor cells relative to the initial chimerism. (d) The contribution of CD45.2 BM donor cells to specific lineages in the peripheral blood of irradiated CD45.1 recipient mice in a competitive transplantation assay following engraftment and subsequent Poly (I:C) treatment. B-cell lineage (B220+), T-cell lineage (CD3+) and myeloid lineage (Mac-1+, Gr-1+). Data represent the percentage of CD45.2 donor cells relative to the initial chimerism. Data represent the average contribution for Rispfl/fl (RISP-WT BM; n = 10 mice) and Rispfl/fl Mx1-Cre (RISP-KO BM; n = 9 mice) in Supplementary Fig. 7c, d. Data are mean ± s.e.m. P ≤ 0.05; P ≤ 0.01. All P values calculated using Student’s t-test.

Supplementary Figure 7 Adult HSC and progenitors gating strategy.

(a) Within the lineage negative population: MPs (myeloid progenitors) were defined as c-kithigh, Sca-1low. Within the MP population: MEPs (Megakaryocyte-erythroid progenitors) were defined as CD34low, FcγRlow, CMPs (common myeloid progenitors) were defined as CD34high, FcγRlow, and GMPs (granulocyte-monocyte progenitors) were defined as CD34high, FcγRhigh. (b) Within the lineage negative population: LSKs were defined as Sca-1high, c-kithigh. Within the LSK population: ST-HSCs (short-term hematopoietic stem cells) were defined as CD48low, CD150low, LT-HSCs (long-term hematopoietic stem cells) were defined as CD48low, CD150high, and MPPs (Multipotent progenitors) were defined as CD48high, CD150low. (c) Adult bone marrow hematopoiesis populations studied, and gating definitions.

Supplementary Figure 8 Full western blot images.

The original western blot images with ladder. Cropped sections are shown in Fig. 1b and Supplementary Fig. 1b.

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Ansó, E., Weinberg, S., Diebold, L. et al. The mitochondrial respiratory chain is essential for haematopoietic stem cell function. Nat Cell Biol 19, 614–625 (2017) doi:10.1038/ncb3529

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