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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Dev Biol. Author manuscript; available in PMC Apr 15, 2011.
Published in final edited form as:
PMCID: PMC2854302
NIHMSID: NIHMS182549

The depletion of skeletal muscle satellite cells with age is concomitant with reduced capacity of single progenitors to produce reserve progeny

Abstract

Satellite cells are myogenic progenitors residing on the myofiber surface that support skeletal muscle repair. We used mice in which satellite cells were detected by GFP expression driven by nestin gene regulatory elements to define age-related changes in both numbers of satellite cells that occupy hindlimb myofibers and their individual performance. We demonstrate a reduction in satellite cells per myofiber with age that is more prominent in females compared to males. Satellite cell loss also persists with age in myostatin-null mice regardless of increased muscle mass. Immunofluorescent analysis of isolated myofibers from nestin-GFP/Myf5nLacZ/+ mice reveals a decline with age in the number of satellite cells that express detectable levels of βgal. Nestin-GFP expression typically diminishes in primary cultures of satellite cells as myogenic progeny proliferate and differentiate, but GFP subsequently reappears in the Pax7+ reserve population. Clonal analysis of sorted GFP+ satellite cells from hindlimb muscles shows heterogeneity in the extent of cell density and myotube formation among colonies. Reserve cells emerge primarily within high-density colonies, and the number of clones that produce reserve cells is reduced with age. Thus, satellite cell depletion with age could be attributed to a reduced capacity to generate a reserve population.

Keywords: Stem cells, satellite cells, reserve cells, aging, myogenesis, skeletal muscle, nestin-GFP, Myf5nLacZ/+, MLC3F-nLacZ, myostatin, Pax7, α7 integrin, Myf5

Introduction

Satellite cells are myogenic progenitors located between the plasma membrane and the basal lamina of the myofiber (Collins et al., 2005; Mauro, 1961). Satellite cells are predominantly quiescent in adult muscle, but are activated upon muscle injury and produce myogenic progeny required to both fortify damaged myofibers and produce new myofibers (Snow, 1978; Zammit et al., 2006). Routine activity of skeletal muscle results in subtle injuries and demands fusion of satellite cell progeny with existing myofibers throughout life. The ability of satellite cells to self-renew also qualifies their definition as muscle stem cells (Collins et al., 2005; Sacco et al., 2008). However, it is unknown if self-renewal capacity is shared by all satellite cells or is a property of a unique subpopulation.

The paired-homeobox transcription factor Pax7 is the most uniformly expressed marker used to identify skeletal muscle satellite cells across species and muscle groups (Seale et al., 2000; Halevy et al., 2004). We demonstrated that satellite cells in young mice could be monitored based on expression of the nestin-GFP transgene (NES-GFP); ~98% of Pax7+ satellite cells on isolated myofibers from extensor digitorum longus (EDL) muscle expressed GFP (Day et al., 2007). NES-GFP expression also permits analysis of gene expression among satellite cells sorted from different muscle groups (Day et al., 2007). Introduction of a nuclear lacZ reporter gene into the locus of the myogenic regulatory factor gene Myf5 (Myf5nLacZ/+) in mice also demonstrated the expression of β-galactosidase (βgal) within quiescent satellite cells (Beauchamp et al., 2000). Analysis of double transgenic NES-GFP/Myf5nLacZ/+ mice showed that ~80% of GFP+ (or Pax7+) satellite cells on EDL myofibers expressed βgal in young mice based on immunofluorescent detection (Day et al., 2007). Using the Myf5-Cre/Rosa26-YFP reporter mouse, it has been further proposed that satellite cells are composed of muscle stem cells (Pax7+/Myf5-YFP-) that contribute to the reservoir of committed myogenic progenitors (Pax7+/Myf5-YFP+), with the latter population constituting the majority of the satellite cells in muscle (Kuang et al., 2007). However, a similar Cre-Lox cell lineage analysis using MyoD-iCre/Rosa26-YFP reporter mice suggests that satellite cells are uniformly committed to myogenesis as they originate from a pool of MyoD+ progenitors during embryonic development (Kanisicak et al., 2009).

Cultures derived from isolated satellite cells or developed from individual myofibers produce a pool of proliferating myoblasts that express both Pax7 and the myogenic regulatory factor MyoD (Shefer et al., 2006). As myoblasts withdraw from the cell cycle and differentiate, Pax7 expression is downregulated while myogenin is upregulated (Day et al., 2009). Together with myoblast differentiation and myotube formation, there is an emergence of mononuclear cells that downregulate MyoD expression and exit the cell cycle, but maintain Pax7 expression; these cells define a reserve population that presumably reflect satellite cell self-renewal (Collins et al., 2005; Halevy et al., 2004; Zammit et al., 2004; Shefer et al., 2006). In cultures of NES-GFP+ satellite cells, GFP expression diminishes in proliferating and differentiating myoblasts. However, NES-GFP expression is subsequently detected in the Pax7+/MyoD- reserve cell progeny positioned between dense myotubes (Day et al., 2007). Therefore, detection of NES-GFP+ cells is associated with the emergence of a quiescent, reserve cell population in culture.

Age-related muscle deterioration (sarcopenia) is characterized by a significant decline in mass, strength, and endurance of skeletal muscles (Thompson, 2009). Studies using rodent models of induced injury have demonstrated that there is an age-linked decline in muscle repair capacity (Carlson and Faulkner, 1989; Conboy et al., 2003; Rader and Faulkner, 2006). This reduced regenerative potential in old animals is due, at least to some extent, to the influence of changes within the muscle niche as exposure to a young systemic environment or transplantation of aging muscle to a young host enhanced regenerative efficiency (Brack et al., 2007; Carlson and Faulkner, 1989; Conboy et al., 2005). Furthermore, populations of satellite cells isolated from old age were shown to progress from proliferation to differentiation and produce reserve cells when cultured in a rich growth-promoting environment or when transplanted into young host muscles (Collins et al., 2007; Shefer et al., 2006). These observations support the hypothesis that at least some satellite cells within aging muscle maintain myogenic stem cell properties. The early phase of myotube formation was also relatively unaltered in some injury models of aging muscle, which further supports the notion that satellite cells from old animals can be recruited into myogenesis (Smythe et al., 2008).

Most assessments of muscle regeneration are based primarily on induction of satellite cell activity en masse induced by injury followed by observation of the dynamics of myogenic cell populations. However, during routine muscle maintenance, satellite cells may be recruited individually for localized repair of subtle injuries. Therefore, it is important to define possible intrinsic changes in satellite cells with age at the single cell level. Indeed, there is evidence that at least in some muscles, satellite cell numbers decline with age (Collins et al., 2007; Gibson and Schultz, 1983; Renault et al., 2002; Shefer et al., 2006; Snow, 1977). Such depletion in satellite cells may reduce the efficiency of routine reparation following subtle myofiber injuries. Gene expression differences among satellite cells such as variation in Myf5-driven βgal or YFP expression may also indicate functional heterogeneity in the satellite cell population (Beauchamp et al., 2000; Day et al., 2007; Kuang et al., 2007). Thus, monitoring myogenic performance of individual cells may identify intrinsic age-associated changes within select cells that will potentially elucidate the significance of satellite cell diminution with age.

Here we used nestin-GFP mice to analyze age-related changes in satellite cell numbers and performance. First, we monitored satellite cells in isolated EDL myofibers and demonstrate a decline in satellite cell numbers with age that is associated with an increase in myofibers devoid of satellite cells and an overall shift to myofibers with fewer satellite cells. We also observed a relative reduction in the number of Myf5-βgal+ satellite cells by βgal immunodetection when analyzing myofibers from old nestin-GFP/Myf5nLacZ+ mice. Second, we sorted GFP+ cells and analyzed their individual capacity to produce reserve cells in culture. Satellite cells isolated from old mice yielded fewer colonies that produced reserve cells compared to young cells. Therefore, aging may impair the intrinsic ability of some progenitors to produce reserve cells. Impairment in reserve cell production could be an underlying factor in the depletion of satellite cells and a potential contribution to the age-related decline in muscle quality and performance.

Materials and Methods

Animals

Muscles were isolated from the hindlimbs of mice. All mice were from colonies maintained at the University of Washington and were housed in micro-isolator cages in a pathogen-free facility under 12-hour light/dark cycle and were fed ad libidum Lab Diet 5053 (Purina Mills). Animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee at the University of Washington. Unless otherwise noted, both males and females were used. Mice ranged in age from 3-8 months (young) to 19-26 months (old) as detailed in the Results section. The following mutant mice (all on C57BL/6 strain background) were used: Nestin-GFP (transgenic, heterozygous; referred below as NES-GFP); developed by Dr. G. Enikolopov (Cold Spring Harbor Laboratory) (Day et al., 2007; Mignone et al., 2004). Myf5nLacZ/+ (knockin, heterozygous); developed by Drs. S. Tajbakhsh and M. Buckingham (Pasteur Institute) (Beauchamp et al., 2000; Tajbakhsh et al., 1996; Tajbakhsh et al., 1997). MLC3F-nLacZ (transgenic, heterozygous); developed by Drs. R. Kelly and M. Buckingham (Pasteur Institute). In these mice regulatory elements of muscle specific myosin light chain 3F (MLC3F) drive LacZ expression in myofiber nuclei (Beauchamp et al., 2000; Kelly et al., 1995; Kirillova et al., 2007). NES-GFP mice were crossed with Myf5nLacZ/+ and MLC3F-nLacZ mice to generate NES-GFP/Myf5nLacZ/+ and NES-GFP/MLC3F-nLacZ double heterozygotes, respectively. Myostatin-/- (null); developed by Dr. S.-J. Lee (Johns Hopkins University) (McPherron et al., 1997). We are also grateful to Dr. K. Wagner (Johns Hopkins University) for a cohort of aging myostatin-/- male mice.

Cell culture medium

DMEM [high glucose, with L-glutamine, 110 mg/L sodium pyruvate, and piridoxine hydrochloride supplemented with 50U/ml penicillin and 50 mg/ml streptomycin; GIBCO-Invitrogen) was used for rinsing muscles, myofibers and cultured cells throughout all procedures. The growth medium for all types of cell cultures detailed below was also DMEM-based and was supplemented with 20% fetal bovine serum (JR Scientific), 10% horse serum (HyClone), and 1% chicken embryo extract prepared from whole 10-day old embryos (Shefer et al., 2006; Shefer and Yablonka-Reuveni, 2005). This growth medium supports both proliferation and differentiation of cultured satellite cells (Day et al., 2007; Shefer et al., 2006; Yablonka-Reuveni, 2004).

Myofiber isolation and recording of myofiber-associated NES-GFP+ satellite cells

Single myofibers were isolated from EDL muscles after collagenase digestion as previously described (Shefer et al., 2004; Shefer and Yablonka-Reuveni, 2005), except that the digested tissue and dissociated myofibers were rinsed more extensively to minimize possible contribution of interstitial cells. In brief, digested muscle bulk was first transferred through a series of three 100-mm Petri dishes filled with warm DMEM. Single myofibers were then dissociated from each EDL muscle by gentle trituration with a fire-polished Pasteur pipet, releasing 10-40 myofibers into successive 100-mm Petri dishes filled with warm DMEM. Intact myofibers from each dish were transferred to a corresponding 60-mm dish with warm DMEM and analyzed closely under the high power magnification on the dissection microscope for the absence of any obvious adhering debris before being selected for plating individually into 24-well trays coated with diluted Matrigel (growth factor reduced formulation). After initial adherence for 3 h, numbers of GFP+ cells on individual myofibers from NES-GFP or NES-GFP/Myf5nLacZ/+ mice were recorded at 40× magnification using green fluorescence filter system. Myofibers that contained debris or clusters of cells attached outside of the myofiber (not observed initially under the dissection microscope) were omitted from the analysis. Myofibers were then fixed for monitoring of GFP+ satellite cells in combination with Pax7 or βgal immunostaining as detailed below. Alternatively, myofibers from NES-GFP/Myf5nLacZ/+ or NES-GFP/MLC3F-nLacZ mice were reacted with X-gal as detailed below to monitor satellite cells or myofiber nuclei that expressed βgal. Myofibers from myostatin-/- mice were monitored for absence of adhering cells by light microscopy and then fixed for Pax7 immunostaining.

Cloning of myofiber-associated NES-GFP+ satellite cells

Cloning of satellite cells from individual EDL myofibers of NES-GFP or NES-GFP/Myf5nLacZ/+ mice was performed following our published procedure (Shefer et al., 2006), except that the number of GFP+ cells on parent myofibers was first recorded. Myofibers were isolated as described above, then placed in uncoated wells and recorded immediately for GFP+ cells. Individual myofibers were then transferred into 1.2 ml of warm growth medium and triturated to disengage associated satellite cells. Each triturated suspension was dispensed in 100 μl aliquots into 12 Matrigel-coated wells in 96-well tissue culture trays. Trays were left undisturbed for 4 days to allow cell adherence and initiation of clonal growth. Each well was then fortified with 100 μl of fresh growth medium followed by replacement of the culture medium with fresh 200 μl growth medium every other day, starting on culture day 5. Clonal growth was first analyzed on culture days 6-7 and all wells were monitored for clonal growth for a total of 12 days. Myogenic clones were identified morphologically by the presence of myotubes, typically found by 7-10 days in culture. Non-myogenic clones were defined by the absence of myotubes and the presence of distinct cells that were characteristically large and flat. These non-myogenic clones were negative for myogenic markers such as MyoD and sarcomeric myosin.

Primary cultures and clones of FACS sorted NES-GFP+ cells

Populations of GFP positive and negative cells were isolated from pooled hindlimb (tibialis anterior and gastrocnemius) muscles from NES-GFP or NES-GFP/Myf5nLacZ/+ mice following our previously published procedure (Day et al., 2007). In brief, muscles were cleaned from connective tissue and major blood vessels. The muscle bulk was then cut into small pieces and digested with Pronase, followed by tissue trituration. The released cells were harvested by low speed centrifugation, resuspended in DMEM containing 10% horse serum and 10 μM Hoechst 33342 (Calbiochem) and the cell suspension was incubated for 30 min at 37 °C prior to cell sorting. An Influx Cell Sorter (Cytopeia Inc.) with UV (351–364 nm) and 488 nm argon lasers was used to sort GFP+ cells from total viable (i.e., Hoechst positive) cells. Gating of GFP positive events was set to at least 10 times the fluorescence intensity of negative events, and GFP+ cells used for all studies were collected from the G0/G1 population (thereby eliminating residual cell aggregates from the analysis).

Primary cultures of sorted GFP+ cells were initiated at densities of 5×103 and 1×104 per well in 24-well dishes (pre-coated with diluted Matrigel) using our growth medium as previously described (Day et al., 2007). Progeny of individual NES-GFP+ satellite cells were analyzed by plating 25, 50, 100, and 200 GFP+ cells using 1.5 ml of standard myogenic growth medium in parallel 35-mm culture dishes pre-coated with Matrigel as in myofiber and primary cultures. This series of low initial cell dilutions were used to optimize numbers of resulting colonies per plate in preparations from both young and old mice in case of differences in cloning efficiency between age groups or overcrowding of colonies at certain initial cell dilutions. After 4 days in culture, 1 ml of fresh medium was added to each plate. After 1 week in culture, individual colonies were identified and their positions were marked to monitor their progress at later time points. Growth medium was replaced henceforth every 3 days. Density of each clone was determined at day 12. Cultures were maintained for up to 3.5 weeks to monitor formation of myotubes and the emergence of GFP+ cells.

Real time RT-PCR

Total RNA was isolated from sorted GFP+ cells using an RNeasy Micro kit (Qiagen) according to the procedure described for less than 1×105 cells as reported previously (Day et al., 2007). RNA integrity and quantity was determined using a 2100 Bioanalyzer with an RNA 6000 Pico Kit (Agilent); all RNA preparations generated RNA integrity numbers between 9 and 10 and typical RNA yields for cells sorted from each mouse were between 50 and 100ng. Synthesis of cDNA was performed using the iScript cDNA synthesis kit (BioRad) and 50ng of input RNA. The relative mRNA expression levels were quantified in triplicate per each RNA preparation using TaqMan gene expression assays with a 7300 Real Time PCR system according to standard PCR cycling conditions (Applied Biosystems). All gene expression assays were purchased from Applied Biosystems: Hs99999901_s1 (18s rRNA), Mm00434400_m1 (α7 integrin), Mm0043125_m1 (Myf5), Mm01354484_m1 (Pax7). Average dCt values were determined by subtracting endogenous 18s control levels according to the relative quantitative method using Applied Biosystems 7300 software. Statistical analyses were performed using ANOVA.

Immunofluorescence

Freshly isolated myofibers, primary cultures, and clones were fixed with 2% paraformaldehyde, permeabilized with Triton X-100, blocked, single or double immunolabeled, and counter-stained with DAPI as performed previously (Day et al., 2007). All antibodies used in the present study were detailed previously (Shefer et al., 2006; Day et al., 2007). The following primary antibodies were used: anti-Pax7 (mouse IgG1, ascites fluid, Developmental Studies Hybridoma Bank [DSHB], 1:2,000 dilution); anti-β-galactosidase (βgal, mouse IgG2a, JIE7 supernatant, DSHB, 1:10); anti-GFP (rabbit IgG, Abcam, 1:20,000). Secondary antibodies used were all produced in goat and conjugated with AlexaFluor (Invitrogen, 1:1,000 dilution) and included: anti-mouse IgG1 − 488, − 568; anti-mouse IgG2a − 568; anti-rabbit IgG − 488; anti-rabbit IgG − 568. Anti-GFP immunostaining was used to enhance GFP fluorescence that was weakened by fixation procedures of primary cultures and even diminished by strong X-gal staining. Controls for immunolabeling included immunostaining with omission of primary antibodies, staining with each of the primary antibodies alone followed by all secondary antibodies, staining with each of the primary antibodies alone followed by the reciprocal secondary antibodies, and immunostaining with the multiple primary antibodies followed by each of the secondary antibodies alone.

X-gal staining for monitoring βgal expression

Freshly isolated myofibers and satellite cell clonal cultures were fixed as described for immunofluorescence except for omitting the permeabilization with Triton X-100. X-gal (Gold Biotechnology, Inc), constituted at 40 mg/ml in N,N dimethylformamide (Sigma-Aldrich), was diluted (40 μl/ml) into yellow staining solution (5mM K3Fe(CN)6, 5mM K4Fe(CN)6.3H20, 2mM MgCl2 in PBS). Clonal cultures initiated from NES-GFP/Myf5nLacZ/+ mice were incubated in X-gal staining solution overnight at 37°C and washed in PBS as performed previously (Kirillova et al., 2007). Single myofibers from NES-GFP/Myf5nLacZ/+ mice were incubated in X-gal solution for up to 16 hours. To define stronger and weaker X-gal+ cells, myofibers were monitored for positive nuclei at 2 hours and subsequently following the overnight incubation. The stained myofibers were then rinsed and reacted with rabbit anti-GFP and counter stained with DAPI. Anti-rabbit IgG − 568 (red) secondary antibody was used to detect anti-GFP reactivity to enable specific GFP signal detection above the overall green fluorescent background produced by prolonged incubation of myofibers in X-gal staining solution. Single myofibers from NES-GFP/MLC3F-nLacZ mice were reacted with X-gal and counter stained with DAPI as described above, except that a 15 min reaction was sufficient to obtain efficient staining of all myofiber nuclei while GFP signal was maintained in satellite cells.

Imaging and data analysis

Observations were made with an inverted fluorescent microscope (Nikon Eclipse, TE2000-S). Images of myofibers and cultured cells stained with X-gal were acquired with a Nikon Coolpix 4500 camera; all other images were acquired with Qimaging Retiga 1300i Fast 1394 monochrome CCD camera. The CCD camera drive and color acquisition were controlled by MetaVue Imaging System (Universal Imaging Corporation). Composites of digitized images were assembled using Adobe Photoshop software. All statistical analyses were performed using ANOVA. Multiple ANOVA was used followed by post-hoc Tukey HSD test for individual comparisons when analysis included greater than 2 groups. Data analysis of clones included square root-transformation prior to performing ANOVA for detection of significant differences between young and old groups.

Results

Age-associated decline in satellite cells on isolated EDL myofibers from NES-GFP mice

We previously reported an age-associated decrease in the number of Pax7+ satellite cells per individual EDL myofibers of male mice (Shefer et al., 2006). We also demonstrated that NES-GFP expression and immunostaining for Pax7 reliably co-labeled satellite cells with 98% agreement on EDL myofibers isolated from 4-10 month NES-GFP mice (Day et al., 2007). In the present study, we extended the analysis to EDL myofibers from old (up to 25 months) NES-GFP mice and confirmed persistent colocalization of Pax7 immunodetection and GFP expression (Fig. 1). There was 96% agreement between Pax7 and GFP expression from 99 myofibers analyzed from old male and female mice. We also quantified the total GFP+ satellite cells per individual myofibers isolated from young and old, male and female NES-GFP mice (Table 1 and Figs. 2A-C). Table 1 summarizes the total numbers of mice and myofibers used, and the average numbers of satellite cells per myofiber for each age group analyzed. The data indicate a significant decline in average numbers of satellite cells per myofiber with age for both males and females, and a reduced number of satellite cells in females compared to males in both young and old age (P <0.001). Comparison of old and young animals reflects an enhanced decline in satellite cells per myofiber in female (~4-fold) compared to male mice (~2-fold) (Table 1).

Figure 1
An EDL myofiber from a 23 month old NES-GFP male mouse depicting a GFP+/Pax7+ satellite cell (A-A′); both the satellite cell nucleus (arrowhead) and the myofiber nuclei counter-stain with DAPI (A).
Figure 2
Quantification of GFP+ satellite cells in EDL myofibers isolated from NES-GFP mice. Satellite cells were recorded per individual myofibers isolated from young and old males (A) and females (B); number of mice used and ages of each group are provided in ...
Table 1
Frequency of satellite cells in myofibers isolated from young and old NES-GFP micea

Individual myofiber data arranged by ascending order of the number of satellite cells per myofiber (x-axis) versus the number of myofibers containing a given number of satellite cells (y-axis) displayed greater numbers of myofibers devoid of satellite cells in old mice compared to young (Figs. 2A,B). Curves that plotted the cumulative myofiber percentile were also generated (Fig. 2A,B); each data point shows the percentage of myofibers (out of total myofibers analyzed) that contained less than or equal to the corresponding number of GFP+ cells per myofiber as indicated on the x-axis. Overlay of cumulative curves show an age-associated shift toward a greater number of myofibers containing fewer satellite cells per myofiber that is relatively more prominent in females compared to males (Fig. 2C).

Our findings suggest that diminution in resident satellite cells is a persistent biomarker of aging EDL muscle. We hypothesized that the increased muscle mass exhibited by myostatin null mice would correlate with sparing EDL myofibers from an age-associated decline in satellite cell numbers. These myostatin mutant mice also showed a reduced progression of sarcopenia and enhanced regeneration in old age compared to wildtype mice (Siriett et al., 2006; Siriett et al., 2007; Wagner et al., 2005). We immunostained single myofibers from myostatin-null male mice for Pax7 to monitor resident satellite cells. Results indicate that myofibers from aged myostatin-null mice also show a reduction of resident satellite cells per myofiber (Table 2 and Fig. 3). The data show an age-associated decline in the average number of satellite cells per myofiber, a shift toward myofibers with a decreased number of satellite cells, and an increase in myofibers lacking satellite cells. Comparison of the satellite cell data between the male groups in myostatin null and wildtype (NES-GFP) mice (Tables 1 and and2,2, Figures 2 and and3)3) indicated a significantly higher number of satellite cells per myofiber in young myostatin null mice versus young wildtype mice (< 0.05), although average values are not overtly different. There was no significant difference between the old myostatin and wildtype groups (Tables 1 and and2,2, Figures 2 and and3).3). Collectively, these results indicate a general decline in satellite cells on EDL myofibers with age.

Figure 3
Quantification of Pax7+ satellite cells in EDL myofibers isolated from young and old myostatin null male mice. The number of mice and ages of each group are detailed in Table 2. The right y-axis represents the cumulative percentile of myofibers analyzed. ...
Table 2
Frequency of Pax7+ satellite cells in myofibers isolated from young and old male myostatin-/- micea

Nestin-GFP/Myf5nLacZ/+ mice display a specific age-associated decline in βgal+ satellite cells in isolated EDL myofibers

We previously showed that 80% of all NES-GFP+ satellite cells on EDL myofibers isolated from young (4-5 months) NES-GFP/Myf5nlacZ/+ mice were βgal+ based on immunostaining (Day et al., 2007). In the present study, we extended this analysis to determine the distribution of βgal+ satellite cells on individual EDL myofibers from old mice. EDL myofibers from old and young NES-GFP/Myf5nlacZ/+ male mice were compared (Table 3 and Figs. 4A-C). For each age group, the average number of GFP+ satellite cells per myofiber observed with NES-GFP/Myf5nlacZ/+ mice was similar to that observed with age-matched NES-GFP male mice (Tables 1 and and3).3). However, in addition to the overall age-associated decline in satellite cell numbers, myofibers from old NES-GFP/Myf5nlacZ/+ mice exhibited an increased disparity between the number of the double-labeled, GFP+/βgal+ versus total GFP+ satellite cells per myofiber compared to young NES-GFP/Myf5nlacZ/+ mice (Fig. 4C). Approximately 79% and 53% of all satellite cells were GFP+/βgal+ on myofibers from young and old NES-GFP/Myf5nlacZ/+ mice, respectively (Table 3). The average number of βgal+ satellite cells per myofiber was significantly reduced (P <0.0001) in myofibers from old mice, while the average number of βgal- satellite cells per myofiber was similar to young mice (Table 3). Hence, our data may indicate that not only is there an overall significant loss of satellite cells on myofibers with age, but there is a specific reduction in the βgal+ satellite cells on freshly isolated myofibers. We also observed a similar decline in βgal+ satellite cells on myofibers from old Myf5nlacZ/+ mice when identifying total satellite cells by Pax7 immunostaining (data not shown).

Figure 4
Quantification of GFP+/βgal- and GFP+/βgal+ satellite cells in EDL myofibers isolated from NES-GFP/Myf5nLacZ/+ male mice. EDL myofibers isolated from an old NES-GFP/Myf5nLacZ/+ mouse immunolabeled for βgal exhibit GFP+/βgal ...
Table 3
Frequency of GFP+/βgal+ and GFP+/βgal- satellite cells in myofibers isolated from young and old male NES-GFP/Myf5nLacZ/+ micea

The observed GFP+/βgal+ and GFP+/βgal- cells potentially represent two distinct satellite cell populations in which the Myf5 locus was possibly active or inactive, respectively. However, it was also possible that the βgal+ and βgal- satellite cells contained higher and lower βgal levels, with the lower level being below detection by immunostaining. To test this hypothesis, we relied upon the sensitivity of X-gal staining of single myofibers isolated from NES-GFP/Myf5nlacZ/+ mice to monitor changes in the detection of βgal by enzymatic activity over time to observe different levels in βgal protein. We first recorded the number of GFP+/X-gal+ cells per myofiber after 2h (young, 10 myofibers; old, 32 myofibers; 2 mice per age group) and found that detection of βgal enzymatic activity closely resembled results obtained by immunostaining with anti-βgal. However, after incubation in substrate overnight, 96% (n=46) and 79% (n=57) of GFP+ satellite cells on myofibers from young and old mice had stronger X-gal staining; remaining satellite cells were also X-gal positive, but with weaker staining. DAPI fluorescence in nuclei was typically quenched by strongly positive nuclear X-gal staining (Fig. 5A-A″), but weakly stained X-gal nuclei showed a range of DAPI fluorescence that validated our assessment of high and low levels of X-gal staining intensity in GFP+/βgal+ satellite cells. Control myofibers from NES-GFP/MLC3F-nLacZ double transgenic mice (in which βgal expression is driven by myosin light chain 3F promoter/enhancer elements) verified strongly positive X-gal reaction in myonuclei (βgal+/DAPI-), while satellite cells were never X-gal positive even after overnight staining (GFP+/DAPI+)(Fig. 5B-B″). Notably, an early publication indeed relied on the maintenance of DAPI staining after X-gal reaction to quantify the number of satellite cell nuclei in myofibers from MLC3F-nLacZ mice (Beauchamp et al., 2000). Altogether, our results suggest that there is a greater ratio of satellite cells on isolated myofibers from old mice that express lower levels of βgal driven by Myf5 gene regulatory elements at the time of myofiber isolation compared to young mice.

Figure 5
Representative images depicting X-gal stained EDL myofibers from adult NES-GFP/Myf5nLacZ/+ and NES-GFP/MLC3FnLacZ/+ mice. The myofiber from NES-GFP/Myf5nLacZ/+ was incubated with X-gal substrate overnight to monitor GFP+/βgal+ nuclei (A-A″). ...

Previous studies that identified non-myogenic cells in cultures emanating from individual myofibers raised the possibility that at least some of the satellite cells might maintain a broader mesenchymal potential rather than restricted commitment to myogenesis (Asakura et al., 2001; Shefer et al., 2004). Recent studies of isolated myofiber cultures showed that a greater number of satellite cell progeny from old mice may convert from myogenic to fibrogenic lineage compared to young (Brack et al., 2007). Therefore, it was attractive to consider that our observed age-associated, relative increase in the proportion of satellite cells expressing lower levels of βgal could reflect an alteration in the number of satellite cells committed to myogenesis during aging. To investigate the possibility that there are greater numbers of satellite cells holding broader potential with age, we analyzed the frequency of myogenic colonies generated by individual satellite cells from young and old NES-GFP and NES-GFP/Myf5nLacZ/+ mice. Satellite cells were harvested from individual EDL myofibers (Fig. 6) and from sorted GFP+ cells isolated from hindlimb muscles (Table 4).

Figure 6
Quantification and classification of clones derived from individual EDL myofibers of young (A) and old (B) mice. Clones were prepared from myofibers isolated from mice detailed in Table 4. Bars represent single myofibers that are arranged in ascending ...
Table 4
Analysis of total colonies generated from sorted NES-GFP+ cells isolated from hindlimb muscles of young and old micea

For the analysis of satellite cells disengaged from individual myofibers, each myofiber was first monitored for the number of NES-GFP+ satellite cells before trituration and dispensation of the resulting cell suspension for clonal growth. Myogenic colonies were identified by the development of myotubes, and non-myogenic colonies were defined by the absence of myotubes and distinct, flattened appearance of the cells that were also negative for myogenic markers such as MyoD and sarcomeric myosin (Shefer et al., 2004; Shefer et al., 2006). Figure 6 depicts the number of GFP+ cells per myofiber before trituration and the number of resulting colonies per each myofiber. Some individual myofibers produced as many myogenic colonies as there were GFP+ satellite cells, but the number of myogenic colonies never exceeded the total number of GFP+ satellite cells recorded. Myofibers devoid of resident GFP+ cells did not produce colonies (Fig. 6, old group). Only two myofibers isolated from old mice yielded non-myogenic colonies, and the total number of colonies derived from these myofibers was greater than the original number of satellite cells recorded (Fig. 6; grey bars depict non-myogenic colonies). Analysis of myofiber-derived colonies also demonstrated that cloning efficiency (the number of myogenic colonies per total number of GFP+ cells recorded on donating myofibers) was the same for preparations from young and old mice.

For analysis of colonies generated from sorted GFP+ satellite cells, we initiated cultures with low cell numbers (25, 50, 100, and 200) per plate to optimize the number of clones for analysis if cloning efficiency differed between young and old groups. We recorded the number of resulting colonies and found only a minor percent of total colonies that yielded non-myogenic progeny (Table 4). There were no significant differences in cloning efficiency of sorted GFP+ cells between young and old groups as shown by the average number of total colonies produced across plates grouped according to the number of initial cells plated (Fig. 7). Notably, all myogenic clones that developed from NES-GFP/Myf5nlacZ/+ mice (when prepared from either single myofibers or from sorted cells) were βgal+ based on overnight X-gal reaction (see Fig. 8 for representative images). Collectively, our analyses of clonal cultures from isolated myofibers and sorted cells show that the number of myogenic colonies generated from single satellite cells is not altered with age.

Figure 7
Cloning efficiency of sorted NES-GFP+ satellite cells isolated from young and old mice. The average number of total colonies produced in plates initiated with the same number of cells was determined for clonal cultures from young (white bars) and old ...
Figure 8
Density classification of colonies generated from individual NES-GFP+ sorted cells from young and old mice. Representative images of low (A-B), intermediate (D-E) and high (G-H) density colonies are depicted at day 12 in culture. Left column (panels A, ...

We previously detected Myf5 transcripts in sorted satellite cells from mouse hindlimb and diaphragm (Day et al., 2007). In view of our findings using immunostaining and X-gal reaction with myofibers from young and old NES-GFP/Myf5nlacZ/+ mice, we also compared endogenous Myf5 mRNA expression levels in freshly sorted GFP+ satellite cells from young versus old mice by real time RT-PCR. We found no significant differences in Myf5 expression levels between young and old mice (n=6, Table 5). We also analyzed mRNA levels of Pax7 and α7 integrin, which are coexpressed in mouse satellite cells (for α7 integrin references see Sacco et al., 2008; Rooney, et al., 2009; Gnocchi et al., 2009). We found no differences in expression levels of Pax7 and α7 integrin when comparing satellite cells of young and old mice (Table 5). Therefore, the age-linked decline in the proportion of satellite cells with stronger expression of βgal in myofibers from nestin-GFP/Myf5nLacZ/+ mice may reflect changes in expression of βgal protein while endogenous Myf5 gene activity is not influenced by age.

Table 5
Average dCt ± SEM values determined by real time RT-PCR comparing relative gene expression levels between sorted NES-GFP+ cells from limb muscles of young and old micea

Satellite cells from old mice produce fewer reserve cells in culture

The reappearance of NES-GFP+ mononuclear cells positioned between dense myotubes of long-term primary cultures and clones was shown by us to indicate a maturation process toward production of Pax7+/MyoD- reserve cells, or self-renewal (Day et al., 2007). We previously observed the emergence of such NES-GFP+/Pax7+/MyoD- reserve cells in a small scale study of clones from young mice. In this initial study, we also demonstrated that these GFP+ cells did not label with BrdU and were negative for differentiation markers myogenin and sacromeric myosin according to immunostaining (Day et al., 2007). In the present study, we hypothesized that colonies produced from individual satellite cells derived from old mice may have a reduced capacity to produce a NES-GFP+ reserve population based on the decrease in satellite cells on myofibers with age. Given that dense myogenic growth appeared to be required for the emergence of reserve cells, we first compared the densities of myogenic colonies produced from the GFP+ cells sorted from hindlimb muscles of young and old NES-GFP and NES-GFP/Myf5nlacZ/+ mice (detailed in Table 4).

The density of myogenic colonies was scored at day 12 by counting the number of cells contained within a 200×200 μm area in the center of each clone at 20× magnification. Colonies that contained a range of 5-7 cells or greater than 20 cells within the described field were scored as “low density” and “high density”, respectively. Colonies with cell numbers falling in the range between low density and high density classifications were scored as “intermediate density”; these colonies typically contained 13-16 cells per field. Phase images depicted in Figs. 8A,D,G show representative colonies exhibiting low, intermediate, and high densities, respectively. Representative colonies of each density were also established from NES-GFP/Myf5nlacZ/+ mice and stained with X-gal (Figs. 8B,E,H). All myogenic colonies from NES-GFP/Myf5nlacZ/+ mice expressed βgal regardless of density or whether they were initiated from satellite cells of young or old mice.

Our analysis showed no significant differences between young and old groups in the average number of low and intermediate density colonies produced across cultures initiated with different dilutions of GFP+ sorted cells (Figs. 8C,F). However, there were generally fewer high-density colonies from old mice, although only the 200 cells/plate dilution was statistically different per the number of high density colonies produced (P <0.02, Fig. 8G). We also analyzed the proportions of low, intermediate, and high density colonies out of total colonies produced from young and old mice. Percentages of each colony density type were: low density (young, 40%; old, 51%), intermediate density (young, 35%; old 35%), and high density (young, 25%; old, 14%). These results further indicate a small decrease in high-density colonies from individual satellite cells derived from old mice. Our observation of similar cloning efficiency between young and old groups (Fig. 7) confirms that this difference in the proportion of colonies displaying various densities with age is not due to differences in cloning efficiency.

We next monitored the emergence of NES-GFP+ reserve cells in individual myogenic colonies from plates that were classified for colony density on day 12. GFP+ cells were not detectable at the time of density classification, but typically emerged within 2-3 weeks in culture only in colonies containing dense myotube networks. GFP+ cells that developed were found associated with the myotube networks (Fig. 9). Compared to individual colonies from young mice, we found fewer or no reserve GFP+ cells within colonies initiated from old mice even though colonies with dense myotube networks were formed (Figs. 9A-J′ and Figs. 10A,B). Images depicted in the latter figures also show a range of GFP intensities observed within GFP+ reserve cells; immunostaining for GFP and Pax7 did not reveal additional GFP+ cells within the clones and all GFP+ cells were confirmed to be Pax7+ (data not shown). According to colony density classification on day 12, the majority of colonies containing reserve GFP+ cells were found in high density colonies, and less frequently within intermediate density colonies from both young and old mice (Table 6). No reserve cells were detected within low density colonies produced from either young or old mice. GFP+ sorted cells from old mice produced fewer high density colonies, and a reduced proportion of intermediate and high density colonies from old mice yielded reserve cells compared to young.

Figure 9
Representative phase and fluorescence images of high density colonies produced from single cells sorted from young (A-E′) and old (F-J′) hindlimb muscles; images were taken at 21 days of culture. Panels A, A′ and F,F′ are ...
Figure 10
Analysis of colonies that generated NES-GFP+ reserve cells in clonal cultures established from freshly sorted nestin-GFP+ cells isolated from young (A) and old (B) mice. Individual plates were grouped across experiments according to number of cells plated ...
Table 6
Distribution of colonies from sorted NES-GFP+ cells classified by density and percent that generated reserve cellsa

A detailed analysis of the total number of colonies that developed GFP+ reserve cells per individual plates demonstrated fewer colonies that contained reserve cells when cultures were initiated from old compared to young mice (Figs. 10A,B). A comparison of average numbers of colonies containing reserve cells per plate showed a specific decline in colonies containing GFP+ cells with age (Fig. 10C). We also found that cloning efficiency of total colonies did not differ between age groups in plates analyzed for reserve cells (data not shown; results as depicted in Fig. 7). Therefore, the difference in the proportion of colonies displaying reserve cells with age is not due to differences in cloning efficiency. We conclude that the total number of colonies initiated from satellite cells of old mice that produce reserve cells within the satellite cells pool is reduced compared to young mice.

Some colonies were maintained up to 26 days to test the possibility that reserve cell development was delayed in high density colonies established from old mice compared to those produced from young. However, with greater time in culture we did not observe an increase in live GFP+ reserve cells or emergence of GFP+ cells in colonies that lacked reserve cells at 3 weeks (data not shown). Immunostaining of these high density colonies from old mice with anti-GFP also did not reveal additional GFP+ cells. Regardless of mouse age, all GFP+ reserve cells developing in clonal cultures from NES-GFP/Myf5nlacZ/+ mice were βgal+ by immunostaining (data not shown).

Primary myogenic cultures produce reserve cells regardless of age

Our analyses showed that NES-GFP+ reserve cells reemerged only in colonies containing dense myotubes, and clones initiated from individual satellite cells from old mice produced fewer reserve cells. To further test the relationships between culture density and the development of reserve cells, we cultured sorted GFP+ satellite cell preparations from hindlimb muscles of young and old NES-GFP mice at our typical high density conditions used to initiate primary cultures. For both age groups, highly dense myogenic cultures developed beginning at 2 weeks that produced mononuclear, Pax7+/GFP+ reserve cells positioned between myotubes at 3 weeks (Fig. 11A-A[triple prime]). We confirmed by immunostaining of parallel cultures that GFP+ reserve cells were typically negative for MyoD, myogenin, and sarcomeric myosin (data not shown; see Day et al., 2007 for a similar analysis). The number of GFP+ reserve cells per 40× field was recorded from 15-20 acquired images of cultures established from sorted cells isolated from 3 individual young and old NES-GFP mice. All GFP+ cells were recorded as reserve cells regardless of GFP signal intensity within the cytoplasm; similar cell numbers were recorded as reserve cells after immunostaining for GFP, although cell signal intensity was more uniform following immunostaining. The average (±SEM) numbers of Pax7+/GFP+ cells per field were similar in cultures from young and old mice (6.3±0.2 and 6.8±0.4 GFP+ cells per field, respectively). These results indicate that the initial high number of sorted cells plated may cooperatively impact the generation of the reserve population. We also found expression of βgal within all GFP+ reserve cells according to immunostaining with anti-βgal of cultures at 3 weeks established from both young and old mice (Figs. 11B-C[triple prime]). This observation that all reserve cells in primary cultures are βgal+ is in agreement with results from our described clonal cultures. Notably, βgal immunoflurescent signal was strong in nuclei of single cells, but weaker and even absent within nuclei in myotubes (Fig. 11). In contrast, using the greater sensitivity of the X-gal staining for detection of Myf5-βgal, we found that nuclei in both single cells and myotubes exhibited strong staining following an overnight reaction (Fig. 8). Overall, comparison of clones and primary cultures indicate that density of myogenic progeny may influence the development of reserve cells; myoblasts may operate together at high density to generate a reserve population regardless of age.

Figure 11
Fluorescence images of 3-week old primary cultures initiated from NES-GFP+ sorted cells from young and old mice. Cultures were immunostained for Pax7 or βgal. A representative culture established from NES-GFP+ satellite cells sorted from hindlimb ...

Discussion

Here we used nestin-GFP mice to analyze age-related changes in satellite cell numbers and performance. First, we monitored satellite cells in isolated myofibers and demonstrate a decline in satellite cell numbers with age that is more striking in female mice and persists in myostatin null mice. Accompanying the age-associated decline in satellite cells, we also detected a decrease in the proportion of satellite cells exhibiting stronger expression of βgal in myofibers from old NES-GFP/Myf5nlacZ/+ mice. Second, we sorted GFP+ satellite cells and analyzed their individual capacity to produce reserve cells in culture. We demonstrated that individual satellite cells vary in their capacity to expand and generate reserve progeny as shown by monitoring colony density and the emergence of NES-GFP+ cells. We identified a reduction in the ability of single satellite cells from hindlimb muscles to generate a reserve population with age. Some satellite cell colonies from old animals produced the apparent density of myotubes required to generate reserve cells, but fewer to no reserve cells were detected. The age-linked decline in the capacity of single satellite cells to generate reserve cells may contribute to satellite cell depletion with age.

Depletion of resident satellite cells with age

Our present analysis of individual EDL myofibers isolated from old mice showed concordance of Pax7 expression with NES-GFP transgene expression, as we previously reported for young male mice (Day et al., 2007). Likewise, the significant decline we observed in the number of NES-GFP+ satellite cells per myofiber with age is similar to our previous findings using Pax7 immunostaining of myofibers from male mice (Shefer et al., 2006). These results demonstrate the reliability of the NES-GFP transgene for live identification of resident satellite cells on myofibers throughout the mouse lifespan. We further compared satellite cell numbers in female versus male mice by following NES-GFP+ cells in isolated EDL myofibers. We observed an increase in the frequency of myofibers devoid of or containing fewer satellite cells and an overall decline in the average number of satellite cells per myofiber in both old male and female mice compared to young animals. Notably, the average number of satellite cells per EDL myofiber was significantly reduced in female compared to male mice with a more accelerated age-linked decline in the average number of satellite cells in females. We found a similar trend of age-associated loss in satellite cells and fewer satellite cells in females compared to males in studies of isolated myofibers from rat gastrocnemius muscle when satellite cells were identified by Pax7 immunostaining (G. Shefer, G. Raunder, Z. Yablonka-Reuveni, D. Benayahu; unpublished). This gender-related distinction in satellite cell profiles could potentially be linked to differential influences of sex steroids. Androgens influence satellite cell number and increase muscle size and strength in humans (Chen et al., 2005; Kadi, 2008). Administration of testosterone led to a dose-dependent increase in satellite cell number, myofiber cross-sectional area, and myofiber nuclei in both young and older men (Sinha-Hikim et al., 2006; Sinha-Hikim et al., 2003). Estrogen treatment of ovariectomized female rats prior to exercise led to enhanced satellite cell numbers and performance (Enns et al., 2008; Enns and Tiidus, 2008). Therefore, differential sex steroid levels that are influenced by aging may contribute to the accelerated diminution in satellite cell numbers in females.

It remains debatable whether the decline in satellite cell numbers with age is a characteristic feature of all muscles (Brack and Rando, 2007). Our study demonstrating an age-associated decline in satellite cells based on direct counts of resident cells in EDL myofibers differs from reports that found no significant reduction in satellite cell numbers in mouse soleus (Schafer et al., 2005) and rat levator ani muscle (Nnodim, 2000). However, other reports also indicated an age-related decrease of satellite cell numbers in rat and mouse TA muscle (Brack et al., 2005; Dedkov et al., 2003) and mouse EDL and soleus muscles (Collins et al., 2007; Gibson and Schultz, 1983; Shefer et al., 2006; Snow, 1977); albeit the onset and magnitude of this age-associated decline in satellite cells may differ among muscles (Brack and Rando, 2007; Shefer et al., 2006). As detailed above, we also found an age-related reduction of satellite cells on myofibers isolated from rat gastrocnemius. Additionally, we observed in the present study that the lack of myostatin had only a subtle influence on satellite cell numbers in myofibers from young mice and no influence on satellite cell numbers in old mice. However, other previous reports showed increased satellite cell numbers in myofibers from young and old myostatin null mice (McCroskery et al., 2003; Siriett et al., 2006). A recent study indicated that hypertrophy in myostatin null mice did not require myogenic progenitor activity and suggests that myostatin may not have an impact on satellite cell numbers (Amthor et al., 2009). Future studies with targeted ablation of satellite cells are required to elucidate the functional relationships between satellite cell numbers and maintenance of muscle mass during aging.

Intrinsic alterations in satellite cells with age

As shown by immunostaining, we found an age-associated diminution in the average number of GFP+/Myf5-βgal+ satellite cells while the numbers of GFP+/Myf5-βgal- cells were unaltered. Consequently, together with the overall decline in NES-GFP+ satellite cells with age, the percentage of Myf5-βgal- cells out of total GFP+ cells were proportionately greater in old mice (~21%, young; ~47%, old). Utilizing the more sensitive βgal detection method by X-gal reaction, we conclude that all satellite cells express βgal, but the proportion of satellite cells with lower βgal expression increases with age. Our observation that fewer satellite cells express Myf5-βgal at higher levels in myofibers from old NES-GFP/Myf5nLacZ/+ mice is not accompanied with a parallel decline in endogenous Myf5 transcript abundance with age as determined by real time RT-PCR of freshly sorted nestin-GFP+ cells isolated from hindlimb muscles. Therefore, age-associated decline in satellite cells expressing higher levels of βgal may be indicative of Myf5 translational differences. Indeed, the nLacZ coding sequence is fused with the first 13 amino acids encoded by exon 1 of the Myf5 gene in phase with the translational start codon in the Myf5nLacZ/+ mouse (Tajbakhsh et al., 1996). The fact that Myf5 protein detection has not been documented by direct immunostaining in satellite cells on isolated myofibers (while Myf5 transcripts are present in quiescent satellite cells) may also be indicative of translational regulation of this myogenic regulatory factor in satellite cells (Yablonka-Reuveni et al., 1999; Shefer et al., 2004). Many reports have also demonstrated dynamic regulation of Myf5 protein at distinct phases of the cell cycle with relatively unaltered mRNA levels in primary myogenic cultures derived from satellite cells and in myogenic cell lines (Day et al., 2009; Doucet et al., 2005; Friday and Pavlath, 2001; Kitzmann et al., 1998; Lindon et al., 2000; Yablonka-Reuveni and Rivera, 1997).

In conclusion, we propose that the age-associated increased proportion of satellite cells with weaker expression of βgal on myofibers may represent a larger pool of progenitors that have remained inactive for an extended period. Indeed, comparisons of single myofiber and satellite cell cultures from young and old rodents demonstrated that satellite cells from old rodents have delayed entry into proliferation that could also be indicative of reduced receptor levels or insensitivity to required mitogens that results from their inactive state (Dodson and Allen, 1987; Schultz and Lipton, 1982; Shefer et al., 2006). Our finding that all GFP+ reserve cells developed in myogenic cultures initiated by satellite cells of young and old nestin-GFP/Myf5nLacZ/+ mice exhibited strong Myf5-βgal signal in our rich culture medium may suggest that strong Myf5-βgal detection in such reserve cells marks cells that have entered quiescence after recent proliferative activity.

Decline in reserve cell output from individual satellite cells with age

We show in this study that individual satellite cells from both young and old mice varied in their performance as reflected by differences in cell density (low, intermediate, or high) and extent of myotube formation in colonies that developed in culture from sorted NES-GFP+ progenitors. These data are consistent with other clonal analyses of progenitors isolated from human and animal models that demonstrated heterogeneity among single satellite cells in their ability to proliferate and differentiate (Baroffio et al., 1995; Quinn et al., 1985; Rouger et al., 2004; Yablonka-Reuveni et al., 1987; Shefer et al., 2006). Our analysis here provides novel insights about heterogeneity in the ability of satellite cells to contribute reserve cells; NES-GFP+ reserve cells typically emerged within high density colonies that also contained a network of dense myotubes. Similarly, the emergence of Pax7+/MyoD- cells in clones has also been reported to require high density (Day et al., 2009; Halevy et al., 2004; Ono et al., 2010). However, the expression of NES-GFP by reserve cells appears to represent a more advanced phase in the maturation toward a satellite cell-like phentoype (Day et al. 2007). Indeed, our recent studies also have demonstrated that these reserve GFP+ cells can be isolated from culture by cell sorting and give rise to a second round of myogenic progeny, myotubes, and GFP+ reserve cells (unpublished).

We found small differences in the number of high density myogenic colonies generated by satellite cells isolated from old mice compared to young, while the numbers of intermediate density colonies were similar. Interestingly, high density colonies produced from satellite cells of old mice contained the same overall morphology and dense myotube networks as colonies from young cells, but nonetheless many of these high density colonies from old mice did not yield reserve cells. However, we found that primary cultures of pooled satellite cells isolated from hindlimb muscles of young and old mice equally generated reserve cells. These data are consistent with our previous study that exhibited similarities in the extent of differentiation and the emergence of Pax7+/MyoD- reserve cells (detected by immunostaining for Pax7 and MyoD) in primary cultures and isolated myofiber cultures of young and old mice; albeit the kinetics of proliferation and differentiation were delayed in cultures from the older mice (Shefer et al., 2006).

Altogether, our data indicate that cultures initiated by a greater number of satellite cells (such as primary cultures) or by single satellite cells with robust proliferative capacity can eventually promote reserve cell development. Thus, high density myogenic progeny produced by satellite cells (either individually or pooled together) may contribute a unique signal(s) that ultimately drives the production of reserve cells. Progeny of select satellite cells from old animals may not produce the same levels of this signal(s) compared to cells from young animals and consequently cannot replenish myogenic progenitors. Furthermore, the declining signal may also be specifically linked to the niche provided by myotubes from aging animals as the reserve NES-GFP+ cells typically develop in close association with the myotubes when GFP+ cells emerge. We are also able to accelerate the appearance of reserve GFP+ cells in primary cultures of satellite cells when initiating cultures at a higher density than that used in the present study. Moreover, using a co-culture model in which freshly sorted “donor” nestin-GFP+ satellite cells were cultured onto “host” cultures containing dense myotube networks, proliferation of donor NES-GFP+ satellite cells was suppressed (shown by lack of BrdU incorporation). Donor cells retained their satellite cell phenotype (GFP+/Pax7+) and intercalated in spaces between myotubes much like the appearance of reserve cells shown in our study here (unpublished). Thus, it is possible that there are also extracellular, cell-to-cell mediated contacts or autocrine/paracrine signals between myofibers/myotubes and satellite cells/reserve cells that may regulate the entry into or exit from the quiescent state. Notably, suspended myofiber cultures where satellite cell progeny are in close contact with the parent myofiber exhibit an early development of the reserve phenotype compared to cultures of dissociated satellite cells (Zammit et al., 2004).

Recent studies have implicated delta/notch, Wnt/β-catenin and Ang1/Tie-2 signaling pathways in the self-renewal of satellite cells (Abou-Khalil et al., 2009; Kuang et al., 2007; Perez-Ruiz et al., 2008), and some of these pathways have been shown to be impacted during aging (Brack et al., 2007; Conboy et al., 2003). Furthermore, a decline in local mitogen production by satellite cell progeny from old animals has been inferred based on the improved proliferation of these cells in response to conditioned medium obtained from primary myogenic cultures from young animals (Mezzogiorno et al., 1993). Likewise, we observed that the delayed entry of satellite cells from old mice into proliferation could be overcome by FGF supplement (Shefer et al., 2006). Such an alteration in the production of growth promoting factors with age or ability to receive signals may be a contributing element to our finding that clones from old age cannot support reserve cell development at the same level as clones from young mice. We are currently using our novel in vitro model with NES-GFP mice to identify signals that may rescue the deficiency in reserve cell production in individual satellite cell cultures established from old mice.

In conclusion, the significance of satellite cell diminution with age and its relationship to sarcopenia awaits future in vivo studies such as development of conditional genetic mouse models to induce ablation of satellite cells from resident myofibers. Our observation of an increased number of myofibers devoid of satellite cells with age maybe reflective of myofibers that completely exhausted their available satellite cell pool; we aim to determine in future studies if such myofibers may also be more advanced with regard to age-associated changes in structure/function. Additionally, age-associated changes in the muscle may influence the intrinsic capacity of individual satellite cells to replenish the reserve pool. Elucidation of density-dependent, paracrine and membrane-bound signals produced by proliferating myogenic cells and myotubes/myofibers may define the mechanisms that drive differentiation or self-renewal, and aid development of therapeutics to replenish myogenic progenitors within aging muscle.

Acknowledgments

We are grateful to Donna Prunkard and Dr. Peter Rabinovitch for their valuable assistance with cell sorting (performed at the core facility of the University of Washington Nathan Shock Center of Excellence). This work was supported by a grant to. Z.Y.R. from the National Institutes of Health (RO1 AG021566). K.D. was supported by the Genetic Approaches to Aging Training Program (T32 AG000057). Z.Y.R. acknowledges additional support during the course of this study from the National Institutes of Health (RO1 AG013798 and RO1 AG035377) and from the Muscular Dystrophy Association (award #135908).

Footnotes

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