Slow-dividing satellite cells retain long-term self-renewal ability in adult muscle

Satellite cells are muscle tissue stem cells located between the basal lamina and plasma lemma of myofibres and play important roles in postnatal muscle growth, as well as adult muscle repair and regeneration (Tedesco et al., 2010). Satellite cells self-renew to maintain a stem cell pool for cell replenishment. In healthy adult muscle, satellite cells are mitotically quiescent and express the paired-box transcription factor Pax7. Satellite cells are activated in response to stimulation such as muscle injury. Activated satellite cells then upregulate MyoD protein expression and proliferate. Following proliferation, the majority of satellite cells downregulate Pax7 expression, maintain MyoD expression and undergo myogenic differentiation to produce new myonuclei. However, a minority of the population downregulates MyoD expression, maintains Pax7 expression and returns to a quiescent state to self-renew (Halevy et al., 2004; Olguin and Olwin, 2004; Zammit et al., 2004).

Transplantation of freshly isolated satellite cells or a single myofibre containing satellite cells significantly generates new muscle and repopulates satellite cells in the host muscle, indicating a potent self-renewal ability of the satellite cell population (Collins et al., 2005; Montarras et al., 2005). Surprisingly, engraftment of only a single muscle stem cell has been shown to give rise to abundant proliferative donor-derived cells and newly formed myofibres in the regenerating muscle (Sacco et al., 2008). Furthermore, a recent study demonstrated that transplantation of single myofibres associated with satellite cells together with muscle injury stimulus prevents age-related muscle wasting and improves muscle force generation throughout life (Hall et al., 2010).

Accumulating evidence indicates that satellite cells within a myofibre are a functionally heterogeneous population (Zammit, 2008; Biressi and Rando, 2010). In vitro studies demonstrated that an undifferentiated non-fused subpopulation self-renews and cells become quiescent during myogenic progression (Baroffio et al., 1996; Yoshida et al., 1998). Recent studies have shown that only a small population of satellite cells possesses stem cell properties (Collins et al., 2005; Kuang et al., 2007; Sacco et al., 2008; Tanaka et al., 2009; Ono et al., 2010). In the postnatal growing rat muscle, approximately 80% of satellite cells are a highly proliferative population that is readily labelled by BrdU pulsing, whereas the remaining cells slowly divide (Schultz, 1996). Similar properties are observed in adult mouse muscle (Shinin et al., 2006; Conboy et al., 2007). It is thought that the slow-dividing minority population reverts to quiescent self-renewing cells and the fast-dividing majority population undergoes limited replication as transiently amplifying cells before myogenesis (Schultz, 1996). However, there is no direct evidence about whether these two populations possess such distinct roles in myogenic progression.

Here we investigated the relationship between proliferation behaviour and satellite cell function. To assess the frequency of cell division, we isolated satellite cells from extensor digitorum longus (EDL) muscle labelled with a fluorescent lipophilic dye (PKH26) and sorted by fluorescence-activated cell sorting (FACS) based on PKH26 fluorescence levels. PKH26^low fast-dividing cells gave rise to a higher number of myogenic differentiating and quiescent-like self-renewing cells compared with PKH26^high slow-dividing cells. However, the slow-dividing-cell-derived cells efficiently generated secondary myogenic colonies containing long-term self-renewing cells when passaged, whereas cells derived from the fast-dividing populaiton rapidly underwent myogenic differentiation after a few rounds of cell division and were eventually exhausted. Furthermore, slow-dividing cells transplanted into damaged muscle, induced by cardiotoxin (CTX) injection, extensively contributed to muscle regeneration in vivo. We also found that the long-term self-renewing cells in the slow-dividing population were restricted to an undifferentiated population that expressed high levels of Id1 protein, which is a negative regulator for bHLH transcriptional factors. Finally, genome-wide gene expression analysis revealed that the PKH26^highId1^high population highly expressed Sdc4, Eya2, Epha2 and Ephb6. Taken together, our results suggest that the undifferentiated slow-dividing cell population in activated satellite cells retain long-term self-renewal ability.

We first investigated whether primary isolated satellite cells could be serially passaged in our culture system (Fig. 1A). Satellite cells were isolated from EDL muscle and induced to differentiate by switching to differentiation medium, and we confirmed the presence of Pax7⁺MyoD^– self-renewing cells (Fig. 1B), as characterised elsewhere (Halevy et al., 2004; Zammit et al., 2004; Ono et al., 2011). Freshly isolated cells were cultured in growth medium for 10 days (Fig. 1C; 5 days in growth medium) and cultured in differentiation medium for 10 days (Fig. 1D). Cells were then re-plated (first passage) and maintained in growth medium. Mononucleated cells were observed (Fig. 1E; 5 days in growth medium). Then, cells were induced to differentiate in differentiation medium for a further 10 days (Fig. 1F). To evaluate whether serially passaged cells were capable of proliferation, cells were again re-plated (second passage) and maintained in growth medium for 10 days (Fig. 1G; 5 days in growth medium) after the first passage. Self-renewed cells generated numerous progeny that formed multinucleated myotubes in differentiation medium (Fig. 1H). These results confirm that isolated satellite cells possess long-term self-renewal ability in our culture system.

Satellite cells exist as two populations: the rapidly dividing majority and slowly dividing minority (Schultz, 1996). To characterise these populations, we monitored proliferating satellite cell proliferation by labelling with the red fluorescent membrane dye PKH26 (Fig. 2A). Time-lapse analysis revealed that fluorescence intensity of PKH26 dye in a parental cell was equally divided into two daughters (Fig. 2B, quantified in 2C). We found that a small number of cells retained a high level of PKH26 dye at 4.5 days in culture in growth medium following PKH26 staining (Fig. 2D,E), suggesting that slow-dividing cells exist amongst activated satellite cells as a minor population. Immunostaining analysis confirmed that plated satellite cells were all Pax7⁺MyoD⁺ activated cells at day 3 after isolation in growth medium and most cells maintained a Pax7⁺ undifferentiated state even at day 7 (Fig. 2F), implying that the PKH26^high slow-dividing cell population is maintained in an activated state.

To investigate the functional differences between PKH26^high slow-dividing and PKH26^low fast-dividing cells, we sorted the two populations by FACS (the top ∼4% was defined as PKH26^high and the bottom ∼30% was defined as PKH26^low) (Fig. 2G–I). Proportions of cells expressing Pax7 and/or MyoD were similar in both populations 1 day after FACS sorting (Fig. 2J). Following sorting, the PKH26^low fast-dividing cells continued to proliferate more rapidly compared with PKH26^high slow-dividing cells (Fig. 2K,L). Indeed, the slow-dividing cells resulted in a lower cell density with fewer differentiated cells expressing myosin heavy chain (MyHC) (Fig. 2M), as well as fewer Pax7⁺MyoD^–Ki67^– self-renewed quiescent-like cells (see Shea et al., 2010) compared with the fast-dividing cells (Fig. 2N,O). Taken together, our results demonstrate that both fast- and slow-dividing cells produce progeny that undergo myogenic differentiation and self-renewal, whereas fast-dividing cells give rise to more progeny capable of differentiation and self-renewal compared with slow-dividing cells.

Having shown that the fast- and slow-dividing cell populations generated self-renewed cells, we next evaluated whether these self-renewed cells are able to generate further myogenic colonies following serial passaging. The FACS-sorted PKH26^high and PKH26^low cell populations were maintained in differentiation medium for 10 days as described in Fig. 2 and then passaged (Fig. 3A). Elongated myotubes were killed by pipetting and the debris was removed by filtration through a 0.45 μm filter at every passage. Single mononucleated cells were observed in both populations after the first passage (Fig. 3B). Interestingly, only PKH26^high cells derived from slow-dividing cells formed secondary myogenic colonies, whereas PKH26^low cells derived from a fast-dividing lineage differentiated after a few rounds of cell division in growth medium and so did not form large colonies (Fig. 3C). These observations were supported by immunostaining showing that PKH26^high slow-dividing cells contained a higher number of Pax7⁺ undifferentiated cells (Fig. 3D,E) and EdU⁺ proliferating cells (Fig. 3F; EdU⁺ nuclei: PKH26^low, 0.9±0.5%, PKH26^high, 66.3±14.5%), compared with PKH26^low fast-dividing cells. Indeed, PKH26^low cells were almost depleted by further passaging (second passage), whereas PKH26^high cells retained the ability to produce myogenic progeny (Fig. 3C). We also found that the PKH26^high population is able to generate PKH26^low fast-dividing cells after passage (supplementary material Fig. S1A–E). These results, therefore, indicate that the slow-dividing cell population is capable of giving rise to highly proliferative progeny after passage and so possesses stem cell potential to reconstitute myogenic colonies. It is important to note that clone sizes varied in colonies derived from PKH26^high cells (data not shown), indicating that self-renewing cells derived from slow-dividing cells are a heterogeneous population.

We next tested whether the PKH26^high slow-dividing cells can effectively regenerate muscles in vivo. Muscle injuries were induced by local CTX injection into the gastrocnemius muscles of recipient Nod/Scid mice (Fig. 4A), as previously described (Ono et al., 2011). PKH26^low or PKH26^high cells, isolated from GFP transgenic mice (Fig. 4B), were then transplanted into injury sites. We found that the PKH26^high cells regenerated muscles more efficiently even in the injured muscles induced by first CTX injection (first CTX), compared with the PKH26^low cells (Fig. 4C,D). This result suggests that the in vivo regenerating environment appears to directly and preferentially favour engraftments with slow-dividing cells. Following transplantation, the effect of secondary CTX injection was also examined further to stimulate muscle regeneration. We found that, after the second CTX injection, more newly regenerated myofibres were observed only in the PKH26^high-cell-transplanted muscle, not in the PKH26^low-cell-transplanted muscle, compared with levels after the first CTX injection (Fig. 4C,D), indicating that self-renewing cells derived from the PKH26^high population underwent a second proliferation and expansion in the injured muscle after the second CTX injection. Thus, our results demonstrate that slow-dividing cells retain stem-cell-like properties that extensively contribute toward regeneration in the host muscle by repetitively producing myogenic progeny.

Because undifferentiated cells and differentiated myotubes were observed within the PKH26^high cell population in growth medium (Fig. 5A), PKH26^high cells might be a functionally heterogeneous population in which some slow-dividing cells immediately differentiate after a few divisions, whereas the remaining cells maintain an undifferentiated state. Therefore, we sorted the two populations by FACS to further characterise slow-dividing cells.

Here we focused on the Id1 protein, which is a potent inhibitor of myogenic differentiation (Benezra et al., 1990). Previously, we demonstrated that the Id1 protein is highly expressed in activated and undifferentiated satellite cells (Ono et al., 2009; Ono et al., 2011). In this study, we used Id1–YFP knock-in mice (Nam and Benezra, 2009), which allowed us to detect endogenous Id1 protein by YFP fluorescence. Immunostaining data showed that all activated satellite cells associated with single myofibres express the Id1–YFP protein (supplementary material Fig. S2A,B) but the level of Id1–YFP expression varied even within Pax7⁺ satellite cells (over 96% of cells were Pax7 positive; Fig. 2F; supplementary material Fig. S2C). To determine whether the level of Id1 expression affects satellite cell fate choice, FACS sorting was performed according to the YFP fluorescence intensity in activated satellite cells (supplementary material Fig. S2D). Notably, Id1^high satellite cells generated a high number of undifferentiated progeny as assessed by morphological observation, whereas Id1^low cells rapidly formed myotubes even under growth conditions (supplementary material Fig. S2E), indicating that the level of Id1 expression correlates with the undifferentiated state of activated satellite cells. Importantly, the Id1^low cells lost self-renewal capacity after passage, whereas the Id1^high cells remained proliferative (supplementary material Fig. S2F). We also found that the level of Id1 expression varied within the PKH26^high cell population (Fig. 5B). Therefore, we further characterised the PKH26^high cells using Id1–YFP knock-in mice. Consistent with the Id1^low cells rapidly differentiating after FACS sorting (supplementary material Fig. S2E), PKH26^highId1^low cells also underwent myogenic differentiation, as indicated by elongated morphological features (Fig. 5D) and increased numbers of myogenin-positive cells (Fig. 5E,F), compared with PKH26^highId1^high cells. Importantly, highly proliferative cells after passage were observed only in the PKH26^highId1^high population (Fig. 5G), suggesting that the undifferentiated slow-dividing cell population is self-renewable over the long term. A PKH26^lowId1^high cell population also maintained an undifferentiated state and was highly proliferative after FACS sorting, and so produced more self-renewed cells compared with the other populations (data not shown). Indeed, cells derived from the PKH26^lowId1^high population were initially in the majority after passage, but most of these cells immediately committed to differentiation and formed myotubes after a few cell divisions (Fig. 5G). Thus, these results imply that the PKH26^lowId1^high cell population is the main source for transit-amplifying cells to provide new myonuclei.

Finally, we determined the gene expression profiles that underlie the long-term self-renewal potential of PKH26^highId1^high cells. Genome-wide gene expression analysis was performed to compare the PKH26^highId1^high population with PKH26^highId1^low, PKH26^lowId1^high and PKH26^lowId1^low cells. This revealed that over 250 genes were upregulated only in PKH26^highId1^high cells (>twofold) compared with levels in the other populations (supplementary material Table S1). Specific genes that were highly expressed only in PKH26^highId1^high cells were also analysed by quantitative RT-PCR (Q-PCR). Q-PCR data confirmed that Sdc4, Eya2, Epha2 and Ephb6 expression were highly upregulated in PKH26^highId1^high cells (Fig. 6).

Tissue stem cells in the adult retain the ability to proliferate, self-renew and differentiate throughout life to maintain tissue homeostasis and repair injuries. How tissue stem cells maintain long-term self-renewal ability and avoid transformation induced by DNA mutation is not fully understood. Stem cells, such as intestinal, hair follicle and hematopoietic stem cells, are considered to divide slowly to avoid DNA mutations and provide a slow-dividing self-renewed daughter and transient amplifying committed progeny that contribute to tissue homeostasis and repair (Fuchs, 2009). Fast-dividing and slow-dividing cell populations within activated satellite cells in growing and regenerating muscles have also been observed (Baroffio et al., 1996; Schultz, 1996; Rouger et al., 2004; Shinin et al., 2006; Conboy et al., 2007). However, the functional differences between the two populations have not yet been characterised.

In this study, we showed that a minority population of activated satellite cells is extremely slow dividing, and both fast-dividing and slow-dividing populations undergo myogenic differentiation and produce self-renewing cells. Although fast-dividing cells generate a higher number of self-renewing cells than slow-dividing cells, most self-renewing cells derived from fast-dividing cells after passage tended to immediately differentiate without producing additional proliferative progeny. These results imply that fast-dividing cells self-renew as committed myogenic progenitors that form myotubes after a few rounds of cell division at the early stage of muscle regeneration. Consistent with these observations, committed myogenic progenitors have been identified by clonal culture analysis showing that approximately 20% of satellite cells isolated from mouse EDL divide minimally before complete differentiation without generating self-renewing daughter cells (Ono et al., 2010). In addition, the fast-dividing self-renewing cells we found in this study might also correspond to the cells that express the myogenin gene immediately after muscle injury and undergo myogenic differentiation with minimal or no cell division (Rantanen et al., 1995).

In hematopoietic stem cells, cell-division frequency inversely correlates with long-term self-renewal potential (Wilson et al., 2008; Foudi et al., 2009). Dormant slow-dividing hematopoietic cells (dividing once every 4–5 months or ∼5 times total) possess a long-term repopulation potential and produce fast-dividing committed cells that repopulate over a short-term. The fast-dividing committed cells give rise to multipotent progenitors that generate nearly a billion circulating blood cells per day. Here we discovered in the skeletal muscle tissue that an undifferentiated slow-dividing satellite cell population retains long-term self-renewal ability. Importantly, the slow-dividing-cell-derived self-renewing cells are capable of generating both slow- and fast-dividing cell populations when reactivated, indicating that slow-dividing cells have more stem-cell-like properties. Previously, single satellite cell clonal experiments demonstrated that large colonies contain numerous self-renewed and differentiated progeny, suggesting that the cells that generate large colonies possess stem cell characteristics (Ono et al., 2010). Unlike small colony-derived cells, we also found that only large colony-derived cells generate highly proliferative progeny with long-term self-renewal ability by single cell clonal analysis (data not shown). Thus, the satellite cell population that generates large colonies probably contains slow-dividing cells. Speculatively, we believe that a small population of satellite cells capable of robust re-population of satellite cells and efficient muscle regeneration in vivo (Collins et al., 2005; Kuang et al., 2007; Sacco et al., 2008; Hall et al., 2010), might also contain the cells that generate slow-dividing cells.

In stem cell fate choice, the balance between self-renewal and differentiation is important for maintenance of stem cell pools and tissue homeostasis. Asymmetric cell division allows a stem cell to generate a daughter cell that self-renews and another that undergoes differentiation. Pax7⁺Myf5^- satellite cells that are 10% of the total satellite cells in mouse EDL muscle (Beauchamp et al., 2000; Kuang et al., 2007), asymmetrically produce a self-renewing Pax7⁺Myf5^- stem cell and a Pax7⁺Myf5^- daughter cell committed to transit-amplifying progeny that undergo limited symmetric proliferation to produce myonuclei (Kuang et al., 2007). It is unclear whether the slow-dividing cells we identified in this study are derived from the Pax7⁺Myf5^- satellite cell population. The ‘immortal strand hypothesis’ has been proposed as a protective mechanism against genomic mutations during DNA replication in stem cells (Cairns, 1975). According to the hypothesis, a stem cell produces a daughter cell that retains older DNA strands and a committed cell with newly synthesised DNA by asymmetric cell division. This phenomenon has also been observed in satellite cells (Shinin et al., 2006; Conboy et al., 2007). Despite the precise mechanism and biological importance being debatable (Zammit, 2008), the presence of a minority population with BrdU- or CIdU-labelled DNA in growing and regenerating muscles (Shinin et al., 2006; Conboy et al., 2007) might support our observation that slow-dividing cells exist in the activated satellite cell population.

We also revealed that transplanted slow-dividing cells extensively contributed to muscle regeneration in vivo. Interestingly, slow-dividing cells gave rise to fewer myotubes and self-renewed cells than fast-dividing cells before passage in culture, whereas transplanted slow-dividing cells effectively produced regenerating myofibres in injured muscles induced by a single CTX injection in vivo. In support of these observations, a previous study has reported that the majority of immortal myoblastic cells quickly died after transplantation into irradiated dystrophic muscles, but a minority, which were slowly dividing in culture in vitro, efficiently survived and were more successfully engrafted in vivo (Beauchamp et al., 1999). Therefore, our results indicate that a regenerative environment has a preferential effect for engraftment of slow-dividing cells.

To further examine the molecular characterisations of long-term self-renewing cells, we performed a genome-wide gene expression analysis and found that Sdc4, Eya2, Epha2 and Ephb6 are highly expressed in the undifferentiated slow-dividing cell population compared with those of other populations. Myoblasts during muscle development and adult satellite cells express Syndecan-4 (Cornelison et al., 2001), and satellite cells derived from Sdc4^–/– mice fail to regenerate injured muscle (Cornelison et al., 2004). Eya2 regulates myogenesis in developing muscle (Heanue et al., 1999; Grifone et al., 2007). However, the role of Eya2 on satellite cell fate choice remains unclear. Epha2 and Ephb6 inhibit cell migration and proliferation, and loss of Epha2 or Ephb6 expression is associated with distant cancer metastasis of several tumour types (Miao et al., 2009; Truitt et al., 2010). Therefore, we assume that Eya2, Syndecan-4, Epha2 and Ephb6 might be involved in stem cell identity and ability, as well as cell cycle frequency in the undifferentiated slow-dividing satellite cell population. Further investigations are needed to clarify the involvement of these highly expressed genes in the functions of long-term self-renewing cells in adult muscle.

In conclusion, we show that the activated satellite cell population contains slow-dividing cells that retain long-term self-renewal ability. Our results therefore provide insight into the mechanisms of satellite cell maintenance by which the risk of DNA mutation is avoided. We show that hierarchical regulation of satellite cell populations exist in adult muscle. Future studies will be valuable in defining the slow-dividing cell properties to further understand the mechanisms of muscle homeostasis and efficient repeated regeneration of adult muscle throughout life.

Mouse anti-Pax7, mouse anti-myogenin and rabbit anti-MyoD antibodies were purchased from Santa Cruz (Santa Cruz, CA). The mouse anti-MyHC (MF20) antibody was purchased from R&D Systems (Minneapolis, MN). The rabbit anti-GFP antibody was purchased from Invitrogen (Carlsbad, CA). The rat anti-Ki67 antibody was purchased from DAKO (Glostrup, Denmark) and rat anti-laminin α2 antibody was purchased from Alexis (San Diego, CA). Mounting medium containing 4,6-diamidino-2-phenylindole (DAPI) for nuclear staining was purchased from Vector Laboratories (Burlingame, CA). The PKH26 dye staining kit was purchased from Sigma (St Louis, MO). The EdU staining kit was purchased from Invitrogen.

Animal experimentation was approved by the Experimental Animal Care and Use Committee of the National Centre of Neurology and Psychiatry, Tokyo, Japan. Adult (8–12 week) male CB7BL/6 mice, C57BL/6-GFP transgenic mice kindly provided by Masaru Okabe (Osaka University, Japan), Id1–YFP knock-in mice (Nam and Benezra, 2009) and immunodeficient Nod/Scid mice were sacrificed by cervical dislocation. EDL muscles isolated and digested in type I collagenase, as previously described (Ono et al., 2010). Satellite cells were obtained from isolated myofibres by trypsinisation in a 0.125% trypsin–EDTA solution for 10 minutes at 37°C with 5% CO₂. Satellite cells were cultured in growth medium (GlutaMax DMEM supplemented with 30% FBS, 1% chicken embryo extract, 10 ng/ml bFGF and 1% penicillin-streptomycin) at 37°C with 5% CO₂. Myogenic differentiation was induced in differentiation medium (GlutaMax DMEM supplemented with 5% horse serum and 1% penicillin-streptomycin) at 37°C with 5% CO₂. For suspension culture, isolated myofibres were cultured in GlutaMax DMEM supplemented with 10% horse serum, 0.5% chicken embryo extract and 1% penicillin-streptomycin at 37°C with 5% CO₂. For PKH26 staining, isolated satellite cells were cultured in growth medium in Matrigel-coated wells for 2.5 days and then labelled with PKH26 dye according to the manufacturer’s instructions. After PKH26 staining, medium was changed every day until sorting using a FACS Aria (BD, Franklin Lakes, NJ).

Total RNA was extracted from isolated satellite cells using an RNAeasy Kit (Qiagen, Hilden, Germany) and cDNA was prepared with a QuantiTect reverse transcription kit with genomic DNA wipeout (Qiagen). Q-PCR was performed using an Applied Biosystems SYBR Green gene expression assay and ABI 7700 Sequence Detection System (Life Technologies, Tokyo, Japan) following the manufacturer’s instructions. Primers were designed using primer blast (NCBI) and are as follows: TATA box binding protein (TBA) as a normaliser (F, 5′-CAGCCTCAGTACAGCAATCAAC-3′ and R, 5′-TAGGGGTCATAGGAGTCATTGG-3′), Syndecan4 (F, 5′-ATACTTCTCTGGAGCCCTCCCCGAC-3′ and R, 5′-ATCCAGTGGCACCAAGGGCTCA-3′), Eya2 (F, 5′-TCGCAACAAGCAGTGACTGGAGCG-3′ and R, 5′-GAGCCGCTGATTTGGCGATGCCTTC-3′), Epha2 (F, 5′-CCGGCAGCAAAGTGCACGAGTT-3′ and R, 5′-CGCAGGTTCCTCCTCCTTCGATGGA-3′) and Ephb6 (F, 5′-AGACAAGAAGGGAAGCAAGCCTGGC-3′ and R, 5′-TCTCTTCGGCACTCCCACCATTGC-3′).

Immunocytochemistry of satellite cells and isolated single fibres was performed as described elsewhere (Ono et al., 2011). Samples were incubated with primary antibodies at 4°C overnight. For immunohistochemistry, frozen muscle cross-sections were blocked with 10% goat serum, incubated with primary antibodies and then visualised using appropriate species-specific Alexa Fluor 488 and Alexa Fluor 568 fluorescence-conjugated secondary antibodies (Invitrogen). Immunostained samples were viewed on an OLYMPUS 1X71 (Olympus, Tokyo, Japan) using Plan-Neofluar lenses. Digital images were recorded with a Hamamatsu ORCA-R2 device using AquaCosmos software version 2.63 (Hamamatsu Photonics, Shizuoka, Japan). Time-lapse images were acquired with an ArrayScan VTI device (Thermo Fisher Scientific Inc., MA).

GFP transgenic mice were used as donors and immunodeficient Nod/Scid mice were used as recipients. Muscle damage was induced by intramuscular injection of 50 μl CTX (10 μM; Sigma) into the left and right gastrocnemius muscles of anaesthetised Nod/Scid mice using a 29 gauge 1/2 insulin syringe, as previously described (Ono et al., 2011). Three days after CTX administration, 5×10³ FACS-sorted PKH26^low or PKH26^high cells derived from GFP transgenic mice in 50 μl growth medium were intramuscularly transplanted into the injury sites in left or right legs, respectively. Two weeks after transplantation, secondary muscle damage was induced by an identical CTX injection. Muscles were removed 10 days after a single CTX injection or 4 weeks after secondary CTX injection and immediately frozen in isopentane cooled in liquid nitrogen for storage at –80°C. Transverse sections of muscle were cut using a cryostat and immunostained for GFP and laminin. The contribution of donor satellite cells derived from GFP transgenic mouse to muscle regeneration was evaluated by counting the number of centrally nucleated GFP-expressing myofibres.

Significant differences were determined using the Student’s t-test, with P<0.05 considered statistically significant.

We thank Naoki Ito for his help. We gratefully acknowledge colleagues who shared antibodies, including through the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa.