Abstract
Haematopoietic stem cells (HSCs), which sustain production of all blood cell lineages1, rely on glycolysis for ATP production2,3, yet little attention has been paid to the role of mitochondria. Here we show in mice that the short isoform of a critical regulator of HSCs, Prdm16 (refs 4, 5), induces mitofusin 2 (Mfn2), a protein involved in mitochondrial fusion and in tethering of mitochondria to the endoplasmic reticulum. Overexpression and deletion studies, including single-cell transplantation assays, revealed that Mfn2 is specifically required for the maintenance of HSCs with extensive lymphoid potential, but not, or less so, for the maintenance of myeloid-dominant HSCs. Mfn2 increased buffering of intracellular Ca2+, an effect mediated through its endoplasmic reticulum–mitochondria tethering activity6,7, thereby negatively regulating nuclear translocation and transcriptional activity of nuclear factor of activated T cells (Nfat). Nfat inhibition rescued the effects of Mfn2 deletion in HSCs, demonstrating that negative regulation of Nfat is the prime downstream mechanism of Mfn2 in the maintenance of HSCs with extensive lymphoid potential. Mitochondria therefore have an important role in HSCs. These findings provide a mechanism underlying clonal heterogeneity among HSCs8,9,10,11 and may lead to the design of approaches to bias HSC differentiation into desired lineages after transplantation.
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Acknowledgements
The pHR-IRES-GFP SIN vector was a gift from R. Creusot. Mfn2−/− MEFs were a gift from D. Chan. We thank S.-H. Ho and E. A. Schon for assistance and discussion. This work was supported by grant NIH RO1 CA167286 and RO1 AG029262 (H.-W.S.), the Druckenmiller Fellowship from the New York Stem Cell Foundation (L.L.L.), the Ruth L. Kirschstein F31 CA196045 fellowship (D.J.C.), the NIH National Center for Research Resources award 1S10RR027050-01 and the NIH Office of the Director award 1S10OD020056-01.
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L.L.L. designed and performed most experiments, contributed to the concept, and co-wrote the manuscript with H.-W.S.; M.J.A. generated the data in Fig. 4e and Extended Data Fig. 8a, and assisted in Figs 2, 4i and Extended Data Figs 6, 7, 8. M.M. cloned the DNDrp1 construct and verified its function (Extended Data Fig. 7d). D.J.C. generated the Mfn2 promoter construct (Extended Data Fig. 3c). H.-W.S. provided concept and guidance, and co-wrote the paper with L.L.L.
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Extended data figures and tables
Extended Data Figure 1 Representative sort gates for isolation of HSCs.
Flow cytometric plots showing the gates used to isolate HSCs (Lin−Sca1+kit+Flt3−CD48−) and CD150hi and CD150lo HSCs.
Extended Data Figure 2 Mitochondrial dynamics in Prdm16−/− MEFs.
a, Frequency distribution (left) and frequency distribution profile (right) of mitochondrial length in WT or Prdm16−/− fetal HSCs. Bars, mean ± s.e.m.; n ≥ 20 fields of cells from three biological replicates; *P < 0.05 within length bins; two-tailed Student’s t-test within length bins. b, Mitochondrial morphology in Prdm16−/− MEFs treated for 24 h with the Drp1 inhibitor, mDivi (30 μM), or vehicle is shown for comparison, Mitotracker Red staining, scale bar 20 μm). c, Frequency distribution (left) and frequency distribution profile (right) of mitochondrial length in WT and Prdm16−/− MEFs treated for 24 h with the Drp1 inhibitor, mDivi (30 μM), or vehicle. Bars, mean ± s.e.m.; n ≥ 16 fields from three biological replicates; *P < 0.05 within length bins; one-way ANOVA with Bonferroni’s post-hoc test within length bins. d, Fluorescence micrographs showing fusion hybrids of WT or Prdm16−/− MEFs transduced with baculovirus expressing mitochondria-tagged GFP and RFP before PEG-mediated fusion (scale bar 50 μm). Fused mitochondria are yellow (arrows), and were only observed in fusions of WT cells. e, western blot (upper, Supplementary Fig. 1 for full scans) and quantification of western blots for Mfn2 of WT and Prdm16−/− MEFs. Bars, mean ± s.e.m.; n = 3 fields biological replicates; *P < 0.05; two-tailed Student’s t-test. f, Mitochondrial morphology in WT, Prdm16−/− and Mfn2−/− MEFs visualized by Tomm20 staining (red) (scale bar 20 μm). (G) Mitochondrial length in WT and Prdm16−/− MEFs transduced with EV or Mfn2-IRES-GFP (Mfn2–GFP). Bars, mean ± s.e.m.; n ≥ 24 fields from three biological replicates; *P < 0.05 compared to Prdm16−/− EV in each length bin; one-way ANOVA with Bonferroni’s post-hoc test within length bins.
Extended Data Figure 3 sPrdm16 interacts with the Mfn2 promoter.
a, Schematic representation of Prdm16 protein domain structure. b, qPCR probe amplification scheme covering the Mfn2 promoter used in chromatin immunoprecipitation assays. c, Proximal Mfn2 promoter luciferase gene reporter assay in WT MEFs transfected 24 h previously with flPrdm16, sPrdm16 or control vectors. Bars, mean ± s.e.m.; n = 3 biological replicates; *P < 0.05; one-way ANOVA with Dunnett’s post-hoc test. d, ChIP quantification in Prdm16−/− MEFs transduced with retroviral Flag–Prdm16 constructs immunoprecipitated using Flag and TF2D antibodies. TFIID positive (GAPDH) and negative (intergenic Chr 6) control probes are also shown. Quantification was performed after establishment of qPCR standard curves for all probes (see Methods). Bars, mean ± s.e.m.; n = 3 technical replicates representative tow biological replicates; *P < 0.05; one-way ANOVA with Dunnett’s post-hoc test. e, Percentage CD45.2 (donor) contribution in PB WBC in CD45.1+CD45.2+ mice reconstituted with 200 transduced (IRES–GFP or Mfn2-IRES–GFP) Prdm16+/− CD45.2+ HSCs and 2 × 105 CD45.1+ competitor bone marrow cells. Plots, mean ± s.e.m.; n = 17 recipients pooled from four independent transplants; *P < 0.05; two-tailed Student’s t-test.
Extended Data Figure 4 Mitochondrial morphology of bone marrow populations.
a, Representative images of mitochondrial morphology in Pham-reporter+ CD150hi and CD150lo HSCs (lin−Sca1+kit+Flt3−CD48−), MPPs (lin−Sca1+kit+CD48+), CMPs (lin−Sca1−kit+), CLPs (lin−Sca1lokitloIL7Ra+Flt3+) and lineage+ cells (scale bar, 5 μm). b, Mitochondrial length frequency distribution in Pham-reporter+ HSCs, progenitors and Lin+ cells. Bars, mean ± s.e.m.; n ≥ 14 fields from three biological replicates; *P < 0.05 within length bins; one-way ANOVA with Bonferroni’s post-hoc test within length bins.
Extended Data Figure 5 Phenotype and function of Mfn2−/− HSCs.
a, Mitochondrial morphology HSCs from Mfn2fl/fl or Mfn2fl/fl-Vav-Cre (Mfn2−/−) mice visualized with immunostaining for Tomm20. b, Frequency distribution of mitochondrial lengths (right panel, compared to WT in each length bin). Bars, mean ± s.e.m.; n ≥ 7 fields from two biological replicates; *P < 0.05 within length bins; two-tailed Student’s t-test within length bins. c, CD150 surface staining mean fluorescence intensity (MFI) of HSCs (Lin−Sca1+kit+CD48−) in 8–12 week old Mfn2fl/fl or Mfn2fl/fl.Vav-Cre (Mfn2−/−) mice. n = 25 biological replicates, *P < 0.05, two-tailed Student’s t-test. d, e, CD150 MFI of Mfn2fl/fl or Mfn2fl/fl.Vav-Cre (Mfn2−/−) donor HSCs 15 weeks after competitive transplantation. Plots in d, mean ± s.e.m.; n ≥ 15 mice from three biological replicates. f, *P < 0.05; two-tailed Student’s t-test. f, Donor (CD45.2) chimaerism 15 weeks after competitive transplantation of 2 × 105 Mfn2fl/fl or Mfn2fl/fl.Vav-Cre (Mfn2−/−) fetal liver cells together with 2 × 105 CD45.1+ competitor bone marrow cells into CD45.1+CD45.2+ recipients Plots, mean ± s.e.m.; n = 10 mice pooled from two independent transplants; *P < 0.05; two-tailed Student’s t-test.
Extended Data Figure 6 Analysis of competitive repopulation experiments and lentiviral vector transduction.
a, Schematic representation of lentiviral constructs used. b, Representative example of transduction efficiency of purified HSCs with IRES–GFP and IRES–mCherry vectors 24–48 h post-transduction. c, Schematic representation of transduction and subsequent competitive repopulation experiments. d, Representative flow cytometric plots of the gates used to analyse recipients of competitive repopulation experiments. The CD45.1/CD45.2 gates were also applied to the myeloid, B and T cell gates to determine donor contribution to individual lineages. In this example, HSCs had been transduced with an IRES–GFP lentiviral vectors (efficiency 80%, see b). Although donor repopulation was high in the periphery (upper left panel, 71.8%) no GFP was detected (lower left panel), suggesting silencing of the vectors. e, Quantification of proviral copy number in donor-derived (CD45.2+) 15 weeks after transduction and competitive transplantation of HSCs. Despite silencing of the vector (see d), approximately 1 proviral copy was present per donor HSC-derived cell. n = 3 biological replicates; *P < 0.05; two-tailed Student’s t-test. f, Representative example of transduction efficiency of purified HSCs with IRES–GFP and Mfn2–IRES–GFP lentiviral vectors. g, Expression of Mfn2 mRNA in CD150hi and CD150lo HSCs 24 h post-transduction relative to EV control (left panel) and in donor-derived HSCs, CMPs and lineage+ cells 15 weeks after competitive transplantation of 200 transduced HSCs together with 2 × 105 CD45.1+ competitor cells (right panel). The data indicate partial silencing of the vector in HSCs, and complete silencing in the progenitors and more mature cells. n = 3 biological replicates; *P < 0.05; two-way analysis of variance (ANOVA) with Bonferroni’s post-hoc test. h, Donor (CD45.2+) chimaerism 15 weeks after transplantation of 200 CD45.2+ HSCs transduced for 24 h with SIN LTR constructs pLVX-IRES-GFP or pLVX-Mfn2-IRES-GFP together with 2 × 105 CD45.1+ competitor fetal liver cells into lethally irradiated CD45.1+CD45.2+ recipients mice. Plots, mean ± s.e.m.; n ≥ 19 recipients from four transplantation experiments; *P < 0.05; two-tailed Student’s t-test. i, Same experiments as in h, but using the non-SIN LTR vector, pHR. Plots, mean ± s.e.m.; n = 5 recipients; *P < 0.05; two-tailed Student’s t-test.
Extended Data Figure 7 Nfat activity in Mfn2−/− and Prdm16−/− MEFs.
a, Mfn2fl/fl (blue) or Mfn2fl/fl-Vav-Cre (Mfn2−/−) (red) HSCs were stained with DCFDA to visualize intracellular reactive oxygen species levels. Cells were treated with oligomycin for 15 min as a positive control. b, HSC apoptosis levels were analysed by surface staining of Annexin V. c, Intracellular staining for the endoplasmic reticulum stress response chaperone GRP78 in Mfn2fl/fl or Mfn2fl/fl-Vav-Cre (Mfn2−/−) HSCs (red colours) MPPs (green colours) and CMPs (blue colours). Histograms; representative of three biological replicates. d, Calcium flux trace (left) and baseline intracellular Ca2+ (right) in WT LSK cells transduced with IRES–GFP or Mfn2–IRES–GFP lentiviral vectors (e) and with WT or Mfn2−/− MEFs stained with Indo1. Bars, mean ± s.e.m.; n = 3 biological replicates; *P < 0.05; two-tailed Student’s t-test. f, Nfat1 staining (left, scale bars, 5 μm) and fraction of nuclear Nfat1 (right, *P < 0.05) in HSCs transduced with IRES–GFP or Mfn2–IRES–GFP lentiviral vector. Bars, mean ± s.e.m.; n ≥ 10 from two biological replicates; *P < 0.05; two-tailed Student’s t-test. g, Nfat1 staining (left; scale bars, 20 μm) and fraction of nuclear Nfat1 (right; *P < 0.05) in WT or Mfn2−/− MEFs. Bars, mean ± s.d.; n ≥ 20 fields from three independent experiments; *P < 0.05; two-tailed Student’s t-test. h, Nfat luciferase gene reporter activity in WT or Mfn2−/− MEFs. Bars, mean ± s.d.; n = 3 experiments; *P < 0.05; two-tailed Student’s t-test. i, Mitochondrial morphology after transfection of dominant negative Drp1 into HeLa cells (scale bars, 5 μm). Note extreme elongation of mitochondria (arrow), confirming that this vector is functional. j, Nfat1 staining (left, scale bars, 5 μm) and fraction of nuclear Nfat1 (right) in HSCs lentivirally transduced with sPRrdm16 and flPRdm16. Plots, mean ± s.e.m.; n ≥ 14 fields of cells pooled from two biological replicates; *P < 0.05; one-way ANOVA with Dunnett’s post-hoc test. k, Nfat luciferase gene reporter activity in WT and in Prdm16−/− MEFs transfected with Mfn2, flPrdm16 or sPrdm16 constructs. Bars, mean ± s.d.; n = 3 experiments; *P < 0.05; one-way ANOVA with Dunnett’s post-hoc test.
Extended Data Figure 8 Effect of VIVIT on WT HSCs.
a, Expression of lymphoid (Sox4 and Il7r) and myeloid/platelet (Vwf and Fli1) genes in HSCs treated with VIVIT or DMSO for 24 h. n = 3 biological replicates; *P < 0.05; Student’s t-test. b, Lymphoid/myeloid ratio of CD45.2+ HSCs cultured for 4 days in DMSO and 500 nM VIVIT, transplanted with 2 × 105 CD45.1+CD45.2+ competitor bone marrow cells into CD45.1+ recipients and analysed 15 weeks post-transplant for lymphoid/myeloid ratio of donor compartment. Plots, mean ± s.e.m.; n = 5–9 recipients from two transplant experiments; *P < 0.05; two-tailed Student’s t-test. c, Donor chimaerism analysis 15 weeks after transplantation of CD45.2+ HSCs transduced with IRES–GFP or VIVIT–GFP with 2 × 105 CD45.1+CD45.2+ competitor bone marrow cells into CD45.1+ recipients Plots, mean ± s.e.m.; n = 9–12 recipients from two independent transplant experiments; *P < 0.05; two-tailed Student’s t-test. Bottom panel shows lymphoid and myeloid donor contribution in each individual recipient.
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Luchsinger, L., de Almeida, M., Corrigan, D. et al. Mitofusin 2 maintains haematopoietic stem cells with extensive lymphoid potential. Nature 529, 528–531 (2016). https://doi.org/10.1038/nature16500
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DOI: https://doi.org/10.1038/nature16500
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