Regeneration of skeletal muscle depends on a population of adult stem cells (satellite cells) that remain quiescent throughout life. Satellite cell regenerative functions decline with ageing. Here we report that geriatric satellite cells are incapable of maintaining their normal quiescent state in muscle homeostatic conditions, and that this irreversibly affects their intrinsic regenerative and self-renewal capacities. In geriatric mice, resting satellite cells lose reversible quiescence by switching to an irreversible pre-senescence state, caused by derepression of p16INK4a (also called Cdkn2a). On injury, these cells fail to activate and expand, undergoing accelerated entry into a full senescence state (geroconversion), even in a youthful environment. p16INK4a silencing in geriatric satellite cells restores quiescence and muscle regenerative functions. Our results demonstrate that maintenance of quiescence in adult life depends on the active repression of senescence pathways. As p16INK4a is dysregulated in human geriatric satellite cells, these findings provide the basis for stem-cell rejuvenation in sarcopenic muscles.
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We are indebted to M. Raya and V. Lukesova for their contributions to this study; J. Martín-Caballero (PRBB Animal Facility), O. Fornas (UPF/CRG FACS Facility) and CRG/UPF Genomic Facility for technical help; A. Consiglio for help in lentivirus obtention; E. Rebollo for advice on imaging; M. van Lohuizen for Bmi1-deficient mice; M. Blasco’s laboratory for help with ageing mice; T. Kawamura and M. Serrano for p16INK4a constructs; A. Sacco, S. Tajbakhsh, B. Gayraud-Morel, D. Montarras, J. Morgan and F. S. Tedesco for advice on cell transplantation; A. Brack and P. Zammit for advice on reserve cells; and Myoage network and tissue bank for support. The authors acknowledge funding from MINECO-Spain (SAF2012-38547, FIS-PS09/01267, FIS-PI13/025, PLE2009-0124), AFM, MDA, E-Rare, Fundació Marató TV3, DuchennePP-NL and EU-FP7 (Myoage, Optistem and Endostem). P.S.-V. and L.G.-P. were supported by predoctoral fellowships from Fundação para a Ciência e a Tecnologia (Portugal) and Programa de Formación de Personal Investigador (Spain), respectively.
The authors declare no competing financial interests.
Extended data figures and tables
Extended Data Figure 1 Muscles of geriatric and progeric mice show signs of sarcopenia and present defective regeneration.
a, Representative images of haematoxylin and eosin (H/E) stained cryosections of tibialis anterior muscle from wild-type mice at different ages: young (2–3 months), adult (5–6 months), old (20–24 months) and geriatric (28–32 months) mice. Asterisks indicate atrophic myofibres. Arrows indicate central-nucleated myofibres. Histograms represent the quantification of myofibre size, evaluated by the cross-sectional area. Frequency distribution of fibres according to size from tibialis anterior muscles from wild-type mice at different ages, and grip strength corrected for body weight. b, Representative pictures of NCAM immunostained cryosections of tibialis anterior muscle from old and geriatric mice. c, As in panel b. Representative pictures of eMHC immunostaining. Histogram represents the quantification of the number of eMHC-positive myofibres per section. d, Fibre-type distribution and quantification of myofibre size, evaluated by the cross-sectional area of the fibres. Quantifications were performed in cryosections stained for specific MHC antibodies. e, Representative images of haematoxylin and eosin staining in cryosections of tibialis anterior muscle from 12-month-old SAMP8 and SAMR1 mice. Asterisks indicate atrophic myofibres. f, Representative images of eMHC staining in cryosections of regenerating tibialis anterior muscle from adult, old and geriatric mice 1 week after cardiotoxin (CTX)-induced injury. Histograms represent the quantification of the number of eMHC+ myofibres and myofibre size, evaluated by the cross-sectional area. g, Immunostaining for dystrophin and nuclear labelling with DAPI was performed in cryosections of extensor digitorum longus muscle from adult and geriatric mice, and SAMR1 and SAMP8 mice 1 week after heterografting onto the tibialis anterior muscle of young mice, and numbers of DAPI-stained nuclei within the dystrophin-positive sarcolemma were quantified. h, Equal numbers of FACS-purified satellite cells from adult, old and geriatric mice, labelled with a lentivirus expressing GFP, were transplanted into the tibialis anterior muscle of young (3 months old) immunodeficient mice. Seven days after transplantation, muscles were collected, sectioned and immunostained for GFP (green). Representative images of transplanted muscles are shown. Graph showing total numbers of GFP+ myofibres scored per muscle. i, Equal numbers of purified satellite cells from SAMP8 and SAMR1 mice were labelled and transplanted into muscles of young immunodeficient mice, and analysed as in panel h. Scale bars, 50 μm. Data are mean ± s.e.m. Two-sided Mann–Whitney U-test was used to assess statistical significance. P values are indicated. a, c, n = 10 mice; d, n = 4 mice; f–i, n = 5 donor mice. f, g, Two muscles grafted per donor mouse. h, i, At least three independent engraftments per donor mouse. n.s., not significant.
Extended Data Figure 2 K-means clustering analysis of quiescent satellite cells and activation capacity.
a, Representative pictures of Pax7 (red) and MyoD (green) immunostaining of FACS isolated satellite cells from adult mice and quantification of activated (Pax7/MyoD double-positive) satellite cells at 14 and 24 h after injury by CTX injection (before the first division) of adult, old and geriatric muscles. Arrows indicate double-positive cells. Scale bar, 50 μm. b, Equal numbers of quiescent satellite cells FACS purified from resting muscle of adult, old and geriatric mice, labelled with a GFP-expressing lentivirus, were transplanted into injured muscle of young wild-type (immunodeficient) mice, and allowed to adapt to the new muscle host for 3 weeks, where they formed new myofibres (see Fig. 1d) and returned to quiescence. After the 3-week adaptation period, the muscle was re-injured to provoke satellite cell reactivation, and GFP+ satellite cells were FACS isolated 24 h after injury, and analysed for activation markers (MyoD and Ki67 immunostaining). MyoD quantification is shown. c, Gene expression microarrays were performed in freshly FACS isolated satellite cells from resting muscle of adult, old and geriatric wild-type mice compared to satellite cells of young wild-type mice (young (n = 3), adult (n = 3), old (n = 3) and geriatric (n = 5) satellite cells). Venn diagrams of overlapping significantly upregulated or downregulated genes in the microarrays (FDR <0.05 and fold change ≥ 1.5). d, K-means clustering analysis of the gene expression microarrays in panel c allowed us to isolate five different gene clusters. One of them was composed of genes with increased expression in geriatric satellite cells (cluster 5, hereafter called cluster G1), and that included genes belonging to our senescence gene set (see Fig. 2d). Heat maps depicting expression levels for genes included in these five clusters. Cluster 2 (not shown) is composed of genes with no changes in expression levels. Red, increased expression; black, neutral expression; green, decreased expression. Expression levels of all the probes present in each cluster were represented. e, Heat maps depicting expression levels for genes included in cluster G2 (generated by K-means clustering of gene expression data from young (n = 3), adult (n = 3), old (n = 3), geriatric (n = 5) and SAMR1 (n = 3) and SAMP8 (n = 3) satellite cells). Red, increased expression; black, neutral expression; green, decreased expression. Venn diagram of overlapping genes between cluster G1 and cluster G2. f, Heat map depicting expression levels for genes included in cluster G3 from young (n = 3), adult (n = 3), old (n = 3), geriatric (n = 5), Bmi1+/+ (n = 3), Bmi1−/− (n = 3), SAMR1 (n = 3) and SAMP8 (n = 3) satellite cells. Red, increased expression; black, neutral expression; green, decreased expression. Functional annotation analysis of the Gene Ontology (GO) was performed in DAVID to identify biological processes enriched in cluster G3 (including Bmi1 data). The top annotation clusters are shown according to their enrichment score. Names are based on enriched GO annotations. g, Venn diagram of the overlap between significantly upregulated genes in geriatric, progeric and Bmi1-deficient satellite cells, genes composing cluster G3 and genes belonging to our senescence gene set (see Supplementary Table 3). h, i, Scheme of transplantation assay of labelled satellite cells into pre-injured young hosts. Equal numbers of FACS-purified satellite cells from resting muscle of Bmi1+/+ and Bmi1−/− mice were transplanted into muscles of young mice (as in Fig. 1d), and activated satellite cells (h) and new regenerating myofibres (i) were analysed at 24 h and 7 days after transplantation, respectively. i, Representative images of transplanted muscles after 7 days are shown. Graph shows numbers of GFP+ myofibres scored per muscle. Scale bar, 50 μm. Data are mean ± s.e.m. Two-sided Mann–Whitney U-test was used to assess statistical significance. P values are indicated. a, n = 6 biological replicates; b, h, n = 4 donor mice; i, n = 5 donor mice. h, i, At least three independent engraftments per donor mouse. n.s., not significant.
Extended Data Figure 3 p16INK4a silencing restores reversible quiescence in geriatric and progeric satellite cells.
a, Number of activated (Pax7+MyoD+) satellite cells, and p16INK4a, p15INK4b and Igfbp5 expression by RT–qPCR in tibialis anterior muscle from geriatric mice transduced with Ad-shRNAp16INK4a or Ad-shScramble, 14 h after CTX injury. Expression values are referred to adult. Similar results were obtained after p16INK4a silencing in satellite cells of geriatric muscle through p16INK4a shRNA (or shScramble) liposome-mediated delivery (not shown). As control, we confirmed that p16INK4a shRNA had no effect in adult or old satellite cells (not shown). b, Scheme of transplantation assay of labelled satellite cells into pre-injured young hosts. Equal numbers of satellite cells from resting muscle of adult old and geriatric mice isolated by FACS were labelled with PKH26 dye, and transplanted into young mice (as in Fig. 2a); senescence-associated markers including p16INK4a, p15INK4b and Igfbp5 were analysed by RT–qPCR of 24-h-activated satellite cells. c, Scheme of transplantation assay of labelled satellite cells into pre-injured young hosts. Equal numbers of satellite cells from resting muscle of SAMP8 and Bmi1 null mice, and their corresponding age-matched control mice, were isolated by FACS, p16INK4a-silenced via liposome-mediated delivery of p16INK4a shRNA (or shScramble) and labelled with PKH26 dye, and immediately transplanted into pre-injured muscle of young mice; satellite cell activation was analysed 24 h later by quantifying the number of sorted MyoD+ or Ki67+ satellite cells (only results of MyoD+ cells are shown); senescence-associated markers including p16INK4a, p15INK4b and Igfbp5 were analysed in sorted labelled cells by RT–qPCR. d, Satellite cells from adult, old or geriatric mice were cultured in differentiation medium, for 96 h to obtain ‘reserve quiescent satellite cells’ (first round myogenesis); subsequently, reserve cells were subjected to a second myogenic round (reactivated with growth medium and cultured in differentiation medium for an extra 96-h period) to obtain secondary reserve quiescent satellite cells (second round myogenesis). Return to quiescence (self-renewal) was defined as Pax7+ reserve satellite cells that could not incorporate BrdU (BrdU negative). Alternatively, geriatric satellite cells cultured in differentiation medium were transduced with Ad-shRNAp16INK4a or Ad-shScramble, and the number of ‘reserve quiescent cells’ was analysed in the two successive myogenesis rounds. e, After quiescence entry, as in panel d, reserve satellite cells were reactivated with growth medium for 48 h, and the increase in number of activated satellite cells (BrdU+ cells after 1 h pulse) was calculated compared to the control. The reactivation capacity of geriatric reserve satellite cells in vitro was assayed after adenoviral infection with Ad-shRNAp16INK4a (or Ad-shScramble). f, Satellite cells from young mice were transduced with vectors (pBabe) expressing p16INK4a (or GFP as control) and cultured in differentiation medium to obtain reserve satellite cells, and reactivated with growth medium as in panel d. The percentage of cells incorporating BrdU after 1-h pulse was calculated. Data are mean ± s.e.m. Two-sided Mann–Whitney U-test was used to assess statistical significance. P values are indicated. a, n = 5 donor mice; b, c, n = 4 donor mice; d, e, n = 4 biological replicates; f, n = 3 biological replicates. b, c, At least three independent engraftments per donor mouse. n.s., not significant.
Extended Data Figure 4 Geroconversion of geriatric, progeric and Bmi1-null satellite cells in vitro and in vivo.
a, Equal number of FACS-isolated satellite cells from adult, old and geriatric mice were cultured in growth medium containing high serum and FGF2 for 4 days and BrdU incorporation was tested after 1 h pulse. b, Sorted satellite cells from adult, old and geriatric mice, cultured in growth medium as in panel a, and immunostained with antibodies against γH2AX. The levels of γH2AX per nucleus were determined. c, Representative pictures of cryosections from tibialis anterior muscles from adult, old and geriatric mice injured with CTX injection and obtained 1 week after injury immunostained for γH2AX. Scale bar, 50 μm. d, Percentage of satellite cells (Pax7+) positive for γH2AX in adult, old and geriatric muscles 1 week after CTX injury. e, An equal number of FACS-isolated satellite cells from adult, old and geriatric mice transplanted into regenerating muscle of young mice, as reported in Fig. 4a, d. Representative images of GFP+ satellite cells co-expressing Pax7 and p16INK4a or γH2AX in 4-day-injured muscles. Scale bar, 25 μm. Data are mean ± s.e.m. Two-sided Mann–Whitney U-test was used to assess statistical significance. P values are indicated. a, d, n = 5 biological replicates; b, n = 6 biological replicates. n.s., not significant.
Extended Data Figure 5 p16INK4a regulates proliferation and promotes geroconversion of geriatric, progeric and Bmi1-null satellite cells in vitro and in vivo.
a, FACS-purified geriatric satellite cells were transduced with Ad-shRNAp16INK4a or Ad-shScramble, cultured in growth medium containing high serum and FGF2 and progeny was quantified over time. b, FACS purified Bmi1−/− satellite cells were transduced with Ad-shRNAp16INK4a or Ad-shScramble, cultured in growth medium for 4 days, and BrdU incorporation was quantified after 1 h pulse. c, Representative pictures of eMHC immunostaining in cryosections from grafted extensor digitorum longus muscle of geriatric wild-type mice transduced (at the moment of grafting) with Ad-shRNAp16INK4a or Ad-shScramble, 1 week after heterografting into the tibialis anterior muscle of young wild-type mice. Number and size of regenerating myofibres (eMHC+ fibres), and relative mRNA levels of p16INK4a, p15INK4b and Igfbp5 are shown. d, Ad-shRNAp16INK4a or Ad-shScramble transduction in SAMP8 extensor digitorum longus muscle heterografts as in panel c. e, Representative pictures of Pax7 (green) immunostaining and DNA counterstained with DAPI (red) in cryosections from extensor digitorum longus muscle from geriatric mice from panel c. Arrows indicate Pax7+ cells. The number of proliferating (Pax7/Ki67 double-positive) satellite cells per section is shown. Scale bar, 50 μm. f, Regenerating eMHC+ myofibre size, p16INK4a expression, number of proliferating (Pax7/Ki67 double-positive) satellite cells and total number of satellite cells (Pax7+) per section in grafted extensor digitorum longus muscle from geriatric (28-month-old) p16INK4a/Arffl/fl mice, transduced (at the moment of grafting) with Ad-Cre or Ad-GFP, 1 week after heterografting onto the tibialis anterior muscle of young, 3-month-old wild-type mice. g, Plasmid vectors expressing p16INK4a or GFP, as control, were electroporated into resting tibialis anterior muscles of young wild-type mice for incorporation into quiescent satellite cells. One day after, injury was induced by CTX injection. After 7 days, satellite cells were FACS isolated, and the number of satellite cells that had expanded from the initial quiescent stem-cell population was determined, and the expression levels of p16INK4a, p15INK4b and Igfbp5 were analysed by RT–qPCR. h, Number of colonies derived from single cells as a measure of the proliferative capacity and relative mRNA levels of p16INK4a, p15INK4b and Igfbp5 evaluated by RT–qPCR from young satellite cells isolated by FACS, transfected with plasmid vectors expressing p16INK4a or GFP (as control) and cultured in proliferative conditions for 96 h. i, As in Fig. 4a, d, equal numbers of FACS-purified satellite cells from adult, old and geriatric mice were transplanted into muscle of young mice. Four days after transplantation, GFP+ satellite cells were re-isolated by FACS and analysed for the expression of Rb/E2F targets (cyclin A, cyclin E, Lmnb1 and Mcm3) and Cdc6. Scale bars, 50 μm. Data are mean ± s.e.m. Two-sided Mann–Whitney U-test was used to assess statistical significance. P values are indicated. a, b, n = 4 biological replicates; c–f, i, n = 4 donor mice; g, n = 6 biological replicates; h, n = 5 biological replicates. d, e, Two muscles grafted per donor mouse. i, At least three independent engraftments per donor mouse. n.s., not significant.
Extended Data Figure 6 Signs of sarcopenia in muscle biopsies of geriatric individuals, and impaired myogenesis and increased senescence of human geriatric satellite cells.
a, Representative images of haematoxylin and eosin (H/E) staining in cryosections of muscles from human donors at different ages: adult (30 years), old (65 years) and geriatric (96 years). Asterisks indicate atrophic myofibres. Arrow indicates a central nucleated myofibre. b, CD56, p16INK4a and Igfbp5 immunostaining in muscle biopsies of geriatric (83 ± 7 years) and adult (28 ± 7 years) individuals. Scale bars, 25 μm. c, Percentage of BrdU incorporation after 1 h pulse in adult and geriatric human satellite cells in proliferation conditions (growth medium). Representative pictures of BrdU immunostaining are shown. d, Representative pictures of eMHC immunostaining of adult and geriatric human satellite cells after culturing for 48 h in differentiation conditions (differentiation medium). The percentage of nuclei inside the myotubes was quantified to determine fusion capacity. Scale bars, 50 μm. Data are mean ± s.e.m. Two-sided Mann–Whitney U-test was used to assess statistical significance. P values are indicated. a, b, Representative pictures from n = 8 for adult, n = 5 for old, and n = 10 for geriatric human donors; c, d, n = 5 biological replicates. n.s., not significant.
Gene list and gene Ontology of Up- and Down-regulated genes in all pairwise comparisons. (XLSX 1773 kb)
Gene list and gene Ontology of the cluster G1, G2 and G3. Gene set enrichment analysis (GSEA) of Cluster G1. (XLSX 298 kb)
Senescence geneset with significantly upregulated genes belonging to Cluster G1 indicated. (DOCX 26 kb)
qPCR and ChIP primers list. (XLSX 11 kb)
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Sousa-Victor, P., Gutarra, S., García-Prat, L. et al. Geriatric muscle stem cells switch reversible quiescence into senescence. Nature 506, 316–321 (2014). https://doi.org/10.1038/nature13013
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