Geriatric muscle stem cells switch reversible quiescence into senescence

ABSTRACT 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. _p16__INK4a_ 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. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS OPTIONS Access through your

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our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS FOXO MAINTAINS A GENUINE MUSCLE STEM-CELL QUIESCENT STATE UNTIL GERIATRIC AGE Article 26 October 2020 CONTROL OF

SATELLITE CELL FUNCTION IN MUSCLE REGENERATION AND ITS DISRUPTION IN AGEING Article 18 October 2021 SENESCENCE ATLAS REVEALS AN AGED-LIKE INFLAMED NICHE THAT BLUNTS MUSCLE REGENERATION

Article Open access 21 December 2022 ACCESSION CODES ACCESSIONS GENE EXPRESSION OMNIBUS * GSE53728 DATA DEPOSITS Microarray data have been deposited into the NCBI Gene Expression Omnibus

under accession number GSE53728. CHANGE HISTORY * _ 19 FEBRUARY 2014 Reference 19 was changed and one reference was updated in the Methods section. _ REFERENCES * Burtner, C. R. &

Kennedy, B. K. Progeria syndromes and ageing: what is the connection? _Nature Rev. Mol. Cell Biol._ 11, 567–578 (2010) Article  CAS  Google Scholar  * Arthur, S. T. & Cooley, I. D. The

effect of physiological stimuli on sarcopenia; impact of Notch and Wnt signaling on impaired aged skeletal muscle repair. _Int. J. Biol. Sci._ 8, 731–760 (2012) Article  Google Scholar  *

Jang, Y. C., Sinha, M., Cerletti, M., Dall’Osso, C. & Wagers, A. J. Skeletal muscle stem cells: effects of aging and metabolism on muscle regenerative function. _Cold Spring Harb. Symp.

Quant. Biol._ 76, 101–111 (2011) Article  CAS  Google Scholar  * Renault, V., Thornell, L. E., Eriksson, P. O., Butler-Browne, G. & Mouly, V. Regenerative potential of human skeletal

muscle during aging. _Aging Cell_ 1, 132–139 (2002) Article  CAS  Google Scholar  * Cheung, T. H. & Rando, T. A. Molecular regulation of stem cell quiescence. _Nature Rev. Mol. Cell

Biol._ 14, 329–340 (2013) Article  CAS  Google Scholar  * Yin, H., Price, F. & Rudnicki, M. A. Satellite cells and the muscle stem cell niche. _Physiol. Rev._ 93, 23–67 (2013) Article 

CAS  Google Scholar  * Shefer, G., Van de Mark, D. P., Richardson, J. B. & Yablonka-Reuveni, Z. Satellite-cell pool size does matter: defining the myogenic potency of aging skeletal

muscle. _Developmental biology_ 294, 50–66 (2006) Article  CAS  Google Scholar  * Carlson, M. E. & Conboy, I. M. Loss of stem cell regenerative capacity within aged niches. _Aging Cell_

6, 371–382 (2007) Article  CAS  Google Scholar  * García-Prat, L., Sousa-Victor, P. & Munoz-Canoves, P. Functional dysregulation of stem cells during aging: a focus on skeletal muscle

stem cells. _FEBS J._ 280, 4051–4062 (2013) Article  Google Scholar  * Liu, L. & Rando, T. A. Manifestations and mechanisms of stem cell aging. _J. Cell Biol._ 193, 257–266 (2011)

Article  CAS  Google Scholar  * Smythe, G. M. et al. Age influences the early events of skeletal muscle regeneration: studies of whole muscle grafts transplanted between young (8 weeks) and

old (13–21 months) mice. _Exp. Gerontol._ 43, 550–562 (2008) Article  CAS  Google Scholar  * Derave, W., Eijnde, B. O., Ramaekers, M. & Hespel, P. No effects of lifelong creatine

supplementation on sarcopenia in senescence-accelerated mice (SAMP8). _Am. J. Physiol. Endocrinol. Metab._ 289, E272–E277 (2005) Article  CAS  Google Scholar  * Dhawan, J. & Rando, T. A.

Stem cells in postnatal myogenesis: molecular mechanisms of satellite cell quiescence, activation and replenishment. _Trends Cell Biol._ 15, 666–673 (2005) Article  CAS  Google Scholar  *

Shea, K. L. et al. Sprouty1 regulates reversible quiescence of a self-renewing adult muscle stem cell pool during regeneration. _Cell Stem Cell_ 6, 117–129 (2010) Article  CAS  Google

Scholar  * Lanigan, F., Geraghty, J. G. & Bracken, A. P. Transcriptional regulation of cellular senescence. _Oncogene_ 30, 2901–2911 (2011) Article  CAS  Google Scholar  * Fridman, A. L.

& Tainsky, M. A. Critical pathways in cellular senescence and immortalization revealed by gene expression profiling. _Oncogene_ 27, 5975–5987 (2008) Article  CAS  Google Scholar  *

Collado, M. et al. Tumour biology: senescence in premalignant tumours. _Nature_ 436, 642 (2005) Article  ADS  CAS  Google Scholar  * Coppé, J. P., Desprez, P. Y., Krtolica, A. & Campisi,

J. The senescence-associated secretory phenotype: the dark side of tumor suppression. _Annu. Rev. Pathol._ 5, 99–118 (2010) Article  Google Scholar  * Gonzalez, S. et al. Oncogenic activity

of Cdc6 through repression of the _INK4/ARF_ locus. _Nature_ 440, 702–706 (2006) Article  ADS  CAS  Google Scholar  * Janzen, V. et al. Stem-cell ageing modified by the cyclin-dependent

kinase inhibitor p16INK4a. _Nature_ 443, 421–426 (2006) Article  ADS  CAS  Google Scholar  * Molofsky, A. V. et al. Increasing _p16__INK4a_ expression decreases forebrain progenitors and

neurogenesis during ageing. _Nature_ 443, 448–452 (2006) Article  ADS  CAS  Google Scholar  * Baker, D. J. et al. Clearance of p16Ink4a-positive senescent cells delays ageing-associated

disorders. _Nature_ 479, 232–236 (2011) Article  ADS  CAS  Google Scholar  * Fukada, S. et al. Molecular signature of quiescent satellite cells in adult skeletal muscle. _Stem Cells_ 25,

2448–2459 (2007) Article  CAS  Google Scholar  * Liu, L. et al. Chromatin modifications as determinants of muscle stem cell quiescence and chronological aging. _Cell Rep._ 4, 189–204 (2013)

Article  ADS  CAS  Google Scholar  * Pallafacchina, G. et al. An adult tissue-specific stem cell in its niche: a gene profiling analysis of _in vivo_ quiescent and activated muscle satellite

cells. _Stem Cell Res._ 4, 77–91 (2010) Article  CAS  Google Scholar  * Jacobs, J. J., Kieboom, K., Marino, S., DePinho, R. A. & van Lohuizen, M. The oncogene and Polycomb-group gene

_bmi-1_ regulates cell proliferation and senescence through the _ink4a_ locus. _Nature_ 397, 164–168 (1999) Article  ADS  CAS  Google Scholar  * Bracken, A. P. et al. The Polycomb group

proteins bind throughout the INK4A-ARF locus and are disassociated in senescent cells. _Genes Dev._ 21, 525–530 (2007) Article  CAS  Google Scholar  * Margueron, R. et al. Ezh1 and Ezh2

maintain repressive chromatin through different mechanisms. _Mol. Cell_ 32, 503–518 (2008) Article  CAS  Google Scholar  * Wang, H. et al. Role of histone H2A ubiquitination in Polycomb

silencing. _Nature_ 431, 873–878 (2004) ADS  CAS  Google Scholar  * Cao, R., Tsukada, Y. & Zhang, Y. Role of Bmi-1 and Ring1A in H2A ubiquitylation and Hox gene silencing. _Mol. Cell_

20, 845–854 (2005) Article  CAS  Google Scholar  * Agherbi, H. et al. Polycomb mediated epigenetic silencing and replication timing at the _INK4a/ARF_ locus during senescence. _PLoS ONE_ 4,

e5622 (2009) Article  ADS  Google Scholar  * Robson, L. G. et al. Bmi1 is expressed in postnatal myogenic satellite cells, controls their maintenance and plays an essential role in repeated

muscle regeneration. _PLoS ONE_ 6, e27116 (2011) Article  ADS  CAS  Google Scholar  * Blagosklonny, M. V. Selective anti-cancer agents as anti-aging drugs. _Cancer Biol. Ther._ 14, 1092–1097

(2013) Article  Google Scholar  * Chicas, A. et al. Dissecting the unique role of the retinoblastoma tumor suppressor during cellular senescence. _Cancer Cell_ 17, 376–387 (2010) Article 

CAS  Google Scholar  * Trimarchi, J. M. & Lees, J. A. Sibling rivalry in the E2F family. _Nature Rev. Mol. Cell Biol._ 3, 11–20 (2002) Article  CAS  Google Scholar  * Krimpenfort, P.,

Quon, K. C., Mooi, W. J., Loonstra, A. & Berns, A. Loss of p16Ink4a confers susceptibility to metastatic melanoma in mice. _Nature_ 413, 83–86 (2001) Article  ADS  CAS  Google Scholar  *

Chakkalakal, J. V., Jones, K. M., Basson, M. A. & Brack, A. S. The aged niche disrupts muscle stem cell quiescence. _Nature_ 490, 355–360 (2012) Article  ADS  CAS  Google Scholar  *

Baker, D. J. et al. Opposing roles for p16Ink4a and p19Arf in senescence and ageing caused by BubR1 insufficiency. _Nature Cell Biol._ 10, 825–836 (2008) Article  CAS  Google Scholar  *

Baker, D. J., Weaver, R. L. & van Deursen, J. M. p21 both attenuates and drives senescence and aging in BubR1 progeroid mice. _Cell Rep._ 3, 1164–1174 (2013) Article  CAS  Google Scholar

  * d’Adda di Fagagna, F. Living on a break: cellular senescence as a DNA-damage response. _Nature Rev. Cancer_ 8, 512–522 (2008) Article  Google Scholar  * Sperka, T., Wang, J. &

Rudolph, K. L. DNA damage checkpoints in stem cells, ageing and cancer. _Nature Rev. Mol. Cell Biol._ 13, 579–590 (2012) Article  CAS  Google Scholar  * Carlson, M. E., Hsu, M. & Conboy,

I. M. Imbalance between pSmad3 and Notch induces CDK inhibitors in old muscle stem cells. _Nature_ 454, 528–532 (2008) Article  ADS  CAS  Google Scholar  * Suelves, M. et al. uPA deficiency

exacerbates muscular dystrophy in MDX mice. _J. Cell Biol._ 178, 1039–1051 (2007) Article  CAS  Google Scholar  * Grounds, M. D., Sorokin, L. & White, J. Strength at the extracellular

matrix-muscle interface. _Scand. J. Med. Sci. Sports_ 15, 381–391 (2005) Article  CAS  Google Scholar  * Vidal, B. et al. Amelioration of Duchenne muscular dystrophy in mdx mice by

elimination of matrix-associated fibrin-driven inflammation coupled to the αMβ2 leukocyte integrin receptor. _Hum. Mol. Genet._ 21, 1989–2004 (2012) Article  CAS  Google Scholar  * Cheung,

T. H. et al. Maintenance of muscle stem-cell quiescence by microRNA-489. _Nature_ 482, 524–528 (2012) Article  ADS  CAS  Google Scholar  * Rocheteau, P., Gayraud-Morel, B.,

Siegl-Cachedenier, I., Blasco, M. A. & Tajbakhsh, S. A subpopulation of adult skeletal muscle stem cells retains all template DNA strands after cell division. _Cell_ 148, 112–125 (2012)

Article  CAS  Google Scholar  * Sacco, A. et al. Short telomeres and stem cell exhaustion model Duchenne muscular dystrophy in mdx/mTR mice. _Cell_ 143, 1059–1071 (2010) Article  CAS  Google

Scholar  * Yoshida, N., Yoshida, S., Koishi, K., Masuda, K. & Nabeshima, Y. Cell heterogeneity upon myogenic differentiation: down-regulation of MyoD and Myf-5 generates ‘reserve

cells’. _J. Cell Sci._ 111, 769–779 (1998) CAS  PubMed  Google Scholar  * Perdiguero, E. et al. Genetic analysis of p38 MAP kinases in myogenesis: fundamental role of p38α in abrogating

myoblast proliferation. _EMBO J._ 26, 1245–1256 (2007) Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS 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. AUTHOR INFORMATION Author notes * Pedro Sousa-Victor Present address: Present address: Buck Institute for Research on Aging, Novato, California

94945, USA., * Susana Gutarra and Laura García-Prat: These authors contributed equally to this work. AUTHORS AND AFFILIATIONS * Department of Experimental and Health Sciences, Cell Biology

Group, Pompeu Fabra University, CIBER on Neurodegenerative diseases, E-08003 Barcelona, Spain, Pedro Sousa-Victor, Susana Gutarra, Laura García-Prat, Laura Ortet, Vanessa Ruiz-Bonilla, Mercè

Jardí, Antonio L. Serrano, Eusebio Perdiguero & Pura Muñoz-Cánoves * Chromatin and Disease Group, Cancer Epigenetics and Biology Programme, Bellvitge Biomedical Research Institute,

L’Hospitalet de Llobregat, E-08907 Barcelona, Spain, Javier Rodriguez-Ubreva & Esteban Ballestar * Stem Cell Aging Group, Centro Nacional de Investigaciones Cardiovasculares, E-28029

Madrid, Spain, Susana González * Institució Catalana de Recerca i Estudis Avançats, E-08010 Barcelona, Spain, Pura Muñoz-Cánoves Authors * Pedro Sousa-Victor View author publications You can

also search for this author inPubMed Google Scholar * Susana Gutarra View author publications You can also search for this author inPubMed Google Scholar * Laura García-Prat View author

publications You can also search for this author inPubMed Google Scholar * Javier Rodriguez-Ubreva View author publications You can also search for this author inPubMed Google Scholar *

Laura Ortet View author publications You can also search for this author inPubMed Google Scholar * Vanessa Ruiz-Bonilla View author publications You can also search for this author inPubMed 

Google Scholar * Mercè Jardí View author publications You can also search for this author inPubMed Google Scholar * Esteban Ballestar View author publications You can also search for this

author inPubMed Google Scholar * Susana González View author publications You can also search for this author inPubMed Google Scholar * Antonio L. Serrano View author publications You can

also search for this author inPubMed Google Scholar * Eusebio Perdiguero View author publications You can also search for this author inPubMed Google Scholar * Pura Muñoz-Cánoves View author

publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS P.S.-V. designed and performed experiments, analysed data, interpreted results and wrote the

manuscript. S.Gu. and L.G.-P. designed and performed experiments, analysed data and interpreted results. L.O., V.R.-B. and M.J. performed experiments and provided technical support. J.R.-U.

and E.B. performed ChIP experiments and edited the manuscript. S.Go. generated transgenic mice and edited the manuscript. A.L.S. and E.P. conceived the project, designed and performed

experiments, interpreted results and wrote the manuscript. P.M.-C. conceived the project, designed experiments, interpreted results and wrote the manuscript. CORRESPONDING AUTHORS

Correspondence to Eusebio Perdiguero or Pura Muñoz-Cánoves. ETHICS DECLARATIONS COMPETING INTERESTS 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

_P16__INK4A_ SILENCING RESTORES REVERSIBLE QUIESCENCE IN GERIATRIC AND PROGERIC SATELLITE CELLS. A, Number of activated (Pax7+MyoD+) satellite cells, and _p16__INK4a_, _p15__INK4b_ 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 _p16__INK4a_ silencing in satellite cells of geriatric muscle through _p16__INK4a_ shRNA (or shScramble) liposome-mediated delivery (not shown). As

control, we confirmed that _p16__INK4a_ 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 _p16__INK4a_, _p15__INK4b_ 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, _p16__INK4a_-silenced via liposome-mediated delivery of _p16__INK4a_ 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 _p16__INK4a_, _p15__INK4b_ 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 _p16__INK4a_, _p15__INK4b_ 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) _p16__INK4a_/_Arf_fl/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 _p16__INK4a_, _p15__INK4b_ 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 _p16__INK4a_, _p15__INK4b_ 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. SUPPLEMENTARY INFORMATION SUPPLEMENTARY TABLE 1 Gene list and

gene Ontology of Up- and Down-regulated genes in all pairwise comparisons. (XLSX 1773 kb) SUPPLEMENTARY TABLE 2 Gene list and gene Ontology of the cluster G1, G2 and G3. Gene set enrichment

analysis (GSEA) of Cluster G1. (XLSX 298 kb) SUPPLEMENTARY TABLE 3 Senescence geneset with significantly upregulated genes belonging to Cluster G1 indicated. (DOCX 26 kb) SUPPLEMENTARY

TABLE 4 qPCR and ChIP primers list. (XLSX 11 kb) POWERPOINT SLIDES POWERPOINT SLIDE FOR FIG. 1 POWERPOINT SLIDE FOR FIG. 2 POWERPOINT SLIDE FOR FIG. 3 POWERPOINT SLIDE FOR FIG. 4 POWERPOINT

SLIDE FOR FIG. 5 POWERPOINT SLIDE FOR FIG. 6 RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE 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 Download citation * Received: 07 June 2013 *

Accepted: 10 January 2014 * Published: 12 February 2014 * Issue Date: 20 February 2014 * DOI: https://doi.org/10.1038/nature13013 SHARE THIS ARTICLE Anyone you share the following link with

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