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First published online January 11, 2007
Stem Cells Vol. 25 No. 4 April 2007, pp. 885 -894
doi:10.1634/stemcells.2006-0372; www.StemCells.com
© 2007 AlphaMed Press

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TISSUE-SPECIFIC STEM CELLS

A Population of Myogenic Stem Cells That Survives Skeletal Muscle Aging

Charlotte A. Collinsa, Peter S. Zammitb, Ana Pérez Ruizb, Jennifer E. Morgana, Terence A. Partridgec

aThe Dubowitz Neuromuscular Unit, Department of Paediatrics, Imperial College London, Hammersmith Hospital, London, United Kingdom;
bRandall Division of Cell and Molecular Biophysics, King's College London, New Hunt's House, Guy's Campus, London, United Kingdom;
cChildren's National Medical Center, Washington, DC, USA

Key Words. Stem cell • Satellite cell • Aging • Skeletal muscle • Self-renewal • Regeneration

Correspondence: Jennifer E. Morgan, Ph.D., The Dubowitz Neuromuscular Unit, Department of Paediatrics, Imperial College London, Hammersmith Hospital, Du Cane Road, London W12 ONN, U.K. Telephone: +44 (0)20 8383 2125; Fax: +44 (0)20 8383 2187; e-mail: jennifer.morgan{at}imperial.ac.uk; or Peter S. Zammit, Ph.D., Randall Division of Cell and Molecular Biophysics, King's College London, New Hunt's House, Guy's Campus, London SE1 1UL, U.K. Telephone: +44 (0)20 7848 8217; Fax: +44 (0)20 7848 6435; e-mail: peter.zammit{at}kcl.ac.uk

Received June 19, 2006; accepted for publication December 22, 2006.
First published online in STEM CELLS EXPRESS   January 11, 2007.


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Disclosure of Potential...
 Acknowledgments
 References
 
Age-related decline in integrity and function of differentiated adult tissues is widely attributed to reduction in number or regenerative potential of resident stem cells. The satellite cell, resident beneath the basal lamina of skeletal muscle myofibers, is the principal myogenic stem cell. Here we have explored the capacity of satellite cells within aged mouse muscle to regenerate skeletal muscle and to self-renew using isolated myofibers in tissue culture and in vivo. Satellite cells expressing Pax7 were depleted from aged muscles, and when aged myofibers were placed in culture, satellite cell myogenic progression resulted in apoptosis and fewer total differentiated progeny. However, a minority of cultured aged satellite cells generated large clusters of progeny containing both differentiated cells and new cells of a quiescent satellite-cell-like phenotype characteristic of self-renewal. Parallel in vivo engraftment assays showed that, despite the reduction in Pax7+ cells, the satellite cell population associated with individual aged myofibers could regenerate muscle and self-renew as effectively as the larger population of satellite cells associated with young myofibers. We conclude that a minority of satellite cells is responsible for adult muscle regeneration, and that these stem cells survive the effects of aging to retain their intrinsic potential throughout life. Thus, the effectiveness of stem-cell-mediated muscle regeneration is determined by both extrinsic environmental influences and diversity in intrinsic potential of the stem cells themselves.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusions
 Disclosure of Potential...
 Acknowledgments
 References
 
Skeletal muscle consists predominantly of a single differentiated cell type, the contractile myofiber. It is supplied with new nuclei during growth and regeneration by the satellite cell, a quiescent stem cell located beneath the basal lamina surrounding each myofiber [1, 2]. In the adult, muscle injury activates satellite cells to proliferate extensively, generating a pool of myoblasts, which subsequently differentiate and fuse to repair or replace damaged myofibers [3]. This satellite cell population is maintained by self-renewal and appears to be self-sufficient as a source of new myonuclei [4–6], but the age-related loss of muscle bulk and regenerative ability may involve an impairment of this mechanism.

There is debate as to the extent to which ineffective muscle regeneration in aged mice is determined by changes in the extrinsic environment that inhibit the regenerative ability of otherwise competent satellite cells [7–9] or by impairment of the satellite cells themselves. The age of the host environment seems to have a major effect on muscle regeneration. Transplants of young or aged muscle tissue into a muscle bed regenerate well in a young host but badly in an aged host [10]. This is at least in part a systemic effect, for in parabiotic pairs of young and aged mice sharing a common circulation, the muscles of the aged partner showed improved regenerative responses [11].

There is no agreement as to whether satellite cell numbers decrease with age. Reports of age-related decline in the numbers of satellite cells in the rat tibialis anterior (TA) muscle [12, 13] and the mouse extensor digitorum longus (EDL) and soleus muscles [14–16] are counterbalanced by findings of no significant reduction in the mouse soleus [17] and the rat levator ani muscles [18]. Most tissue culture studies have been conducted on the total myogenic population extractable from muscle, which would include all cells with myogenic potential [19–22] in addition to satellite cells. However, a recent study of satellite cells on isolated myofibers in tissue culture has shown that, although number and proliferative ability of satellite cells decline with age, the latter can be partly restored by exposure to high levels of ß-fibroblast growth factor [15]. This study, however, provides no information on satellite cell behavior in vivo.

Here, we show that aging affects myofiber-associated satellite cells differentially. Although the number of satellite cells expressing the paired-box transcription factor Pax7 declines with age, this is partly counterbalanced by cells lacking myogenic markers that become associated with the satellite cell niche. In culture, the nonmyogenic cells undergo apoptosis, and the total yield of progeny from satellite cells associated with aged compared with young myofibers is reduced. Notably, however, a few satellite cells associated with aged myofibers do generate large clusters of progeny that comprise both cells undergoing myogenic differentiation and cells of a quiescent satellite cell phenotype characteristic of self-renewal. Moreover, this smaller population of satellite cells associated with individual aged myofibers can, when grafted, regenerate and self-renew as effectively as the larger population of satellite cells associated with young myofibers. These findings lend support to the idea that adult muscle regeneration is largely mediated by a minor subset of stem-like satellite cells that survive the effects of aging.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusions
 Disclosure of Potential...
 Acknowledgments
 References
 
Single Myofiber Isolation
Single myofibers were prepared from the EDL muscles of young and aged 3F-nLacZ-2E [23] and Myf5nLacZ/+ mice [23] and from the engrafted TA muscles of mdx (X chromosome-linked muscular dystrophy)-nude mice [24] as described previously[5, 25]. "Aged" mice were used between 22 and 30 months of age, and "young" mice were used between 1 and 2 months of age.

Isolation of Satellite Cells from Single Fibers
Single myofibers were prepared from the EDL muscles of young and aged 3F-nLacZ-2E [23] mice. Satellite cells were separated from them by physical trituration [5] and sorted by flow cytometry on the basis of size and granularity using satellite cells prepared from the diaphragm of Pax3GFP/+ mice as a reference [26]. We resuspended 200 small, nongranular cells (sng) and 200 cells from the remaining population in 4 µl of growth medium for grafting. An aliquot of each preparation was cytospun onto gelatin-coated slides for immunostaining [26].

Culture of Myofibers in Suspension
Batches of 20–30 myofibers were incubated in plating medium [4] at 37°C and 5% CO2.

Culture of Myofibers on Matrigel
We coated 8-well Lab-Tek Chamber Slides (Nunc, Rochester, NY, http://www.nuncbrand.com) with Matrigel (BD Biosciences, San Diego, http://www.bdbiosciences.com) (1 mg/ml in Dulbecco's modified Eagle's medium). One myofiber with 250 µl of plating medium was placed in each well, and cultures were maintained at 37°C/5% CO2. A 50% medium change was performed every 48 hours from 72 hours after plating.

Immunocytochemistry of Isolated Myofibers and Satellite Cells
We fixed 3F-nLacZ-2E myofibers or adherent cultures with 4% paraformaldehyde and incubated in 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal) solution or immunostained as previously described [27]. Primary antibodies used were mouse monoclonal anti-Pax7 (Developmental Studies Hybridoma Bank, Iowa City, IA, http://www.uiowa.edu/~dshbwww), mouse monoclonal anti-MyoD1 (clone 5.8A; DakoCytomation, Glostrup, Denmark, http://www.dakocytomation.com), rabbit polyclonal anti-MyoD (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), rabbit polyclonal anti-myogenin (Santa Cruz Biotechnology Inc.), rabbit polyclonal anti-ß-gal (Molecular Probes, Carlsbad, CA, http://probes.invitrogen.com), and rabbit polyclonal anti-laminin (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). For secondary detection, AlexaFluor 488-conjugated or AlexaFluor 594-conjugated goat anti-mouse Ig (Molecular Probes), tetramethylrhodamine isothiocyanate-conjugated swine anti-rabbit Ig (DakoCytomation), and AlexaFluor 488-conjugated goat anti-rabbit Ig were used. We used 4',6-diamidino-2-phenylindole (DAPI) (10 µg/ml) to counterstain nuclei. Myofibers isolated from Myf5nLacZ/+ myofiber-engrafted mdx-nude TA muscles were fixed as above and then incubated overnight in X-gal solution or permeabilized in 0.5% Triton X-100 and incubated overnight with antibodies to Pax7 and ß-galactosidase (ß-gal). For TdT-mediated dUTP-biotin nick end labeling (TUNEL) assays, an APO-BrdU TUNEL Assay Kit (Molecular Probes) was used according to the manufacturer's protocol, with minor modifications. We detected bromodeoxyuridine (BrdU) incorporation at DNA break sites using an AlexaFluor 488-labeled mouse anti-BrdU antibody.

Grafting into Mouse Muscles
Individual donor myofibers were grafted into the preirradiated TA muscles of mdx-nude host mice as previously described [5]. In one experiment, 200 satellite cells sorted by flow cytometry on the basis of size and granularity were grafted into preirradiated mdx-nude host mouse muscles.

Model of Muscle Injury
To investigate the ability of donor cells to regenerate tissue after acute injury, 4 weeks after grafting mice were reanesthetized, and 10 µl of Notechis scutatus scutatus notexin (Latoxan, Valence, France, http://www.latoxan.com) (10 µg/ml) was injected into each engrafted TA muscle [28]. Vetergesic (buprenorphine; Alstoe Ltd Animal Health, York, U.K., http://www.alstoe.co.uk) 0.05 mg/kg was administered for postoperative analgesia.

Immunohistochemistry of Tissue Sections
Engrafted muscles were frozen in isopentane cooled in liquid nitrogen. Three or four serial 7-µm cryosections were collected at 100-µm intervals throughout the entire muscle. Sections were immunostained with P7 rabbit polyclonal anti-dystrophin and BF34 mouse monoclonal anti-neonatal myosin antibodies and counterstained with DAPI [5]. Staining was preceded by blocking with 10% goat serum and 10% swine serum. X-gal and H&E staining were performed as previously described [28].

Microscopy
Fluorescent and bright-field microscopy and image capture were performed using an epifluorescence microscope (model Axiophot; Carl Zeiss, Jena, Germany, http://www.zeiss.com). Digital images were acquired with a Charge-Coupled device (model RTE/CCD-1300-Y; Princeton Instruments Inc., Trenton, NJ, http://www.piacton.com) at –10°C using MetaMorph version 4.5r5 software (Molecular Devices Corporation, Sunnyvale, CA, http://www.moleculardevices.com). Images were optimized globally for brightness and contrast and assembled into figures using Adobe Photoshop CS (Adobe Systems Incorporated, San Jose, CA, http://www.adobe.com).

Quantification
In myofiber suspension and adherent cultures, counts were made of cells that contained the blue reaction product of X-gal staining (which quenches DAPI fluorescence) and of DAPI+ cells that had been categorized by immunostaining. Data from multiple myofibers or myofiber cultures were pooled to give a population mean ± SEM for cells in each category.

To analyze the numbers of new myofibers generated from single myofiber grafts, counts were made of the maximum number of donor dystrophin+ myofibers in immunostained sections and ß-gal+ myofibers or nuclei in adjacent X-gal-stained sections of host muscles. Data in each plot were pooled from multiple donor animals as indicated in figure legends.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusions
 Disclosure of Potential...
 Acknowledgments
 References
 
Aging Results in Reduced Numbers of Pax7+ Satellite Cells
The number and phenotype of satellite cells were compared on single myofibers from EDL muscles of six aged (mean age 735 ± 42 days) (n = 107) and seven young (mean age 43 ± 5 days) (n = 77) 3F-nLacZ-2E mice, in which ß-gal reports expression of the myosin light chain MLC3F gene in fast myofibers [29]. Myofibers were stained in X-gal such that all myonuclei contained the blue reaction product (which quenches fluorescence), and other associated cells could be identified using the fluorescent nuclear counterstain DAPI [27]. Coimmunostaining for Pax7, which is expressed by the majority of quiescent adult satellite cells [4, 30], and the basal lamina component laminin identified sublaminal satellite cells, whereas any other associated cells appeared as DAPI+ nuclei beneath or outside the basal lamina (Fig. 1A–1D).


Figure 1
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  		Figure 1. Myogenic progression of aged satellite cells in myofiber suspension culture. (A, B): Myofibers isolated from the extensor digitorum longus (EDL) muscles of aged 3F-nLacZ-2E mice, stained in 5-bromo-4-chloro-3-indolyl-ß-D-galactoside (X-gal) solution and coimmunostained for Pax7 and laminin. Myonuclei contain the blue reaction product of X-gal staining, which quenches DAPI immunofluorescence. (A): Example of a Pax7+ satellite cell beneath the basal lamina of a myofiber. (B): Example of a Pax7– cell that appears to be encapsulated by the basal lamina. Scale bar, 10 µm. (C): Bar graph depicting the mean numbers of 3F(ß-gal)+ myonuclei in myofibers from seven young and six aged mice. (D): Bar graph depicting the mean numbers of Pax7+ satellite cells and Pax7– basal lamina-associated cells in myofibers from young and aged mice. A total of 107 aged and 77 young myofibers were analyzed. Each error bar represents a standard error between the mean counts obtained from six or seven animals. (E–G): Myofibers isolated from the EDL muscles of an aged 3F-nLacZ-2E mouse and cultured in suspension for 96 hours, stained in X-gal, and then immunostained. The blue reaction product of X-gal staining marks expression of 3F(ß-gal) in both myonuclei and differentiated satellite cell progeny. (E): Myofiber immunostained for Pax7 and MyoD. A rare cluster of cells that contains a Pax7+ MyoD– cell of a quiescent satellite-cell-like phenotype (arrow) as well as differentiating 3F(ß-gal)+ and MyoD+ progeny. (F): Myofiber stained for MyoD and myogenin. A typically small cluster of cells contains two myogenin+ MyoD+ cells in an early-stage of differentiation (arrows) as well as DAPI+ cells, which do not express either myogenin or MyoD. (G): Myofiber immunostained for Pax7 and myogenin. An unusually large cluster contains 3F(ß-gal)+ cells, differentiation-committed myogenin+ cells, and undifferentiated Pax7+ cells. Scale bar, 10 µm. Abbreviations: ß-gal, ß-galactosidase; DAPI, 4,6-diamidino-2-phenylindole.

 
Counts of 3F(ß-gal)+ myonuclei in young and aged myofibers were not significantly different (Mann-Whitney, p > .05) (Fig. 1C). Counts of Pax7+ cells, seen in an exclusively sublaminal satellite cell position (Fig. 1 A), revealed means of 4.6 ± 0.6 per young myofiber and 2.4 ± 0.3 per aged myofiber (Fig. 1 D), a significant difference (Mann-Whitney, p < .001). Whereas all sampled young myofibers bore at least one Pax7+ satellite cell, 9.8% of aged myofibers bore no Pax7+ cells at all. In preparations from young mice, only 0.2 ± 0.0 sublaminal DAPI+ cells per myofiber were negative for Pax7. Preparations from aged mice bore 1.1 ± 0.1 Pax7– DAPI+ cells either below, or apparently encapsulated by, the basal lamina of each aged myofiber, a significant difference (Mann-Whitney, p < .001) (Fig. 1B). Immediately after isolation, no myofiber-associated cells contained myogenin (data not shown), and MyoD staining was confined to a small minority of Pax7+ cells, confirming that, irrespective of anatomical location, Pax7– cells were not participating in myogenic differentiation. Thus, satellite cells are predominantly Pax7+ in young muscles, but cells with immunodetectable Pax7 decline in frequency with age, and their place is occupied by cells within or beneath the basal lamina, which do not express markers of myogenesis (3F(ß-gal)– Pax7– MyoD– myogenin–).

Aging Perturbs Myogenic Progression of Activated Satellite Cells
To investigate the functional potential of aged satellite cells, we cultured isolated myofibers in suspension, preserving a near-normal relationship between satellite cell and myofiber [4]. Satellite cells initially activate MyoD and then proliferate to generate small clusters of progeny, within which some cells downregulate Pax7, express myogenin, and terminally differentiate, and others reversibly exit the cell cycle and reacquire the Pax7+ MyoD– phenotype characteristic of quiescent satellite cells [4].

Total numbers of progeny associated with aged and young myofibers at 0-, 24-, and 48-hour time points were not significantly different (Kolmogorov-Smirnov two sample test, p > .05 in each case), but in cultures from aged mice a proportion of the total DAPI+ cells expressed no myogenic markers. Whereas at 0 hours most Pax7+ cells were MyoD–, when sampled at 24 hours and 48 hours the majority of stained cells in cultures from both young and aged mice coexpressed Pax7 and MyoD, and by 48 hours occasional couplets of dividing Pax7+ MyoD+ cells were observed. Thus, a proportion of aged satellite cells retained the ability to initiate an active myogenic program, remaining responsive to the same mitogenic environment as young satellite cells.

From 48 hours onward, satellite cells proliferated to generate clusters of progeny. At both 72 hours and 96 hours, myofibers from aged mice had fewer associated large clusters of cells and significantly fewer total cells than myofibers from young mice (Kolmogorov-Smirnov two sample test, p < .001 at each time point), as might be expected from the smaller starting number of Pax7+ cells. Interestingly, however, by 96 hours the maximum number of cells surrounding any one myofiber was similar in both age groups (Fig. 2), the differences in mean populations mainly reflecting the large proportion of aged myofibers with few or no associated cells.


Figure 2
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  		Figure 2. Quantitative and phenotypic analysis of satellite cell progeny in myofiber suspension cultures. (A, B): Bar charts depicting phenotypic characterization of satellite cell progeny in myofiber suspension cultures. Myofibers were fixed and analyzed after 0, 24, 48, 72, or 96 hours in suspension culture. Batches of myofibers from the same mice were stained in 5-bromo-4-chloro-3-indolyl-ß-D-galactoside solution to reveal ß-gal activity and then coimmunostained for Pax7 and MyoD, MyoD and myogenin, or Pax7 and myogenin. Values are population means of the number of satellite cells in each category (± SEM) per myofiber isolated from two young mice (A) (n = 15–21 per category) or three aged mice (B) (n = 30 per category). (C, D): Bar charts depicting individual counts of the total number of cells associated with each cultured young or aged myofiber. Counts are ranked in order of the total number of cells associated with each myofiber. Abbreviations: ß-gal, ß-galactosidase; DAPI, 4,6-diamidino-2-phenylindole; h, hours; myog., myogenin.

 
Phenotypically, many of the progeny of young satellite cells were in various stages of myogenic differentiation by 72 hours, expressing either myogenin or 3F(ß-gal), but in aged fiber cultures, few satellite cell progeny reached this stage until 96 hours (Figs. 1E–1G, 2). Concomitant with the appearance of differentiated myogenin+ and 3F(ß-gal)+ cells, a subset of progeny downregulated MyoD but maintained robust Pax7 expression, thus opting out of immediate differentiation and acquiring the phenotype characteristic of quiescent satellite cells (Fig. 1E–1G). In cultures from young muscles, Pax7+ MyoD– cells formed on average 13.5% ± 1.7% of the total population at 72 hours and 13.1% ± 1.9% of the total population at 96 hours, significantly more than in cultures from aged muscles (1.3% ± 0.6% of the total progeny at 72 hours and 2.7% ± 0.7% at 96 hours; Mann-Whitney, p < .001 at each time point; Fig. 2). In both age groups, the majority of Pax7+ MyoD– cells in 96-hour cultures (88.9% in aged and 90.5% in young myofiber cultures) were located in clusters together with other differentiated cells (Fig. 1E–1G), favoring the view that, despite their rarity in aged cultures, they arose, as in young cultures, from Pax7+ MyoD+ progeny rather than from satellite cells that had failed ever to activate MyoD. Thus, the subpopulation of myofiber-associated satellite cells retaining the ability to generate new cells of the quiescent satellite cell-like phenotype is much lower in aged than in young muscles.

Apoptosis of Nonmyogenic Cells During Satellite Cell Activation and Proliferation
We compared the rate of apoptosis between the progeny of cells associated with young and aged myofibers using the TUNEL assay, which specifically labels DNA breaks in apoptotic nuclei [31]. Since, in cultures from aged mice, almost all myogenic cells (those expressing at least one myogenic marker) are MyoD+ between 24 hours and 72 hours (Fig. 2 B), we prepared suspension cultures of myofibers from three young and three aged mice, fixed samples at intervals between 0 hours and 72 hours, and carried out TUNEL and MyoD immunostaining. Very few TUNEL+ cells were associated with young myofibers at any time point (Fig. 3B), whereas cultures from aged muscles showed a progressive increase in the number of TUNEL+ cells concomitant with the period of satellite cell activation and proliferation (Fig. 3A, 3B). TUNEL staining persists for only 1–3 hours during apoptosis in vitro [31], so counts of TUNEL+ nuclei mainly represent cells dying at the time of observation. At 24 hours, when most satellite cells have activated to express MyoD but little or no division has occurred, occasional TUNEL+ cells appeared MyoD+, although we cannot exclude nonspecific immunostaining of apoptotic cells. Most TUNEL+ cells were MyoD– and equivalent in number to the nonmyogenic cells that we have described at this time point, implying that it is this class of cells that apoptose within 24 hours of exposure to serum mitogens. Couplets of dividing myogenic cells first appear at about 48 hours in both young and aged cultures, and the population expands rapidly between 48 hours and 72 hours in cultures from young muscles but far less rapidly in cultures from aged muscles (Fig. 2). Many TUNEL+ cells appeared in aged cultures during this proliferative phase, most being negative for MyoD (Fig. 3A, 3B). Some instances were noted, however, of clusters containing TUNEL+ nuclei (Fig. 3A[iii] and 3A[iv]), suggesting apoptosis of previously proliferating myogenic cells. Thus, the nonmyogenic cells associated with aged myofibers appear highly vulnerable to apoptosis, and it also seems likely that, at least in vitro, a significant proportion of the progeny of aged satellite cells lose their myogenic potential and succumb to apoptosis.


Figure 3
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  		Figure 3. Apoptosis of nonmyogenic cells during myogenic progression of aged satellite cell progeny. (A): TUNEL assay to identify apoptotic nuclei on extensor digitorum longus myofibers from young and aged 3F-nLacZ-2E mice after culture in suspension. (i, ii): 72-h aged muscle-derived myofiber with two TUNEL+ cells (green, one arrowed). (iii, iv): 72-h aged muscle-derived myofiber bearing a cluster of cells in which five nuclei are TUNEL+ (green, arrows mark two examples). Scale bars, 25 µm. (B): Bar chart showing the numbers of TUNEL+ and TUNEL+ MyoD+ cells associated with myofibers isolated from three young or three aged mice after 0 h, 24 h, 48 h, and 72 h in suspension culture. Values are population means of the number of satellite cells in each category (± SEM) per myofiber. A total of 30 myofibers was analyzed per time point per age group. Each error bar represents a standard error. (C, D): Progeny of young and aged satellite cells in long-term adherent myofiber cultures. Single myofibers were isolated from three young and three aged 3F-nLacZ-2E mice and cultured on matrigel for 96 h or 216 h. Fixed cultures were stained in 5-bromo-4-chloro-3-indolyl-ß-D-galactoside and then immunostained for Pax7 and MyoD. (C): Culture from aged mouse analyzed at 96 h. (D): Culture from aged mouse analyzed at 216 h. Scale bars, 10 µm. (E): Bar chart depicting mean counts of phenotypically characterized nuclei per freshly isolated myofiber at 0 h (n = 20–22) or myofiber culture at 96 h (n = 23–43) or 216 h (n = 20–27) in cultures from young or aged mice. Each error bar represents a standard error. (F): Bar charts depicting individual counts of the total numbers of nuclei generated in each myofiber culture. Data are ranked in order of the total number of nuclei per culture. Abbreviations: ß-gal, ß-galactosidase; DAPI, 4,6-diamidino-2-phenylindole; h, hours; TUNEL, TdT-mediated dUTP-biotin nick end labeling.

 
Myogenic Potential of Aged Satellite Cells in Long-Term Culture
To examine longer time points, we maintained myofibers isolated from three young and three aged 3F-nLacZ-2E mice as individual adherent cultures on Matrigel, which permits cells to migrate from the myofiber and proliferate extensively. By 96 hours, the majority of satellite cell progeny in both young and aged cultures coexpressed Pax7 and MyoD, a phenotype characteristic of proliferating myogenic cells (Fig. 3C). By 216 hours, most nuclei had undergone myogenic differentiation, expressing 3F(ß-gal) and/or incorporation into multinucleate myotubes (Fig. 3D). The percentage of total differentiated nuclei was higher in cultures derived from young mice (82.2% ± 4.0%) than in cultures derived from aged mice (66.1% ± 4.2%). In cultures from both young and aged mice, most of the remaining mononucleate cells expressed Pax7 and/or MyoD, indicating their myogenic character. Aged myofiber cultures contained significantly fewer total progeny than young myofiber cultures both at the 96-hour (Mann-Whitney, p < .001) and the 216-hour time point (Mann-Whitney, p < .001) (Fig. 3F, 3G). Even by 216 hours, aged single myofiber cultures contained a mean of only 121.0 ± 31.5 differentiated nuclei (Fig. 3E), less than half the nuclei needed to completely regenerate one myofiber (Fig. 1C). At this time point, only 3.9% of 4,446 total nuclei counted in aged myofiber cultures were both undifferentiated and negative for Pax7 and MyoD (Fig. 3E, 3F). This lack of nonmyogenic cells in long-term cultures of aged myofibers suggests that such cells are largely eliminated by apoptosis at earlier times, and that satellite cells from aged muscle do not propagate nonmyogenic progeny.

Satellite Cells Associated with Aged Myofibers Mediate Extensive Regeneration After Engraftment
To assay the myogenic potential of aged satellite cells in vivo, we isolated myofibers from the EDL muscles of three aged 3F-nLacZ-2E mice (mean age 735 ± 48 days) and grafted a single myofiber, with its associated satellite cells, into each of the irradiated TA muscles (n = 32) of 16 24-day-old mdx-nude mice. Examination of muscles 4 weeks after grafting revealed that 40.1% (13/32) of them contained substantial clusters of donor-derived myofibers identifiable by both 3F(ß-gal) and dystrophin expression (Fig. 4). In two cases, more than 100 new dystrophin+ myofibers were formed, requiring extensive proliferation of donor cells to give rise to an estimated 10,000–20,000 differentiated donor myonuclei [5]. Both the proportion of grafts which generated new muscle and the quantities of muscle formed were comparable to our previous experiments employing grafts of myofibers from young muscles, [5] despite the fact that the aged myofibers had significantly fewer associated Pax7+ satellite cells.


Figure 4
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  		Figure 4. Myogenic potential of aged satellite cells in single myofiber grafts. (A): Sections of an irradiated mdx-nude tibialis anterior (TA) muscle that had been engrafted with a single myofiber isolated from the extensor digitorum longus (EDL) muscle of an aged 3F-nLacZ-2E mouse 4 weeks previously. The satellite cells (average 2.4) present in the graft proliferated and gave rise to a large cluster of donor muscle identified by 5-bromo-4-chloro-3-indolyl-ß-D-galactoside staining, locating expression of 3F(ß-gal) (i) and, in a serial section, immunohistochemistry for dystrophin protein (ii), which is not expressed by the host. Asterisks mark the same myofiber in each section. Scale bar, 100 µm. (B): Bar chart depicting the numbers of donor ß-gal+ and dystrophin+ myofibers generated from 32 individual grafts of single myofibers derived from the EDL muscles of three aged 3F-nLacZ-2E mice and grafted into the irradiated TA muscles of 16 host mdx-nude mice. Four-week time point. Ranked in order of the number of dystrophin+ myofibers. Abbreviation: ß-gal, ß-galactosidase.

 
To compare the regenerative potential of equal numbers of satellite cells from young and aged muscles, we sorted 3F-nLacZ-2E EDL myofiber-derived satellite cells on the basis of size and granularity [26]. We grafted 200 sng satellite cells into the preirradiated right TA muscle and 200 cells from the residual satellite cell population into the irradiated left TA of host mice. Five mice were grafted with satellite cells from aged donors and five mice with satellite cells from young donors. Muscles were removed for analysis 4 weeks after grafting.

Preparations of sng cells from young muscle were 82% Pax7+, comparable to the 93% Pax7+ in our previous preparations from diaphragm [26], whereas only 40% of the sng cells from aged muscle were Pax7 +. Interestingly, 43% of the residual satellite cell population from young and 44% of the residual satellite cell population from aged EDL muscles were Pax7+.

Donor muscle regeneration at graft sites of sng and residual satellite cell populations showed no significant age-related difference in performance (p > .1 in all cases). Donor muscle was found in four out of five muscles grafted with aged sng cells (41 ± 17.7 [SEM] dystrophin+ fibers; 8 ± 3.99 ß-gal+ fibers) and in all five muscles grafted with young sng cells (42.4 ± 16.8 dystrophin+ fibers; 8 ± 2.66 ß-gal+ fibers). Residual cells formed donor muscle in only one out of five muscles grafted with cells from each age group, probably reflecting contamination of this population with a few stem-like satellite cells, as flow cytometry does not separate cell populations with 100% accuracy.

The apparent better performance of sng cells over the remainder of the population was significantly different for the young (p = .015 for both ß-gal and dystrophin) but not the aged (p > .1 in all cases) group, the latter probably because of the small sample size, and was highly significant when the two age groups were combined (p = .005 for ß-gal; p = .002 for dystrophin). Thus, satellite cells from young and aged donors show equivalent capacity to regenerate skeletal muscle, and this property is associated with the sng rather than the Pax7+ phenotype.

Satellite Cells Associated with Aged Myofibers Generate New Satellite Cells In Vivo
To investigate the ability of aged satellite cells to self-renew and generate new satellite cells in vivo, we grafted single myofibers derived from the EDL muscles of Myf5nLacZ/+ mice, in which ß-gal reports expression of Myf5 in the nuclei of satellite cells and also in newly-formed myofibers [27, 32]. Myofibers isolated from the EDL muscles of 4 aged Myf5nLacZ/+ mice were grafted individually into irradiated TA muscles of 11 mdx-nude mice. Engrafted muscles were removed after 4–5 weeks and disaggregated into myofibers, which were stained in X-gal to localize expression of Myf5(ß-gal). Myofibers isolated from 50% (6/12) of engrafted muscles bore ß-gal+ nuclei in the satellite cell position (Fig. 5A[i]). To confirm that the ß-gal+ cells were indeed satellite cells, myofibers from 7 engrafted muscles were examined for their content of satellite cells (Pax7+) of donor origin (Myf5 ß-gal+). Myofibers isolated from four out of seven mice contained ß-gal+ Pax7+ satellite cells (Fig. 5A[ii]–5A[iv]; Table 1). Thus, aged satellite cells had reconstituted the satellite cell pool to a similar extent to young satellite cells [5].


Figure 5
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  		Figure 5. The reduced population of satellite cells on each aged myofiber contains equivalent in vivo myogenic and self-renewal potential to the larger population of satellite cells on each young myofiber. (A): Myofibers isolated from an irradiated mdx-nude (tibialis anterior) TA muscle 5 weeks after grafting with a single myofiber, with its associated satellite cells, derived from the extensor digitorum longus (EDL) muscle of an aged Myf5nLacZ/+ mouse. (i): Staining with 5-bromo-4-chloro-3-indolyl-ß-D-galactoside (X-gal) reveals Myf5(ß-gal)+ nuclei in a satellite cell position (arrowed). (ii–iv): Immunostaining with antibodies to Pax7 and ß-gal (counterstained with DAPI) shows a donor-derived satellite cell (arrowed). (B, C): Transverse sections of irradiated mdx-nude TA muscles 5 weeks after engraftment with a single myofiber, and its associated satellite cells, derived from the EDL muscle of an aged Myf5nLacZ/+ mouse. Serial sections stained in X-gal (i), immunostained for dystrophin and neoMyHC (ii), or stained with H&E (iii). (B) is a noninjured control, and (C) was injured by injection of notexin 1 week before analysis. (B): The inset shows a Myf5(ß-gal)+ nucleus in a peripheral satellite cell position (i). Arrows mark the same myofiber in each series. Scale bars, 200 µm (B) and 100 µm (C). (D–G): Bar charts depicting numbers of dystrophin+ myofibers, dystrophin+ neoMyHC+ myofibers, and ß-gal+ nuclei generated from single Myf5nLacZ/+ myofibers 5 weeks after grafting. Ranked in order of the number of dystrophin+ myofibers. (D, F): The left TA muscles of host mice were engrafted with single myofibers derived from four young donor mice. (E, G): The contralateral right TA muscles were engrafted with single myofibers derived from four aged donor mice. (D, E): The engrafted muscles of 12 host mice functioned as noninjured controls. (F, G): The engrafted muscles of 14 host mice were injected with notexin 4 weeks after grafting and analyzed 1 week later. Abbreviations: ß-gal, ß-galactosidase; DAPI, 4,6-diamidino-2-phenylindole.

 


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  		Table 1. Counts of satellite cells generated from individual grafts of Myf5nLacZ/+ extensor digitorum longus single myofibers

 
Myogenic and Self-Renewal Potential of Aged and Young Satellite Cells Are Equivalent
To directly compare the myogenic potential of satellite cells from young and aged muscles and their daughter satellite cell progeny generated from these transplanted satellite cells in vivo, we grafted single myofibers derived from the EDL muscles of four young (mean age 51 ± 5 days) and four aged (mean age 703 ± 11 days) donor Myf5nLacZ/+ mice into the irradiated TA muscles of 26 host mdx-nude mice. Each left TA was grafted with a myofiber from a young donor and each contralateral TA with a myofiber derived from an aged donor. Four weeks later, both TA muscles of 14 host mice were injured by injection of the notexin, the remaining 12 host mice functioning as noninjured controls. Notexin destroys 75%–80% of myofibers in injected muscle within 24 hours, sparing blood vessels and satellite cells [33]. Muscles were removed 1 week later (5 weeks after grafting) to assess the regenerative response.

In noninjured muscles, similar numbers of donor dystrophin+ myofibers were generated from young and aged myofiber grafts. Most dystrophin+ myofibers did not express neoMyHC or myonuclear Myf5(ß-gal) and were large and mainly peripherally nucleated, a phenotype characteristic of mature nonregenerating muscle (Fig. 5B). Occasional Myf5(ß-gal)+ nuclei were located predominantly at the periphery of myofibers, consistent with a satellite cell phenotype (Fig. 5B[i], inset). Several engrafted muscles that had been injected with notexin 1 week before removal contained compact clusters of donor dystrophin+ myofibers, which were small and centrally nucleated and contained cytoplasmic neoMyHC and myonuclear Myf5(ß-gal), showing them to have been newly regenerated by donor-derived myogenic progenitors during the week following injury (Fig. 5C). Grafts derived from young muscles and from aged muscles were very similar with regard to both the proportion of engrafted muscles that contained donor muscle and the total numbers of newly-regenerated donor myofibers formed (Fig. 5D–5G). Thus, in vivo, aged satellite cells underwent self-renewal to generate new satellite cells, which, after experimental injury of the engrafted muscle, exhibited equivalent regenerative potential to satellite cells formed by self-renewal of young satellite cells.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusions
 Disclosure of Potential...
 Acknowledgments
 References
 
Skeletal muscle in the aging individual manifests a progressive failure to maintain itself, leading to a loss of tissue mass and function. Satellite cells are reduced in number in aged muscle and exhibit a range of functional impairments in tissue culture [12–15]; but, significantly, the ability of aged muscle to regenerate can be at least partially restored by exposure to a young systemic environment [11]. This apparent paradox led us to investigate directly the function of aged satellite cells themselves, using parallel culture and engraftment assays as measures of regenerative potential.

We show that the number of Pax7+ myofiber-adherent satellite cells is reduced in preparations from aged muscles, in agreement with the findings of another recent tissue culture study [15]. We extend these observations by showing that this decline is associated with the presence of cells that do not express either Pax7 or other myogenic markers. Our tissue culture data show that such nonmyogenic cells are highly vulnerable to apoptosis and that the total yield of progeny from satellite cells derived from aged muscle is significantly lower than that of satellite cells derived from young muscle. Importantly, however, a subpopulation of satellite cells on aged myofibers remains capable of generating large clusters of progeny that contained both differentiating cells and other cells with a quiescent satellite cell phenotype (Pax7+ and MyoD– and myogenin–), signifying the retention of both regenerative and self-renewal potential.

As a direct assay of regenerative potential in vivo, we grafted single myofibers with their associated satellite cells into irradiated mdx-nude mouse muscles. This model provides an optimized environment for assessing the contribution of stem cell populations to muscle regeneration [5, 26, 34] and parallels methods used to assess bone marrow stem cells in that it reveals stem cell function that is manifest only in an environment whose intrinsic stem cell population has been depleted [35]. Our grafts of EDL myofibers provide a sensitive assay, because whereas all myofibers derived from young soleus or TA muscles carry a stem cell capable of engraftment, only a proportion (50%–75%) of myofibers derived from young EDL muscles do so [5]. Moreover, recent work has suggested that the age-related depletion of myofiber-associated satellite cells is more pronounced in the EDL than the soleus muscle [15].

We found that the smaller population of satellite cells associated with each aged myofiber (mean 2.4) had equivalent regenerative and self-renewal potential to the larger population of satellite cells associated with each young myofiber (mean 4.6). As shown previously [5], injury of the recipient muscle provokes an increase in the frequency of donor-derived muscle regeneration. On the basis of this, we speculate that, in a proportion of grafts, the donor satellite cells are viable but not recruited into an active myogenic program until later stimulation by notexin-induced local muscle damage. Overall, our data show that, in vivo, a reduction in aged satellite cell population size does not result in any significant functional deficit. These findings support the idea that a stem cell subset of adult satellite cells is primarily responsible for muscle regeneration and, moreover, suggest that such cells are selectively retained within the aged satellite cell niche.

In grafts of EDL satellite cells sorted on the basis of size and granularity [26], the amount of muscle formed by sng cells from the two age groups was similar, despite the number of Pax7+ satellite cells in this fraction prepared from aged muscle being far smaller. In young muscles, the stem-cell-like cells must be Pax7+, since the frequency of Pax7– cells on young myofibers is far below the frequency of young myofiber grafts that successfully regenerate muscle. However, a high proportion of aged myofibers have both Pax7+ and Pax7– cells associated with them, and we cannot directly demonstrate any relationship between Pax7 expression and stem cell properties. Notably, although the young and aged residual cell populations contained a similar percentage of Pax7+ cells to the aged sng fraction, they formed less muscle. This suggests that size and granularity is the best predictor of high regenerative capacity, leaving Pax7 positivity as perhaps essential but not sufficient.

We and others [12–15] have shown that total satellite cell number declines with aging, and data from our culture experiments suggest that failure of effective self-renewal is a likely explanation for this. Although a subset of satellite cells in myofiber preparations from aged muscles remains capable of generating large clusters of progeny, containing both differentiated cells and new quiescent satellite-cell-like cells, the majority of the population generates few progeny, of which very few reacquire the satellite-cell-like phenotype associated with self-renewal.

The Pax7– population of aged myofiber-associated cells may either be the product of the aberrant myogenic progression of aged satellite cells or interlopers from outside the myogenic lineage that have penetrated the basement membrane and, thus, acquired the same anatomical situation as satellite cells [36, 37]. Their vulnerability to apoptosis suggests that they are unlikely to constitute an alternative myogenic population (for example, blood-vessel-associated mesangioblasts [38]), but they bear interesting comparison to the satellite cells of the Pax7-null mouse, which are similarly susceptible to apoptosis following activation [39].

Phenotypically homogeneous populations of hematopoietic stem cells are known to exhibit significant functional heterogeneity, the basis of which remains incompletely understood [40]. Both we [5, 27, 41] and others [42–46] have shown that the adult muscle stem cell compartment is diverse in both phenotype and function. Our data suggest that this diversity becomes more pronounced with age, such that some myofiber-associated cells have little or no regenerative potential, and others have the same stem cell characteristics as satellite cells from young muscles. Engraftment studies demonstrated that stem cell properties were inherited by the new satellite cells generated by self-renewal in aged-muscle-derived grafts, resulting in their ability to mediate robust regeneration upon subsequent injury. Previous demonstrations that a young systemic environment improved the regeneration of young muscles [11] implied that muscle stem cells can be instructed to adapt their function. Our engraftment experiments support this view by showing that satellite cells from young and aged muscles behave similarly in response to the same optimized in vivo environment. However, our data show that only a minority of the satellite cell population possesses this stem cell function. If all satellite cells responded similarly to the same environment, then the greater numbers of satellite cells associated with young myofibers would generate new muscle in greater quantity and with greater frequency than the smaller numbers of satellite cells associated with aged myofibers, whereas in fact there was no such difference.


   CONCLUSIONS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
Disclosure of Potential...
 Acknowledgments
 References
 
We demonstrate that a subset of functional myogenic stem cells persists in aged skeletal muscle and provide evidence that these stem cells are present with similar frequency in the satellite cell compartments of young and aged muscles. This indicates that muscle stem cell potential is as much a product of the intrinsic properties of the cell as of the extrinsic influences of the muscle environment. The stem cell subpopulation of muscle satellite cells represents a prominent and specific target for any therapeutic regime that might be applied to regeneration-defective aging, sarcopenic, or dystrophic muscle.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Disclosure of Potential...
 Acknowledgments
References
 
C.A.C. was funded by the Muscular Dystrophy Campaign (RA1/685 and RA3/711) and MYORES Network of Excellence, contract 511978, from the European Commission 6th Framework Programme. P.S.Z. is funded by the Medical Research Council (MRC). A.P.R. is funded by a Fundación Ramón Areces postdoctoral fellowship. J.E.M. is funded by an MRC Joint Collaborative Career Development Award in Stem Cell Research. T.A.P. was funded by the MRC and a Blaise Pascal chair awarded by the Ecole Normale Supérieure. The Pax7 antibody developed by A. Kawakami was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA. The authors are grateful to Dr. Margaret Buckingham for providing the 3F-nLacZ-2E and Myf5nLacZ/+ mouse strains and to Dr. Didier Montarras and Dr. Margaret Buckingham for advice on fluorescence-activated cell sorting and providing the Pax3 (green fluorescent protein) reference cells.

C.A.C. is currently affiliated with the Wellcome Trust Centre for Stem Cell Research, University of Cambridge, U.K.


   REFERENCES
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 Disclosure of Potential...
 Acknowledgments
 References
 

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