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FoxO maintains a genuine muscle stem-cell quiescent state until geriatric age

Nature Cell Biology volume 22, pages 1307–1318 (2020)Cite this article

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Abstract

Tissue regeneration declines with ageing but little is known about whether this arises from changes in stem-cell heterogeneity. Here, in homeostatic skeletal muscle, we identify two quiescent stem-cell states distinguished by relative CD34 expression: CD34High, with stemness properties (genuine state), and CD34Low, committed to myogenic differentiation (primed state). The genuine-quiescent state is unexpectedly preserved into later life, succumbing only in extreme old age due to the acquisition of primed-state traits. Niche-derived IGF1-dependent Akt activation debilitates the genuine stem-cell state by imposing primed-state features via FoxO inhibition. Interventions to neutralize Akt and promote FoxO activity drive a primed-to-genuine state conversion, whereas FoxO inactivation deteriorates the genuine state at a young age, causing regenerative failure of muscle, as occurs in geriatric mice. These findings reveal transcriptional determinants of stem-cell heterogeneity that resist ageing more than previously anticipated and are only lost in extreme old age, with implications for the repair of geriatric muscle.

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Fig. 1: CD34 identifies QSC states with distinct engraftment and self-renewal capabilities.
Fig. 2: CD34 identifies distinct quiescent stem-cell states in genetically marked satellite cells.
Fig. 3: Analysis of the quiescent stem-cell states from birth to old age.
Fig. 4: The QSCgenuine state is maintained by the FoxO family of TFs.
Fig. 5: Loss of FoxO signalling causes spontaneous activation and reduced regenerative potential of satellite cells.
Fig. 6: FoxO prevents the inappropriate expression of myogenic-differentiation genes in the QSCgenuine state.
Fig. 7: Early establishment and life persistence of the genuine stem-cell state requires FoxO activity.
Fig. 8: A Markov-chain-based computational method predicts reinforcement and rejuvenation of the genuine state by Igf–Akt pathway inhibition.

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Data availability

The RNA-seq and ATAC-seq data that support the findings of this study have been deposited in GEO under the accession code GSE155642. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

Code availability

Codes for RNA-seq and ATAC-seq analysis (GSE155642) are available from the corresponding author on request. NicheHotSpotter is available at https://gitlab.com/srikanth.ravichandran/signalingfactorscd34positive. Automated counting macros for Fiji are available at https://github.com/MolecularImagingPlatformIBMB.

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Acknowledgements

We thank C. Keller and M. Capecchi for the Pax-cre mouse lines; A. Webb and A. Brunet for LV-hFoxO3a; E. Andrés, J. Segalés, V. Lukesova, A. Martín, A. Pollán, A. Navarro, J. M. Ballesteros, M. Raya and L. Barberi for their technical contributions; A. Serrano, J. Isern and the members of the P.M.-C. laboratory for their helpful discussions; J. Martín-Caballero and S. Rodríguez-Colilla (PRBB/CNIC Animal Facilities); O. Fornas and M. Montoya (UPF/CRG/CNIC FACS Facilities); A. Dopazo (CNIC-Genomics Facility); J. Rodríguez (qGenomics); Leica Microsystems for IBMB-imaging-platform support; S. Bartlett for editing and W. Link (IIB-CSIC) for FoxO-activation advice. The authors acknowledge funding from MINECO-Spain (grant no. RTI2018-096068), ERC-2016-AdG-741966, LaCaixa-HEALTH-HR17-00040, MDA, UPGRADE-H2020-825825, AFM and DPP-Spain to P.M.-C; María-de-Maeztu-Program for Units of Excellence to UPF (grant no. MDM-2014-0370) and the Severo-Ochoa-Program for Centers of Excellence to CNIC (grant no. SEV-2015-0505). This work was also supported by NIAMS IRP through NIH grants nos AR041126 and AR041164 to V.S. and utilized computational resources of the NIH HPC Biowulf cluster (http://hpc.nih.gov); ASI, Ricerca Finalizzata, Ateneo Sapienza to A.M.; AIRC (grant no. 23257); ASI (grant no. MARS-PRE, DC-VUM-2017-006); H2020-MSCA-RISE-2014 (645648) to M.S. and a FNR core grant (grant no. C15/BM/10397420) to A.d.S. L.G.P. was partially supported by an FPI fellowship and an EMBO fellowship (grant no. ALTF 420-2017); and S.C., X.H. and V.M. by FI, Severo-Ochoa and PFI Fellowships (Spain), respectively.

Author information

Author notes

  1. Laura García-Prat

    Present address: Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada

  2. Sonia Alonso-Martín

    Present address: Neurosciences Area, Biodonostia Health Research Institute, Donostia-San Sebastián, San Sebastián, Spain

  3. Marta Flández

    Present address: Grupo de Investigación en Oncología Clínico Traslacional, Instituto de Investigación Hospital 12 de Octubre, Madrid, Spain

  4. These authors contributed equally: Laura García-Prat, Eusebio Perdiguero, Sonia Alonso-Martín.

Authors and Affiliations

  1. Department of Experimental and Health Sciences, Pompeu Fabra University (UPF), CIBER on Neurodegenerative Diseases (CIBERNED), Barcelona, Spain

    Laura García-Prat, Eusebio Perdiguero, Silvia Campanario, Laura Ortet, Vanessa Ruiz-Bonilla, Victoria Moiseeva, Mercè Jardí & Pura Muñoz-Cánoves

  2. Spanish National Center on Cardiovascular Research (CNIC), Madrid, Spain

    Laura García-Prat, Sonia Alonso-Martín, Silvia Campanario, Xiaotong Hong, Marta Flández & Pura Muñoz-Cánoves

  3. Laboratory of Muscle Stem Cells and Gene Regulation, National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), NIH Bethesda, Bethesda, MD, USA

    Stefania Dell’Orso, Aster H. Juan & Vittorio Sartorelli

  4. Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg

    Srikanth Ravichandran & Antonio del Sol

  5. Biodata Mining and Discovery Section, NIAMS, NIH Bethesda, Bethesda, MD, USA

    Stephen R. Brooks, Kan Jiang & Hong-Wei Sun

  6. Molecular Imaging Platform, Molecular Biology Institute of Barcelona (IBMB-CSIC), Barcelona, Spain

    Elena Rebollo

  7. DAHFMO-Unit of Histology and Medical Embryology, Laboratory Affiliated to Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Sapienza University of Rome, Rome, Italy

    Antonio Musarò

  8. Department of Biomedical Science, University of Padova, Padova, Italy

    Marco Sandri

  9. CIC bioGUNE, Derio, Spain

    Antonio del Sol

  10. IKERBASQUE, Basque Foundation for Science, Bilbao, Spain

    Antonio del Sol

  11. ICREA, Barcelona, Spain

    Pura Muñoz-Cánoves

Authors
  1. Laura García-Prat
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Contributions

S.D.’O., S.R. and S.R.B. contributed equally as co-second authors. L.G.-P. and P.M.-C. conceived the study, conceptualized ideas and wrote the manuscript. L.G.-P., E.P. and S.A.-M. designed and performed most experiments, helped with the interpretion of results and revising/editing the manuscript. S.D.’O., A.H.J. and L.G.P. performed the RNA-seq, ChIP and ATAC-seq analyses. S.R.B., K.J., and H.-W.S. designed the bioinformatics pipelines and performed bioinformatics analyses for RNA-seq and ATAC-seq. L.G.-P. and E.P. partially contributed to the RNA-seq bioinformatics. S.R. and A.d.S. designed and generated the NicheHotSpotter model and performed computational analysis of RNA-seq data. S.C. performed the RNAscope analysis. V.M. performed single-cell RNA-seq. L.O., V.R.-B., M.F., X.H. and M.J. performed some cell/mouse experiments and/or provided technical support. E.R. provided technical support for the microscopy. A.M. and M.S. provided essential mouse lines. L.G.-P. and E.P. prepared the final figures. V.S. and P.M.-C. discussed and interpreted results, and supervised, managed and obtained funding for the study.

Corresponding authors

Correspondence to Vittorio Sartorelli or Pura Muñoz-Cánoves.

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Extended data

Extended Data Fig. 1 CD34 stratifies quiescent stem-cell states in satellite-cell reporter mice.

a, Heatmap generated by ImaGEO algorithm using the indicated data sets (from gene expression omnibus, GEO) and which defined the “Quiescence Core” list of genes (see Supplementary Table 1). b, Gating strategy used to isolate quiescent satellite cells by FACS from young WT mice. c, Expression correlation plots showing inverse correlation between Cd34 and Myod1 or Myog in individual satellite cells, by scRNA-seq analysis from young mouse muscle (Myod1, 860 cells; Myog, 1191 cells). d, Representative immunofluorescence images of EdU staining and (bottom) quantification of cell area in FACS-isolated satellite cells from young resting muscle and after 3 days of cardiotoxin injection into TA muscle (2-3 mo, n=46 CD34H, n=62 CD34L and n=83 3dCTX cells from 3 mice). Scale bar, 5 μm. e, Representative immunofluorescence images and quantification of CD34 on muscle sections from Pax7CRE-ER;ROSA26YFP mice (2-3 mo, n=183 total cells from 4 mice). The sum of CD34 fluorescence intensity per cell is represented: black line across show the median; black short lines represent individual data points; bean plot represent the estimated density of the data. Scale bar, 5 μm. f, Representative immunofluorescence images and correlation of Ryr3 and CD34 staining in freshly isolated QSCs from young resting muscle (2-3 mo, n=30 cells from 3 mice). Scale bar, 5 μm. g, Volcano plot showing the most differentially expressed genes identified by RNA-seq between CD34H and CD34L from young Pax7CRE-ER;ROSA26YFP mice (2-3 mo, 4 samples, 3 mice pooled per sample). N numbers provided in source data. h, RNA-seq PCA analysis comparing CD34H and CD34L satellite cells from both WT and Pax7CRE-ER;ROSA26YFP mice. Average distance analysis among CD34H or between CD34H and CD34L is shown (right) (2-3 mo, "to High” n=5 samples, 3 mice pooled per sample, Min 9.8, Median 11.8, Max 16.9 and “to Low” n=5 samples, 3 mice pooled per sample, Min 23.2, Median 27.3, Max 33.6; centre lines show the medians; box limits indicate maximum and minimum). i, Heatmap illustrating average differential gene expression profiles between CD34H and CD34L QSC from WT and Pax7CRE-ER;ROSA26YFP mice (2-4 mo, n=3 mice). j, RNA-seq PCA analysis comparing CD34H, CD34L and CD34N satellite cells from Pax7CRE-ER;ROSA26YFP mice (2-4 mo, "to High” n=3 samples, 3 mice pooled per sample, Min 25.7, Median 26.4, Max 29.7 and “to Low” n=3 samples, 3 mice pooled per sample, Min 9.5, Median 12.7, Max 16; centre lines show the medians; box limits indicate maximum and minimum). Average distance analysis between CD34H and CD34L or between CD34H and CD34N is shown (right). Means ± s.e.m.; two-tailed unpaired t-test with Welch’s correction unless otherwise indicated.

Source data

Extended Data Fig. 2 Distinct functional properties of genuine and primed quiescent satellite-cell states.

a, Representative example of FACS strategy and gating scheme to isolate CD34H, CD34L and CD34N QSCs from young Pax7nGFP mice. b, (left) Quantification of self-renewal capacity of CD34H, CD34L and CD34N QSCs from Pax7-nGFP mice (2-4 mo, n=8 CD34H/CD34L and n=6 CD34N transplanted muscles from 3 mice) transplanted into pre-injured TA of recipient immunodeficient mice for 21 days. Equal number of freshly isolated cells were used. (right) Quantification of self-renewal in vitro of freshly sorted CD34H, CD34L and CD34N QSCs (2-4 mo, n=4 mice for all groups). Cells were expanded and cultured in differentiation medium (DM) for 72-96 h to obtain ‘reserve QSCs’ (that is self-renewal), defined as Pax7+Ki67- satellite cells (from the total number of DAPI+ cells). Paired t-test. c, Clonogenic assay of freshly sorted CD34H, CD34L and CD34N satellite cells from Pax7-nGFP mice. Number of colonies and cells were quantified (2-4 mo, n=32 wells from 8 mice, 4 wells/mouse). d, QSCs GFP+ from young Pax7-nGFP mice were isolated based on CD34 expression (2 mo, n=3 CD34H and n=4 CD34L transplanted muscles from 6 mice). Equal number of freshly isolated quiescent CD34H and CD34L satellite cells were transplanted for 21 days into pre-injured TA muscle of recipient immunodeficient mice. After 21 days, cardiotoxin (CTX) was injected into the transplanted TAs and muscles were recovered 21 days later. Pax7+GFP+ satellite cells were quantified within the regenerated area. e, Enrichment map of gene sets differentially enriched in CD34H vs CD34L QSCs from resting muscle of young WT and Pax7CRE-ER;ROSA26YFP mice (FDR q value ≤ 0.05). Node size is proportional to the number of genes identified in each gene set (minimum 10 genes/gene set). Grey edges indicate gene overlap. Blue node: enrichment in CD34H (positive NES) (NES: normalized enrichment score). Orange node: enrichment in CD34L (negative NES). Clusters were automatically annotated using Autoannotate app in Cytoscape. (OXPHOS: Oxidative Phosphorylation, TCA: Tricarboxylic acid). Means ± s.e.m.; two-tailed unpaired t-test with Welch’s correction unless otherwise indicated.

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Extended Data Fig. 3 Evolution of the genuine and primed stem-cell states with ageing.

a, Quantification of satellite cells from young (2-3 mo, n=18 mice, Min 103.3, Median 170.1, Max 294.4), old (22-24 mo, n=13 mice, Min 60.1, Median 90, Max 165) and geriatric (>28 mo, n=5 mice, Min 46.1, Median 101.2, Max 136.8) mice by FACS. centre lines show the medians; box limits indicate the 25th and 75th percentiles. b, QSCs isolated from young (2-3 mo, n=10 transplanted muscles from 6 mice), old (22-24 mo, n=7 transplanted muscles from 3 mice) and geriatric (>28 mo, n=7 transplanted muscles from 8 mice) WT mice were transduced with GFP-expressing lentivirus and transplanted for 21 days into pre-injured TA muscles of recipient immunodeficient mice. Equal number of freshly-isolated cells were used. Representative images and quantification of GFP+ fibres are shown. Scale bar, 50 μm. c, Representative images and frequency distribution analysis of positive embryonic myosin heavy chain (eMHC) fibre size in cryosections of regenerating TA muscles from young (2-3 mo, n=3 mice), old (22-24 mo, n=3 mice) and geriatric mice (>28 mo, n=3 mice), one week after cardiotoxin (CTX)-induced injury. Scale bar, 50 μm. For the frequency distribution representation. 500 fibres per muscle were measured. d, Percentage of QSCsgenuine and QSCsprimed in young (2-3 mo, n=18 mice), old (22-24 mo, n=18 mice) and geriatric (>28 mo, n=17 mice) WT mice. Percentages were set at 15% for each population in one young WT mice from each FACS experiment. e, Enrichment map of gene sets enriched in QSCsgenuine from geriatric vs young WT mice (FDR q value ≤ 0.05). Node size is proportional to the number of genes identified in each gene set (minimum 10 genes/gene set). Grey edges indicate gene overlap. Clusters were automatically annotated using Autoannotate app in Cytoscape. (OXPHOS: Oxidative Phosphorylation, TCA: Tricarboxylic acid, NO: Nitric oxide). Means ± s.e.m.; two-tailed unpaired t-test with Welch’s correction unless otherwise indicated.

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Extended Data Fig. 4 Loss of FoxO transcription factors provokes loss of genuine quiescent satellite cells and regenerative decline.

a, Transcription-factor binding-site enrichment analysis in QSCgenuine and QSCprimed gene signatures, using GSEA. FoxO binding-site enrichment highlighted in red. b, FoxO motif enrichment analysis in ATAC-seq libraries from YFP+ CD34H, CD34L and CD34N QSCs isolated from Pax7CRE-ER;ROSA26YFP mice (2-3 mo, n=6 mice). c, Representative images for FoxO1, 3a and 4 immunostaining of satellite cells (Pax7+) on single myofibre explants from WT resting muscle. This experiment has been repeated 3 times. Scale bar, 50 μm. d, Generation of satellite cell-specific FoxO3aΔPax7ER mice. RT-qPCR of FoxO3a in freshly-isolated satellite cells from FoxO3aWT and FoxO3aΔPax7ER mice (2-3 mo, n=2 mice) 7-days after the last tamoxifen injection. e, Western blotting of FoxO3a in satellite cells from (d). Graph shows quantification of FoxO3a normalized to Tubulin (2-3 mo, n=6 mice) (see Unprocessed Blots Extended Data Fig. 4 for original blot). f, Representative flow-cytometry plots for QSCs from FoxO3aWT and FoxO3aΔPax7ER mice (2-3 mo, n=3 mice) 7-days after the last tamoxifen injection. CD34 MFI quantified by flow cytometry. g, Fibre-size average and frequency distribution of eMHC+ fibres in regenerating TA from FoxO1WT and FoxO1ΔPax7ER mice (2-3 mo, n=3 WT and n=4 FoxO1ΔPax7ER mice) . For the frequency distribution representation. 500 fibres per muscle were measured. 7 days post-cardiotoxin (CTX) injection. h, RT-qPCR of FoxO1, FoxO3a and FoxO4 in satellite cells from WT and FoxO,1,3a,4ΔPax7ER mice (2-3 mo, n=3 mice) 7-days after tamoxifen injection. i, Representative immunofluorescence images and quantification of proliferating satellite cells (Pax7+Ki67+) in resting muscle from FoxO1,3a,4WT and FoxO1,3a,4ΔPax7ER mice 7-days after tamoxifen treatment (2-4 mo, n=4 mice). Scale bar, 10 μm. j, Efficiency of Cre recombinase in FoxO1,3a;4WT;ROSA26YFP mice 30-days after tamoxifen treatment. Percentage of YFP+ satellite cells within the α7-integrin+CD34+ population by flow cytometry. Representative dot plots are shown. k, RT-qPCR of FoxO1, FoxO3a and FoxO4 genes in YFP+-isolated satellite cells from FoxO1,3a,4WT;ROSA26YFP, FoxO,1,3a,4ΔPax7ER;ROSA26YFP or FoxO3aΔPax7ER;ROSA26YFP mice at 1 and 3 days after the last tamoxifen injection (2-4 mo, n=3 mice for all groups except FoxO,1,3a,4ΔPax7ER;ROSA26YFPat day 1 n=6 mice). Means ± s.e.m.; two-tailed unpaired t-test with Welch’s correction unless otherwise indicated.

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Extended Data Fig. 5 Regulation of the genuine and primed gene expression programmes by FoxO transcription factors.

a, Venn-diagram showing overlapping genes between the “QSCprimed signature” and the downregulated genes in FoxO1,3a,4ΔPax7ER vs FoxO1,3a,4WT mice (2-4 mo, n=3 mice) 30 days after the last tamoxifen injection. Actc1, Cdkn1c, Myog and Tnnt2 genes are highlighted. b, Venn-diagram showing overlapping genes between the “QSCgenuine signature” and the upregulated genes in FoxO1,3a,4ΔPax7ER vs FoxO1,3a,4WT mice (2-4 mo, n=3 mice) 30 days after the last tamoxifen injection. Cd34, Dmd, Igfbp4 and Notch3 genes are highlighted. c, RT-qPCR of FoxO1, FoxO3a and FoxO4 in YFP+ satellite cells submitted for RNA-seq from FoxO1,3a,4WT;ROSA26YFP and FoxO,1,3a,4ΔPax7ER; ROSA26YFP mice 4 days after the last tamoxifen injection (2-4 mo, n=4 mice). d, Volcano-plot showing the most differentially-expressed genes by RNA-seq between FoxO1,3a,4WT and FoxO1,3a,4ΔPax7ER mice (2-4 mo, 2 mice) 4 days after the last tamoxifen injection. N numbers provided in source data. e, Venn-diagram showing overlapping genes between differentially-expressed genes in FoxO1,3a,4WT and FoxO1,3a,4ΔPax7ER mice 4 (2-4 mo, n=2 mice) and 30 days (2-3 mo, n=3 mice) after the last tamoxifen injection. f, Enrichment map of gene-sets enriched in FoxO1,3a,4WT and FoxO1,3a,4ΔPax7ER mice 30 days after the last tamoxifen injection (FDR q value ≤ 0.05). NES (Normalized Enrichment Score) values for the comparison between FoxO1,3a,4ΔPax7ER vs FoxO1,3a,4WT mice 4 days after the last tamoxifen injection are shown. Red node: positive NES (downregulated in FoxO1,3a,4ΔPax7ER cells). Blue node: negative NES (upregulated in FoxO1,3a,4ΔPax7ER cells). Node size is proportional to the number of genes in each gene-set (minimum 10 genes/gene-set). Black-edges indicate gene overlap. Clusters were automatically annotated using Autoannotate app in Cytoscape; (OXPHOS: Oxidative Phosphorylation). g, Normalized ATAC-seq signal profiles in the indicate promoters (left) and quantification of ATAC-seq normalized peak-tags 1Kb upstream and downstream the transcription start site (TSS) for the indicated promoters (right) from FoxO1,3a,4ΔPax7ER vs FoxO1,3a,4WT mice (2-4 mo, n=4 mice). Means ± s.e.m.; two-tailed unpaired t-test with Welch’s correction unless otherwise indicated.

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Extended Data Fig. 6 Loss of FoxO signalling imposes the primed-state gene expression programme and depletes the genuine state.

a, Analysis of the association of FoxO3a to the promoter of the following genes: Myog, Cd34, Tnnt2, Igfbp4 and Cdkn1b (as positive control) by chromatin-immunoprecipitation (ChIP) analysis. Satellite cells were cultured and treated for 2 h with LY294002 (20 μM) to inhibit PI3K/Akt activation and induce FoxO nuclear translocation. Relative recruitment versus input is shown for each gene analysed by qPCR (2-4 mo, n=5 Negative; n=10 Myog-IgG and n=11 Myog-FoxO3a; n=4 Cd34; n=2 Tnnt-2-IgG and n=3 Tnnt2-FoxO3a; n=5 Igfbp4; n=11 Cdkn1b(p27) mice). b, Confirmation of reduction of Myog mRNA expression by qPCR in freshly isolated satellite cells from FoxO1,3a,4ΔPax7ER;ROSA26YFP mice (2-4 mo, n=3 mice) transfected 3 h with siScramble or siMyog. c, Confirmation of the overexpression of FoxO3a in cultured WT satellite cells after transduction with LV-hFoxO3a virus (2-4 mo, n=3 mice). d, Confirmation of the deletion of FoxO1, 3a and 4 in cultured FoxO1,3a,4f/f satellite cells after transduction with Adeno-Cre virus compared to Adeno-GFP (2-4 mo, n=3 mice). e, RT-qPCR analysis of FoxO1, 3a and 4 expression in freshly-isolated satellite cells from FoxO1,3a,4WT and FoxO1,3a,4ΔPax7 mice (2-4 mo, n=6 mice). f, Expression of Myod1, Ccnd1 and Myog genes in satellite cells from juvenile FoxO1,3a,4WT and FoxO1,3a,4ΔPax7 mice (21d, n=3 mice). g, Juvenile FoxO1,3a,4WT and FoxO1,3a,4ΔPax7 mice were treated with BrdU in drinking water for 10 days (from 21 to 31 days of age, n=3 mice). (Left) Representative immunofluorescence images of BrdU+ satellite cells/myonuclei outside or inside the myofibre labelled with Dystrophin. Scale bar, 50 μm. (Right) Representative immunofluorescence images and quantification of BrdU+ myonuclei in freshly-isolated single fibres. Scale bar, 50 μm. Means ± s.e.m.; two-tailed unpaired t-test with Welch’s correction unless otherwise indicated.

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Extended Data Fig. 7 The molecular properties of the genuine stem-cell state are maintained by FoxO signalling until geriatric age.

a, Enrichment map of gene sets enriched in young FoxO1,3a,4ΔPax7ER vs FoxO1,3a,4WT mice (FDR q value ≤ 0.05) 30 days after the last tamoxifen injection. The enrichment (FDR q-values) of those gene sets in FoxO1,3a,4WT and FoxO1,3a,4ΔPax7 mice and FoxO1,3a,4ΔPax7ER vs FoxO1,3a,4WT at different ages are also represented. Node size is proportional to the number of genes identified in each gene set (minimum 10 genes/gene set). Black edges indicate gene overlap. Clusters were automatically annotated using Autoannotate app in Cytoscape. (OXPHOS: Oxidative Phosphorylation; TCA: Tricarboxylic acid). b, Quantification of the FoxO1 and FoxO4 MFI analysed by flow cytometry of freshly isolated QSCsgenuine and QSCsprimed from young (2-4 mo, n=5 mice) and geriatric (>28mo, n=5 mice) mice. c, Quantification of the percentage of YFP+ satellite cells from Lin- cells analysed by flow cytometry and isolated from FoxO1,3a,4ΔPax7ER vs FoxO1,3a,4WT mice at young (2-4 mo, n=3 mice) and geriatric (>28mo, n=3 mice) age. Data was normalized to one young control mouse (100%). d, Representative flow cytometry plots of freshly isolated QSCs (YFP+) from FoxO1,3a,4WT;ROSA26YFP and FoxO1,3a,4ΔPax7ER;ROSA26YFP mice at geriatric (>28 mo, n=3 mice) age after tamoxifen administration. e, GSEA analysis of the QSCgenuine (right) and QSCprimed (left) signatures in QSCs isolated form FoxO1,3a,4WT;ROSA26YFP and FoxO1,3a,4ΔPax7ER;ROSA26YFP mice at geriatric (>28 mo, n=3 mice). Means ± s.e.m.; two-tailed unpaired t-test with Welch’s correction unless otherwise indicated.

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Extended Data Fig. 8 NicheHotSpotter predicts niche-derived Ins/Igf-driven Akt signalling to inhibit FoxO in the young primed stem-cell state.

a, Complete predicted signalling network around Akt1 for young QSCsgenuine in comparison to QSCsprimed. Akt1 and FoxO TFs are shown as orange and purple coloured nodes, the green and red triangles are upregulated and downregulated downstream TFs respectively. All other grey nodes are intermediate molecules that are expressed. Green and red edges indicate activation and inhibition interactions, respectively. The network depicts potential flow of external signals via the predicted signalling hotspots to the differentially expressed TFs. b, Quantification of p-Akt signal in freshly-isolated QSCgenuine and QSCprimed cells from young mice (2-3 mo, n=1 mice, 35 total cells). Representative immunofluorescence images and quantification of p-Akt signal of freshly-isolated QSCprimed cells from young mice 2-weeks after treatment with vehicle (control) or wortmannin (2-3 mo, n=3, 135 total cells). Scale bar, 5 μm. c, Quantification of FoxO3a nuclear signal in freshly-isolated QSCgenuine and QSCprimed cells from young mice (2-3 mo, n=3, 232 total cells). Representative immunofluorescence images and quantification of FoxO3a nuclear signal of freshly isolated QSCprimed cells from young mice 2-weeks after treatment with vehicle (control) or wortmannin (2-3 mo, n=1, 63 total cells). Scale bar, 5 μm. Means ± s.e.m.; two-tailed unpaired t-test with Welch’s correction unless otherwise indicated.

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Extended Data Fig. 9 NicheHotSpotter predicts the Igf–Igfr–Akt pathway to be more active in geriatric than in young genuine satellite cells.

a, Clonogenic assay of freshly-sorted QSCsgenuine and QSCsprimed from young mice 2-weeks after treatment with vehicle (control) (n=20 wells from 4 mice, 5 replicates/mouse) or wortmannin (n=18 wells from 4 mice, 4-5 replicates/mouse). Number of colonies and cells were quantified. b, Complete predicted signalling network around Akt1 for young QSCsgenuine in comparison to geriatric QSCsgenuine. The network colours representation is the same as in Extended Data Fig. 8a. c, Representative immunofluorescence images and quantification of FoxO3a on QSCs freshly isolated from young WT and MLC-IGF1 (Tg-IGF1) mice (2-3 mo, n=88 cells from 2 WT mice and n=135 cells from 4 MLC-IGF1 mice. Scale bar, 5 μm. Means ± s.e.m.; two-tailed unpaired t-test with Welch’s correction unless otherwise indicated.

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Extended Data Fig. 10 Analysis of the quiescent stem-cell state in IGF1-overexpressing mice and proposed model of quiescent satellite-cell heterogeneity.

a, Representative immunofluorescence images and quantification of p-Akt immunostaining on freshly-isolated QSCs from young WT and Tg-IGF1 (MLC-IGF1) mice (2-3 mo, n=37 WT and n=32 MLC-IGF1 cells from 2 mice). Scale bar, 5 μm. b, Clonogenic assay of freshly-sorted QSCs from (a). Number of colonies and cells were quantified (n=22 WT and n=24 MLC-IGF1 wells from 4 mice, 4-6 replicates/mouse). c, GSEA analysis of the “QSCgenuine signature” in CD34High satellite cells (QSCsGenuine) isolated from young WT and MLC-IGF1 (Tg-IGF1) mice. d, Scheme showing that the quiescent satellite cell (QSC) state is molecularly and functionally heterogeneous, encompassing a genuine state (QSCgenuine) (in blue), with inferred stemness features, and a primed state (QSCprimed) (in orange), which is more prone to myogenic differentiation. FoxO signalling is required first to establish the QSC pool early in postnatal life and second to preserve the QSCgenuine state throughout adult and late life. Niche-derived IGF1-mediated Akt activity inhibits FoxO signalling, thus deteriorating the genuine state through inappropriate expression of the primed-state gene programme. Interventions inhibiting Akt and reactivating FoxO were computationally predicted and experimentally confirmed to be capable of converting the primed into a more functional genuine-like state. Means ± s.e.m.; two-tailed unpaired t-test with Welch’s correction unless otherwise indicated.

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Supplementary information

Reporting Summary

Supplementary Tables

Supplementary Table 1. List of genes that compose the quiescence core in Fig. 1a. Supplementary Table 2. Lists of genes included in the QSCgenuine and QSCprimed core signatures of Fig. 2e. Supplementary Table 3. Supplementary information on reagents—antibodies, oligos, primers and so on−used in the paper.

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García-Prat, L., Perdiguero, E., Alonso-Martín, S. et al. FoxO maintains a genuine muscle stem-cell quiescent state until geriatric age. Nat Cell Biol 22, 1307–1318 (2020). https://doi.org/10.1038/s41556-020-00593-7

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