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Stem Cell Reports

Ar ticle

The Satellite Cell Niche Regulates the Balance between Myoblast

Differentiation and Self-Renewal via p53

Valentina Flamini,

Rachel S. Ghadiali,

Philipp Antczak,

2,3

Amy Rothwell,

Jeremy E. Turnbull,

and Addolorata Pisconti

Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB, UK

Department of Functional Genomics, Institute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB, UK

Computational Biology Facility, Institute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB, UK

*Correspondence: pisconti@liverpool.ac.uk

https://doi.org/10.1016/j.stemcr.2018.01.007

SUMMARY

Satellite cells are adult muscle stem cells residing in a specialized niche that regulates their homeostasis. How niche-generated sig-

nals integrate to regulate gene expression in satellite cell-derived myoblasts is poorly understood. We undertook an unbiased

approach to study the effect of the satellite cell niche on satellite cell-derived myoblast transcriptional regulation and identiﬁed

the tumor suppressor p53 as a key player in the regulation of myoblast quiescence. After activation and proliferation, a subpopu-

lation of myoblasts cultured in the presence of the niche upregulates p53 and fails to differentiate. When satellite cell self-renewal

is modeled ex vivo in a reserve cell assay, myoblasts treated with Nutlin-3, which increases p53 levels in the cell, fail to differ-

entiate and instead become quiescent. Since both these Nutlin-3 effects are rescued by small interfering RNA-mediated p53

knockdown, we conclude that a tight control of p53 levels in myoblasts regulates the balance between differentiation and return

to quiescence.

INTRODUCTION

Satellite cells (SCs) are quiescent muscle stem cells

residing in a specialized anatomical niche located be-

tween the plasma membrane of the muscle ﬁber and

the surrounding basal lamina (Mashinchian et al.,

2017). In response to muscle damage, SCs become acti-

vated and re-enter the cell cycle. After one or more

rounds of proliferation, the vast majority of SC-derived

muscle progenitors (called myoblasts) exit the cell cycle

and enter a terminal G0 phase that leads to differentia-

tion, followed by fusion to existing damaged muscle

ﬁbers to repair them or one-another to generate new

muscle ﬁbers. During this process, a small portion of

myoblasts do not differentiate and rather enters a revers-

ible G0 phase of the cell cycle, effectively replenishing

the pool of quiescent SCs (Olguı

´n and Pisconti, 2012).

The mechanisms that regulate these fate decisions have

been investigated, and at least two models have been pro-

posed. In the ﬁrst model, activated SCs divide asymmet-

rically upon activation giving rise to a daughter cell

that self-renews and another daughter cell that becomes

a myoblast and gives rise to a myogenic progeny (Du-

mont et al., 2015; Kuang et al., 2007; Troy et al., 2012).

In a second model, proliferating myoblasts are induced

to overexpress Pax7, which in turn inhibits myogenin

expression and promotes entry into a mitotically quies-

cent state (Olguı

´n and Olwin, 2004; Wen et al., 2012).

In both cases, a key role appears to be played by the extra-

cellular environment, called the SC niche (Mashinchian

et al., 2017).

The SC niche is the complex set of molecules surround-

ing the SC in its anatomical location and the receptors

that are expressed on its surface. Several of these molecules

play important roles in driving SC fate (Mashinchian et al.,

2017). However, it is also well established that when SCs

are completely stripped of their native niche and cultured

on a proteinaceous substrate, usually collagen, laminin,

or gelatin, they retain the capacity to recapitulate the

fate choices normally made in the presence of the niche,

including proliferation, differentiation, fusion, and gener-

ation of a population of quiescent cells resembling self-

renewed SCs (Olguı

´n and Olwin, 2004). These observations

raise two questions: (1) Are SCs primed to follow the

myogenic program regardless of the presence of the niche?

(2) Is the transcriptional program that drives these fate

decisions in SCs the same in the presence of the niche

and its absence?

Here we attempt to answer these questions by investi-

gating gene expression in SC-derived myoblasts cultured

under two different conditions: in the presence of their

native niche (on isolated myoﬁbers) or in its absence (on

gelatin-coated dishes). We show how myoblast gene expres-

sion is affected by the presence of the niche and identify the

p53 gene network as a key regulator of myoblast fate in the

presence of the SC niche. Lastly, we show that a sustained

increase in p53 during myoblast cell-cycle exit inhibits

myoblast differentiation while promoting quiescence.

970 Stem Cell Reports jVol. 10 j970–983 jMarch 13, 2018 jª2018 The Authors.

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

RESULTS

Dispersed and Myoﬁber-Associated Myoblasts Exit the

Cell Cycle and Initiate Differentiation with Similar

Timing

When SCs are isolated from the muscle tissue and cultured

on gelatin-coated dishes, they extensively proliferate for

the ﬁrst 2–3 days in culture (Figures 1A, 1B, and S1). Prolifer-

ating SCs express Pax7,Myf5,andMyoD1 and are often

referred to as myoblasts. On the fourth day in culture, a few

myotubes can be already observed (Figure S1). Indeed, myo-

genin-positive (MYOG+) cells are occasionally observed on

the third day in culture (Figure 1B), suggesting that SC-

derived myoblasts in dispersed cultures begin to exit the

cell cycle and undergo terminal differentiation between

48 and 72 hr after isolation. Similarly, on day 3 in culture,

MYOG+ cells are observed amongst myoﬁber-associated

myoblasts (Figures 1C and 1D), which are cultured in the

same medium as dispersed myoblasts. This suggests that

the timing ofmyoblast cell-cycle exit and entryinto terminal

differentiation are comparable regardless of the presence of

the niche. To test whether these comparable timings were

driven by comparable transcriptional programs, we carried

out a globalgene expression analysis of SC-derived myoblasts

cultured either in dispersed cultures or on explanted myoﬁb-

ers. We proﬁled gene expression in myoblasts from both cell

culture types at 48 and 72 hr after isolation, when cell-cycle

exit and commitment to terminal differentiation appear to

occur under both culture conditions (Figures 1A–1D).

Myoblast Cell-Cycle Exit Is Associated with Different

Transcriptional Signatures in the Presence or Absence

of the SC Niche

We collected four biological replicates for each time point

(48 and 72 hr) in each culture condition and analyzed

gene expression by microarray technology. The extent of

reproducibility across replicates was excellent (Figures S2A

and S2B). By contrast, the myoblast transcriptome at 48 hr

was remarkably different from the transcriptome at 72 hr

under both culture conditions, as evidenced by the large

Figure 1. Cell-Cycle Exit and Terminal

Differentiation Are Induced in Both Myo-

ﬁber-Associated and Dispersed Myoblasts

between 48 and 72 hr after Isolation

(A and B) Dispersed myoblasts cultured

on gelatin-coated plates show a rounded

morphology (A) and proliferate extensively

in the ﬁrst 2–3 days as revealed by positive

staining for the cell-cycle marker KI67+.

No differentiating cells are detected at 48 hr

after isolation (B). As early as 72 hr post-

isolation occasionally MYOG+ cells are de-

tected in dispersed cultures (B), arrow.

(C and D) For the ﬁrst 2 days myoﬁber-

associated myoblasts (C) proliferate as re-

vealed by positive staining for KI67+ and

absence of differentiating (MYOG+) cells

(D). At 72 hr after isolation a few MYOG+

cells are occasionally detected (D), arrow.

(E and F) Genes differentially expressed

between 48 and 72 hr in dispersed (E)

and myoﬁber-associated (F) myoblasts were

mapped to canonical gene networks using

IPA, revealing that the top most enriched

gene network in dispersed myoblasts is

centered around Erk1/2 downregulation (E),

while the top most enriched network in

myoﬁber-associated myoblasts is centered

around Trp53 upregulation (F). Genes labeled

in green are downregulated, genes labeled in

red are upregulated at 72 hr compared to

48 hr. The color intensity is proportional to

the extent of up- or downregulation.

Stem Cell Reports jVol. 10 j970–983 jMarch 13, 2018 971

number of differentially expressed genes (at q < 0.01) de-

tected between 48 and 72 hr undereither culture conditions:

1,810 in dispersed myoblasts and 1,999 in myoﬁber-associ-

ated myoblasts. Interestingly, when we compared the 72 hr/

48 hr fold changes between the two culture conditions,

it appeared evident that gene expression changes between

48 and 72 hr were different in the two culture conditions

(Figure S2C). To gain insightinto the molecular mechanisms

that were associated with these dramatic changes in the

transcriptional signature of myoblasts between 48 and

72 hr in either dispersed or myoﬁber-associated cultures,

we mapped the differentially expressed genes to known

gene networks using Ingenuity Pathway Analysis (IPA).

The top most enriched network to which differentially

expressed genes from dispersed myoblasts mapped, was

centered around a decrease in the intracellular kinases Erk1

and Erk2 (Figure 1E). In contrast, the top most enriched

network to which differentially expressed genes from myo-

ﬁber-associated myoblasts mapped, was centered around

an increase in the tumor suppressor Trp53 (p53) (Figure 1F).

ERK1/2 are key promoters of myoblast proliferation (Jones

et al., 2001) and, similarly, an increase in p53 levels is

expected to lead to cell-cycle arrest (Levine, 1997). Thus,

these results are consistent with our initial hypothesis

that between 48 and 72 hr both dispersed and myoﬁber-

associated myoblasts prepare to exit the cell cycle, though

via different molecular mechanisms.

The Signaling Pathways that Regulate Cell-Cycle Exit

in the Presence or Absence of the Niche Are Different

To further our understanding of the molecular mechanisms

regulating SC gene expression in the presence and absence

of the SC niche, we analyzed the canonical signaling path-

ways that were enriched between 48 and 72 hr in dispersed

and myoﬁber-associated myoblasts using IPA, which as-

signs an activation score to a signaling pathway based on

the direction and extent of change of the differentially

expressed genes mapped to that signaling pathway. The

ﬁrst observation was that the vast majority of canonical

signaling pathways moved in different directions between

48 and 72 hr in myoﬁber-associated versus dispersed myo-

blasts (Figure 2A).

When we then analyzed in more detail the IPA category

Cell Cycle, the most striking feature was a strong activation

of Cdk5 signaling (Figure 2B) observed in both culture condi-

tions, indicating cell-cycle arrest and preparation to differ-

entiate (Lazaro et al., 1997; Sarker and Lee, 2004). In myo-

ﬁber-associated myoblasts this was accompanied by an

even stronger activation of ATM signaling, whichis upstream

of p53 and also induces cell-cycle arrest (Figure 2B). Consis-

tently, signaling pathways that promote proliferation, such

as Ceramide signaling (Gangoiti et al., 2012), G2/M transition

signaling,Cyclin signaling, and Aryl hydrocarbon receptor

signaling (Barouki et al., 2007; Yin et al., 2016), were inacti-

vated in myoﬁber-associated myoblasts (Figure 2B). Simi-

larly, dispersed myoblasts showed a marked inactivation

of Integrin signaling, also pointing toward inhibition of pro-

liferation (Figure 2B). Thus, canonical signaling pathways

related to cell-cycle regulation are activated or deactivated

in both culture conditions in a manner that supports prepa-

ration to exit the cell cycle. The only exception to this was

the G1/S checkpoint regulation signaling pathway, which

was activated in myoﬁber-associated but not dispersed myo-

blasts (Figure 2B). Similarly, the IPA category Cell Growth and

Proliferation showed a series of pro-proliferative signaling

pathways (ILK, HGF, PDGF, CREB, P70S6K, and CDC42

signaling pathways) that were activated in myoﬁber-associ-

ated myoblasts but deactivated in dispersed myoblasts (Fig-

ure 2C). Since proliferation and cell-cycle exit are mutually

exclusive, the fact that both pro-proliferation and pro-cell-

cycle exit pathways are activated in myoﬁber-associated

myoblasts, supports previous ﬁndings that myoﬁber-associ-

ated myoblasts are heterogeneous (Ono et al., 2010).

Lastly, we analyzed the IPA category Growth Factor

Signaling (Figure 2D). Amongst the signaling pathways that

were differentially activated between dispersed and myo-

ﬁber-associated myoblasts we found activation of pro-prolif-

eration signaling (HGF) and self-renewal signaling (GDNF

signaling, which requires syndecan-3 (SDC3), and therefore

is expected to promote self-renewal, Bespalov et al., 2011;

Pisconti et al., 2010) in myoﬁber-associated but not in

dispersed myoblasts.

Comparative Analysis of Gene Expression Suggests a

Role for the Niche in Driving Self-Renewal

To understand the differences in myoblast gene expression

changes due to culture conditions we compared ratios of

72 hr/48 hr mRNA levels within each culture condition

with each other (Figure S2C) and generated a fold-change

ratio using the formula:

Comparative ratioðCRÞ=ðmRNA at 72 hr=mRNA at 48 hrÞdispersed

ðmRNA at 72 hr=mRNA at 48 hrÞmyofiber-associated

972 Stem Cell Reports jVol. 10 j970–983 jMarch 13, 2018

Genes that score high CR values (>1) are genes that in-

crease in dispersed but not in myoﬁber-associated myoblasts

between 48 and 72 hr, while genes that score low CR values

(<1) are genes that increase in myoﬁber-associated but not in

dispersed myoblasts between 48 and 72 hr. We identiﬁed

2,583 genes with CR > 1 and 3,120 genes with CR < 1 at

1% false discovery rate (FDR), suggesting very different

time-dependent responses between the two culture condi-

tions. After ﬁltering the resulting list of genes to include

only genes with a q value < 0.01, we then functionally

analyzed these genes by mapping them to the gene ontology

(GO) category Biological Process using the online tool

DAVID (Tables S1 and S2). Table S1 shows the GO annota-

tion of genes that had CR > 2, while Table S2 shows the

GO annotation of genes that had CR < 0.5.

The most signiﬁcant GO terms to which genes that in-

crease in dispersed but not in myoﬁber-associated myo-

blasts (CR > 2) map are associated with cell movement

(Taxis) and muscle differentiation (Striated Muscle Differen-

tiation)(Table S1). Interestingly, the term Negative regulation

of transport also included genes that promote muscle differ-

entiation, such as Il-6 and Nos1 (De Palma et al., 2010;

Hoene et al., 2013). In contrast, the genes that increase

between 48 and 72 hr in myoﬁber-associated myoblasts

but not in dispersed myoblasts (CR < 0.05) map to

Cell Adhesion, Differentiation, and Cell Fate Commitment

(Table S2). The latter appears to be related mostly to main-

tenance of stemness as it contains genes that are associated

with stemness in SCs (Notch1, Notch3, Pax7, and Sox8;

Bjornson et al., 2012; Gopinath et al., 2014; Olguı

´n and

Olwin, 2004; Olguı

´n and Pisconti, 2012; Pisconti et al.,

2010; Schmidt et al., 2003).

Lastly, we organized the differentially expressed genes

according to their CR and ﬁltered the gene list through

a manually curated list of gene families that have been

shown to play a role in myogenesis (see Experimental

Procedures for details). This analysis showed that genes

associated with muscle differentiation were increased

Figure 2. Canonical Signaling Pathways

Are Differentially Activated in Dispersed

and Myoﬁber-Associated Myoblasts

(A) Genes differentially expressed between

48 and 72 hr post-isolation in dispersed

(Dis) and myoﬁber-associated (Mf-A) myo-

blasts were functionally mapped to all ca-

nonical signaling pathways listed by IPA.

For each signaling pathway, an enrichment

p value and a Zscore of activation were

calculated and the pathways with enrich-

ment p value < 0.01 (–log[p value] > 1.3) are

plotted as heatmaps, were orange repre-

sents a positive Zscore (= activation), blue

is a negative Zscore (deactivation), and

white is Z= 0. Canonical signaling pathways

that were not enriched enough to show a

Zscore were excluded. Canonical signaling

pathways that change in opposite direction

in dispersed and myoﬁber-associated myo-

blasts are highlighted by a green box.

(B–D) Heatmaps obtained as in (A) for ca-

nonical signaling pathways mapping to the

IPA categories: Cell Cycle (B), Cell Growth

and Proliferation (C), and Growth Factor

Signaling (D).

(E) Heatmap distribution of the comparative

ratio (CR) = ([72 hr/48 hr]

dispersed

/[72 hr/

48 hr]

myoﬁber-associated

) for gene families

that are involved in myogenesis (manual

annotation, see Experimental Procedures

section).

Stem Cell Reports jVol. 10 j970–983 jMarch 13, 2018 973

in dispersed but not in myoﬁber-associated myoblasts

(Table S3;Figure 2E: Myog, Myl3, Myom3, Lmod3, Postn,

Ttn, and Colq). By contrast, genes associated with self-

renewal were increased in myoﬁber-associated but not

dispersed myoblasts (Table S3;Figure 2E: Cxcr4, Col18a1,

Notch1, Notch3, Cdc42, Pax7, Hey1, Jam2, and Sdc3).

To summarize the data shown so far: Erk1/2 downregula-

tion in dispersed myoblasts is mostly associated with acti-

vation of signaling pathways that lead to cell-cycle arrest

and the onset of differentiation. In contrast, Trp53 upregu-

lation in myoﬁber-associated myoblasts is associated with

a more heterogeneous transcriptomic signature in which

signaling leading to cell-cycle arrest, differentiation, self-

renewal, and proliferation co-exist. Since the role of p53

in myogenesis is complex and not well understood, we

decided to focus on understanding how an increase in

p53 levels might affect myoblast cell fate.

An Asymmetric Increase in p53 Protein Levels Is

Incompatible with Myogenic Differentiation in

Myoﬁber-Associated Myoblasts

The expression of p53 is rapidly and transiently upregu-

lated in the ﬁrst hours of C2C12 myoblast differentiation

induced by serum deprivation (Halevy, 1993), and is impor-

tant for myogenic differentiation, as expression of a p53

dominant-negative mutant leads to impaired differentia-

tion (Soddu et al., 1996), but is not involved in differentia-

tion-induced apoptosis (Cerone et al., 2000). Thus, it could

be speculated that the upregulation of p53 that we observe

in myoﬁber-associated myoblasts drives differentiation.

However, it has been recently shown that a sustained in-

crease in p53 levels, such as that due to genotoxic stress,

or treatment with the MDM2 inhibitor Nutlin-3, impairs

myogenic differentiation, possibly via direct inhibition of

myogenin expression (Walsh et al., 2015; Yang et al.,

2015). Moreover, p53 mediates hypoxia-induced inhibi-

tion of myoblast differentiation (Wang et al., 2015).

To investigate the role of the increase in p53 gene expres-

sion detected by microarray in myoﬁber-associated myo-

blasts, we ﬁrst tested whether such an increase in mRNA

levels was accompanied by an increase in p53 protein levels

and whether it was associated with differentiation. Indeed,

p53 levels appeared to increase in myoﬁber-associated

myoblasts, identiﬁed by the general myoblast marker

SDC3 (Cornelison et al., 2001) between 48 and 72 hr in cul-

ture, as measured by an increase in the proportion of p53+/

SDC3+ myoblasts (Figures 3A and 3B). Intriguingly, we oc-

casionally observed an asymmetric distribution of p53 in

myoblast doublets (Figure 3C), which was more prominent

at 72 hr (Figure 3D), and was accompanied by an almost

perfect inverse correlation with myogenin (MYOG) expres-

sion at 96 hr (Figure 3E). When we measured p53 protein

levels across the same 3-day time course in dispersed myo-

blasts, we found only a small and not signiﬁcant increase in

p53 protein levels over time (Figure S3). Moreover, immu-

noﬂuorescence analysis of primary dispersed myoblasts

showed that p53 protein was still present in MYOG+

dispersed myoblasts, although often at lower levels than

in MYOG– cells (Figure 3F). These results suggest that,

when myoblasts are cultured in the presence of their native

niche, p53 protein levels increase over time dramatically

and selectively in some cells, and in these cells myogenic

differentiation is inhibited (Figure 3E). When the niche is

absent, such a dramatic and sustained increase occurs in

fewer cells, while the majority of cells maintain lower levels

of p53, which are permissive of myogenic differentiation

(Figure 3F).

If it is true that the presence of the SC niche promotes

p53 upregulation in a subpopulation of myoﬁber-associ-

ated myoblasts and that this in turn inhibits myogenic dif-

ferentiation in these cells, then decreasing p53 in myoﬁber-

associated myoblasts via RNAi should promote myoblast

differentiation in myoﬁber cultures. To test this hypothe-

sis, we transfected myoﬁber cultures with either p53 small

interfering RNA (siRNA) or with control (scrambled) siRNA

at 48 hr post-isolation. Transfection of C2C12 myoblasts

was used to validate p53 knockdown efﬁciency (Figure S4).

Two days after transfection, we ﬁxed and immunostained

the myoﬁbers to detect and quantify MYOG+ myoblasts

(Figure 4A). As predicted, the percentage of MYOG+/

SDC3+ cells was increased in p53 siRNA-transfected cul-

tures compared with control siRNA-transfected cultures,

although the difference did not reach statistical signiﬁ-

cance (Figure 4B). The fact that the difference was small,

could be due to the high propensity of differentiated myo-

ﬁber-associated myoblasts to immediately fuse with the

underlying ﬁber (Kuang et al., 2007), which removes

them from the equation when MYOG+/SDC3+ cells are

scored as a measure of differentiation. Indeed, the number

of myonuclei per unit length was signiﬁcantly increased

(Figure 4C), while the frequency of myoblasts (SDC3+ cells)

per unit length was decreased (Figure 4D) upon p53 knock-

down, supporting the idea that p53 inhibits differentiation

in a subpopulation of myoﬁber-associated myoblasts.

The next question was: what is the fate of these myo-

blasts that show high p53 protein levels and fail to differen-

tiate? Since high levels of p53 are incompatible with prolif-

eration, we hypothesized that a sustained increase in p53

levels in myoblasts would lead to apoptosis, senescence,

or quiescence.

An Increase in p53 Protein Levels Promotes Myoblast

Quiescence

Nutlin-3, a small molecule compound that blocks the inter-

action between p53 and the E3 ligase MDM2, is often used

to increase the levels of p53 protein in the cell (Vassilev,

974 Stem Cell Reports jVol. 10 j970–983 jMarch 13, 2018

2005). Nutlin-3 treatment of C2C12 myoblasts leads to a

generalized increase in p53 protein levels and both prolifer-

ation and differentiation inhibition (Walsh et al., 2015),

thus supporting our hypothesis that a sustained increase

in p53 levels in primary myoblasts might lead to apoptosis,

senescence, or quiescence, as well as differentiation inhibi-

tion. To test this hypothesis we treated primary dispersed

myoblasts with increasing concentrations of Nutlin-3 and

veriﬁed that indeed Nutlin-3 inhibits proliferation (Fig-

ure S5A). However, Nutlin-3 treatment does not seem to in-

crease apoptosis in primary myoblasts (Figure S5B), which

is consistent with the absence of a role for p53 in serum

deprivation-induced myoblast apoptosis (Cerone et al.,

2000). Since Nutlin-3 does not promote differentiation,

in fact it inhibits differentiation (Figure S6), the lack of

apoptosis suggests that Nutlin-3-mediated inhibition of

Figure 3. p53 Increases over Time in a

Subset of Myoﬁber-Associated Myoblasts

and Is Asymmetrically Distributed

(A) Individual myoﬁbers were isolated and

cultured in suspension for 48 and 72 hr prior

to ﬁxation and immunostaining to detect

p53 (green), the myoblast marker synde-

can-3 (SDC3, red), and DNA (DAPI, blue).

Arrows indicate SDC3+ SCs.

(B) Quantiﬁcation of (A) where at least

15 myoﬁbers across 3 independent experi-

ments had been scored, and the percentage

of p53+ cells over the total number of SDC3+

cells plotted as average ±SEM. **p < 0.01.

cultured as in (A) then immunostained

to detect p53 (green), SDC3 (white), and

DNA (PI, red). Arrows indicate doublets of

dividing cells where p53 is distributed

either symmetrically (48 hr, left panels) or

asymmetrically (72 hr, right panels).

(D) Quantiﬁcation of (C) where at least 15

ﬁbers across 3 independent experiments

had been scored.

(E and F) p53+/MYOG– myoblasts are very

abundant in myoﬁber cultures (E) but less in

dispersed cultures (F). Individual myoﬁbers

or primary myoblasts were isolated and

cultured for 96 hr, then ﬁxed and immuno-

stained to detect p53 (green), myogenin

(MYOG, red), and DNA (DAPI, blue). Arrows

in (E) indicate p53+/MYOG+ cells, while

all the other cells are either p53+/MYOG–

or p53-/MYOG+. Arrowheads in (F) indicate

p53+/MYOG– cells. All the other cells are

p53+/MYOG+.

Stem Cell Reports jVol. 10 j970–983 jMarch 13, 2018 975

proliferation is due to induction of either quiescence or

senescence.

To discriminate between quiescence and senescence we

performed a reserve cell assay. Cultures of both primary

dispersed myoblasts and C2C12 myoblasts provide a

validated ex vivo model of SC self-renewal: upon serum

lowering, the vast majority of myoblasts exit the cell cycle

with a portion of them downregulating PAX7 and undergo-

ing terminal differentiation, and another portion maintain-

ing high levels of PAX7 and becoming ‘‘reserve cells’’ (Yosh-

ida et al., 1998). In reserve cell assays (Figure 5A), Nutlin-3

treatment of both primary and C2C12 myoblast cultures

dramatically increased the percentage of putative reserve

cells (PAX7+/KI67–) generated by serum lowering (Figures

5B, 5C, 5E, and 5F). This was accompanied by a decrease

in the percentage of proliferating myoblasts (PAX7+/

KI67+) in C2C12 cultures (Figures 5E and 5G), although

no signiﬁcant changes in PAX7+/KI67+ cells were detected

in primary myoblast cultures (Figures 5B and 5D).

In addition to being mitotically quiescent, reserve cells

also show decreased levels of MYOD (Yoshida et al.,

1998), thus ‘‘true’’ reserve cells are PAX7+/KI67–/MYOD–

(Yue et al., 2016). Although we could effectively label

C2C12 myoblasts to simultaneously detect PAX7, KI67,

and MYOD, this could not be achieved with primary myo-

blasts (triple PAX7/KI67/MYOD staining of primary myo-

blasts showed cytoplasmic positivity for MYOD and/or

KI67, even at very low antibody concentration, which is

not observed when either KI67 or MYOD antibodies are

used in a double staining of C2C12 cells with PAX7).

Thus, we were forced to label primary myoblasts separately

for PAX7 and KI67 (as discussed above) and PAX7 and

MYOD. Nutlin-3 treatment dramatically reduced MYOD

protein levels and led to an increase in the frequency

of true reserve cells (PAX7+/KI67–/MYOD–) in C2C12

myoblast cultures (Figures 5K–5L), which was accompa-

nied by a decrease in proliferating myoblasts (PAX7+/

MYOD+/KI67+; Figures 5K and 5M). Similarly, Nutlin-3

treatment produced a 20-fold increase in the numbers of

uncommitted myoblasts (PAX7+/MYOD– cells; Figures

5H and 5I), but only a 6-fold increase in the percentage of

committed myoblasts (PAX7+/MYOD+; Figures 5H and

5J) in primary myoblast cultures. Although in the absence

of a triple PAX7/MYOD/KI67 staining it is not possible to

establish whether Nutlin-3 promotes true reserve cell gen-

eration in primary myoblast cultures as it does in C2C12

cultures, the observations that: (1) the increase in uncom-

mitted myoblasts produced by Nutlin-3 is over 3 times

greater than the increase in committed myoblasts (20-

versus 6-fold), and (2) PAX7+/KI67+ myoblasts do not

increase in response to Nutlin-3 in primary cultures (Fig-

ure 5D), while PAX7+/KI67– cells increase 7-fold (Fig-

ure 5C), suggest that the percentage of true reserve cells

(PAX7+/KI67–/MYOD–) is likely to signiﬁcantly increase

in primary myoblast cultures as it does in C2C12 cultures

in response to Nutlin-3 treatment.

In contrast to differentiated and senescent cells, which

are in an irreversible G0 phase, reserve cells are quiescent,

Figure 4. p53 Knockdown Promotes Myoblast Differentiation in Myoﬁber Cultures

(A) Individual myoﬁbers were isolated and cultured in suspension for 48 hr prior to transfection with either a speciﬁc p53 siRNA or

a scrambled (ctrl) siRNA and 4 hr later ﬁxed and immunostained to detect myogenin (MYOG, green), syndecan-3 (SDC3, red), and DNA

(DAPI, blue).

(B) Differentiated myoblasts were scored as percentage of MyoG+/SDC3+ myoblasts over total nuclei per unit length across at least eight

myoﬁbers/experiment in three independent experiments (N > 24), and plotted as average ±SEM.

(C) The number of myonuclei per unit length was measured as total number of DAPI+ nuclei minus the number of nuclei contained in SDC3+

cells (since SDC3 marks SCs and myoblasts at all stages in myogenesis) per unit length across at least eight myoﬁbers/experiment in three

independent experiments (N > 24), and plotted as average ±SEM.

(D) The percentage of myoblasts was calculated as percentage of nuclei contained in SDC3+ cells over the total number of DAPI+ nuclei per

unit length across at least eight myoﬁbers/experiment in three independent experiments (N > 24), and plotted as average ±SEM.

**p < 0.01 when comparing the indicated population scored in control siRNA-transfected cultures with the same population scored in p53

siRNA-transfected cultures.

976 Stem Cell Reports jVol. 10 j970–983 jMarch 13, 2018

Figure 5. Nutlin-3 Promotes Reserve Cell Generation in Serum-Deprived Myoblasts

(A) Schematic representation of the reserve cell assay experimental design. Primary and C2C12 myoblast cultures were switched to low

serum medium to induce cell-cycle exit in the presence of either 20 mM Nutlin-3 (Nut3) or vehicle (DMSO). To distinguish between

quiescence and senescence, myoblast cultures that had been maintained in low serum supplemented with either DMSO or Nutlin-3 for

3 days were re-exposed to high serum for 2 days. The experiment was repeated 3 times independently and each time 10–15 technical

replicates were scored.

(B–D) Primary myoblast cultures were treated as in (A) and, after 3 days in low serum, ﬁxed, immunostained to detect PAX7 (green), KI67

(red), and DNA (DAPI, blue), and scored for the percentages of PAX7+/KI67– (C) and PAX7+/KI67+ (D) cells. In (B), one representative

image for each treatment is shown. In (C) and (D) quantitative analyses of the indicated cell subpopulations across all three independent

experiments were plotted as average ±SEM. The arrows in (B) indicate PAX7+/KI67+ cells.

(E–G) C2C12 myoblasts cultures were treated as in (A) and, after 3 days in low serum, ﬁxed, and immunostained as in (B). In (E) one

representative image for each treatment is shown. In (F) and (G) quantitative analyses of the indicated cell subpopulations across all three

independent experiments were plotted as average ±SEM.

(H–M) Primary (H and J) and C2C12 (K–M) myoblasts that had been maintained in low serum supplemented with either DMSO or Nutlin-3

for 3 days were ﬁxed and immunostained to detect PAX7 (green), KI67, and/or MYOD (red) and DNA (DAPI, blue). In (H) and (K) one

representative image for each treatment is shown. Insets are enlarged on the side of the main image to show mutual exclusion or

co-localization of PAX7 and MYOD (H) or PAX7 and MYOD+ KI67 (K). In (I), (J), (L), and (M) quantitative analyses of the indicated cell

subpopulations across all three independent experiments were plotted as average ±SEM.

(N–Q) In order to quantify true reserve cells, after 3 days in low serum, primary (N and O) and C2C12 (P and Q) myoblast cultures were

re-exposed to high serum as depicted in (A) for 2 days, then ﬁxed and immunostained to detect PAX7, KI67, and DNA. The percentages of

PAX7+/KI67– (N and P) and PAX7+/KI67+ (O and Q) were calculated and plotted as fold change of the same population in cultures that had

been maintained in low serum and then re-exposed to high serum (we will call it ‘‘post-wash’’ for simplicity) versus the same population in

cultures maintained for only 3 days in low serum (indicated as ‘‘pre-wash’’). The average of the post-wash/pre-wash fold change for each

(legend continued on next page)

Stem Cell Reports jVol. 10 j970–983 jMarch 13, 2018 977

in a reversible G0 phase, and therefore can be induced to re-

enter the cell cycle by adding serum to the culture medium

(Yoshida et al., 1998). To discriminate whether the putative

reserve cells generated in myoblast cultures in response to

Nutlin-3 treatment were truly quiescent or senescent, we

exposed again to high serum the cultures that had been

maintained for 3 days in low serum supplemented with

either Nutlin-3 or DMSO, and 2 days later we measured

the percentages of cells that had re-entered the cell cycle

(Figure 5A). In both C2C12 and primary myoblast cultures

washed and re-exposed to high serum, the percentage of

PAX7+/KI67+ cells increased more dramatically if the cul-

tures had been previously treated with Nutlin-3 compared

with vehicle (Figures 5O and 5Q). Interestingly, the per-

centage of PAX7+/KI67– cells did not signiﬁcantly change

upon serum re-exposure in cultures that had been previ-

ously treated with vehicle, but signiﬁcantly decreased

in cell cultures that had been previously treated with

Nutlin-3 (Figures 5N and 5P). These results indicate that

the percentage of true reserve cells is normally very low in

our reserve cell assay, regardless of the origin of the myo-

blasts used (primary or C2C12). However, this percentage

of true reserve cells is dramatically increased by Nutlin-3

treatment.

An Increase in p53 Levels Is Directly Responsible for

the Increased Reserve Cell Generation in Response to

Nutlin-3

To test whether Nutlin-3 promoted reserve cell generation

and inhibited differentiation directly via p53, we trans-

fected C2C12 myoblasts with either a control siRNA or

with an anti-p53 siRNA, and 3 hr later we replaced the

transfection medium with low serum medium (to induce

cell-cycle exit followed by myotube and reserve cell

generation) supplemented with either Nutlin-3 or vehicle.

Although p53 siRNA transfection did not appear to alter

the differentiation capacity of C2C12 myoblasts treated

with vehicle, it partly rescued the differentiation levels of

cells treated with Nutlin-3 (Figure 6A). More importantly,

the percentage of PAX7+/KI67– reserve cells that was

increased by Nutlin-3 in cultures transfected with control

siRNA, was then rescued by transfection with a speciﬁc

anti-p53 siRNA (Figures 6B and 6C), further indicating

a direct role for p53 in regulating the balance between dif-

ferentiation and reserve cell generation. Interestingly, the

decrease in the percentage of PAX7+/KI67– reserve cells

caused by p53 knockdown in Nutiln-3-treated cultures

was not due to a rescue of the number of proliferating

(PAX7+/KI67+) myoblasts (Figure 6B), but to a decrease

in differentiation. This suggests that Nutlin-3 promotes

reserve cell generation via p53 by affecting directly the

cell fate decision to either differentiate or become quies-

cent, rather than the earlier decision to either divide or

exit the cell cycle.

DISCUSSION

In this study, we show that primary myoblasts cultured

ex vivo carry out different transcriptional programs to regu-

late their cell fate transitions according to whether they

are cultured in the presence or absence of their native

niche. In the absence of the niche, the vast majority of

myoblasts activate a transcriptional program dominated

by Erk1/2 downregulation and differentiate. In the pres-

ence of the niche, the transcriptional program is domi-

nated by an upregulation of Trp53. Validation experiments

revealed that a sustained p53 increase in myoﬁber-associ-

ated myoblasts is restricted to a subpopulation of cells

that fail to differentiate. These myoblasts showing high

levels of p53 are more rare in the absence of the niche,

which is likely why p53 upregulation is not signiﬁcantly

detected in the transcriptomic analysis of the dispersed

myoblasts. Thus, our data suggest that the presence of

the niche leads to a greater percentage of myoblasts

showing a sustained increase in p53 levels and associated

fail to differentiate. We then used gain- and loss-of-

function experiments in a culture context that promotes

cell-cycle exit to determine the fate of these high p53

myoblasts that fail to differentiate, and showed that for

the most part these myoblasts become quiescent. Our

data therefore point toward p53 signaling as a regulator

of the balance between differentiation and self-renewal:

although a transient increase in p53 levels occurs in the

early hours that precede differentiation and is likely neces-

sary for cell-cycle exit, p53 must quickly return to basal

levels upon cell-cycle exit in order for differentiation to

occur (Figure 7). However, in some cells, p53 levels further

increase and remain high over time, leading to differenti-

ation inhibition and promotion of self-renewal (Figure 7).

The latter process appears to be promoted when myogen-

esis occurs in the presence of the SC niche.

subpopulation across 3 independent biological replicates (each one scored for 10–15 technical replicates) was calculated and

plotted ±SEM.

In (C) and (D), (F) and (G), (I) and (J), and (L) and (M): **p < 0.01, where p is the p value of the average percentage of each cell

subpopulation in Nut-3-treated cultures versus DMSO-treated cultures. In (N)–(Q),

p < 0.05 and

p < 0.01, where p is the p value of the

fold change calculated as ‘‘subpopulation percentage in post-wash/subpopulation percentage in pre-wash’’ within each treatment (DMSO

or Nut-3).

978 Stem Cell Reports jVol. 10 j970–983 jMarch 13, 2018

Our ﬁndings open up new questions regarding the mo-

lecular mechanisms that underlie p53-mediated regulation

of the balance between differentiation and self-renewal.

One possibility is that p53 regulates such balance via direct

inhibition of myogenin expression (Yang et al., 2015) and

consequently inhibition of differentiation, upregulation

of PAX7, and activation of a program that resembles in vivo

quiescence (Olguı

´n and Olwin, 2004). By contrast, a direct

upregulation of PAX7 by p53 appears unlikely (Yang et al.,

2015). Interestingly, mice lacking the p53 target gene PW1

are born with fewer quiescent SCs (Nicolas et al., 2005).

Since it is established that neonatal SCs are derived during

development form MYOD+ myoblasts (Kanisicak et al.,

2009), the ﬁnding that PW1 mutants lack quiescent SCs

at birth further supports our view that p53 promotes gener-

ation of a population of quiescent SCs from proliferating

myoblasts.

Although it had been previously shown that p53 inacti-

vation via expression of a dominant-negative mutant de-

creases myoblast differentiation (Soddu et al., 1996) and

that differentiation is decreased in primary Trp53

/

myo-

blasts (Porrello et al., 2000), when we decreased p53 levels

in myoblasts by transfecting a speciﬁc anti-p53 siRNA we

did not observe altered differentiation in the absence of

Nutlin-3. This might be due to the fact that we lowered

serum levels prior to an effective knock down of p53 (the

medium was switched only 3 hr after transfection). Indeed,

also reserve cell generation was not affected by p53 knock-

down in cultures that had not been treated with Nutlin-3.

Thus, it is possible that a transient peak of p53 expression

still occurred in cultures that had not been treated with

Nutlin-3 prior to effective p53 knockdown by siRNA,

thus ensuring a normal distribution between differentia-

tion and quiescence. Vice versa, in cultures treated with

Nutlin-3, p53 levels either rapidly increased in response

to Nutlin-3 above the normal transient peak and remained

high for the entire duration of the experiment (control

siRNA), or, in the absence of Nutlin-3, were prevented

from increasing further than the normal transient peak

by p53 siRNA, and in this second case the distribution

between differentiation and quiescence was maintained

at basal levels. Thus, our data suggest that, after cell-cycle

exit, the levels of p53 in the cell determine whether the

cell is going to enter a terminal G0 phase and differentiate

(p53 levels returning low; Figure 7), or whether the cell is

going to remain in a reversible G0 phase and effectively

renew the pool of undifferentiated, quiescent SCs (p53

levels further increasing and remaining high; Figure 7).

Overall our study supports a model whereby myogenic

differentiation is the preferred fate choice once SCs have

been removed from their anatomical niche and cultured

as myoblasts, whereas the presence of the niche provides

signals that promote quiescence and self-renewal via mul-

tiple mechanisms, one of which leads to p53 upregulation.

Further research is necessary in order to identify the molec-

ular mechanisms through which the SC niche regulates

Figure 6. p53 Knockdown Rescues

Reduced Differentiation and Increased

Reserve Cell Generation in Myoblasts

Treated with Nutlin-3

(A) Differentiation of C2C12 myoblasts is

reduced by Nutlin-3 treatment and rescued

by p53 knockdown. C2C12 myoblasts were

transfected with either control or p53

siRNA, then maintained for 3 days in low

serum supplemented with either DMSO

or 20 mM Nutlin-3 prior to ﬁxation and

immunostaining to detect myosin heavy

chain (MYHC, green) and DNA (DAPI,

blue).

(B) Reserve cell (PAX7+/KI67–) generation

is increased by Nutlin-3 treatment and

rescued by p53 knockdown. C2C12 myo-

blasts were transfected and cultured as in

(A) prior to ﬁxation and immunostaining to

detect PAX7 (green), KI67 (red), and DNA

(DAPI, blue). Arrows point to PAX7+/KI67+

cells. All PAX7+ cells not labeled by an

arrow are KI67–. All KI67+ cells that are not

labeled by an arrow are PAX7–.

(C) Quantiﬁcation of (B) where the average percentage of PAX7+/KI67– cells (over total DAPI+ cells) is calculated across 10–15 technical

replicates for 3 independent experiments (N = 35) and plotted. Error bars are SEM. **p < 0.01.

Stem Cell Reports jVol. 10 j970–983 jMarch 13, 2018 979

p53 protein levels in myoblasts; however, our results

strongly support the notion that p53 plays an important

physiological role in muscle stem cell biology.

EXPERIMENTAL PROCEDURES

Mice

Male C57Bl/6J mice were purchased from Charles River and

housed until used (at 12–13 weeks of age) in a pathogen-free facil-

ity at the University of Liverpool, in accordance with the Animals

(Scientiﬁc Procedures) Act 1986 and the EU Directive 2010/63/EU,

and after local ethical review and approval by the Animal Welfare

and Ethical Review Body.

Myoﬁber Cultures

Single myoﬁbers were isolated and cultured as described previously

(Pisconti et al., 2016). Further details can be found in Supplemental

Experimental Procedures.

Primary Dispersed Myoblast Cultures

Dispersed myoblasts were prepared and cultured as described

previously (Pisconti et al., 2010). Further details can be found in

Supplemental Experimental Procedures.

C2C12 Cell Cultures

C2C12 myoblasts (Yaffe and Saxel, 1977) were cultured as

described previously (Arecco et al., 2016). Further details can be

found in Supplemental Experimental Procedures.

Immunoﬂuorescence

Myoﬁbers, dispersed primary, and C2C12 myoblasts were ﬁxed

for either 20 min (myoﬁbers) or 10 min (primary and C2C12

myoblasts) at room temperature with 4% paraformaldehyde prior

to being processed for immunoﬂuorescence as described previ-

ously (Pisconti et al., 2016). For a list of primary and secondary

antibodies, the concentrations used and microscopy details, refer

to Supplemental Experimental Procedures.

Isolation of Myoblasts from Myoﬁber Cultures

Forty-eight and 72 hr after isolation, myoﬁbers were collected,

washed twice with PBS, always collecting the supernatant which

contained loosely attached myoblasts, then treated with 0.05%

trypsin for 10 min at 37C. The action of trypsin was then

stopped by addition of 2 volumes of primary SC growth medium

and the myoﬁbers separated from stripped myoblasts by centrifu-

gation (3 min at 100 3g): the supernatant was collected and

combined with the two previous PBS washes. Myoblasts were

then collected by centrifugation at 500 3g, the pellet washed

once in PBS, and then immediately lysed in RLT buffer (RNeasy

Kit, QIAGEN). This method yielded highly pure preparations of

myoﬁber-associated myoblasts (Figures S6A–S6E) and high-qual-

ity RNA (Figures S6F and S6G), which did not contain contami-

nant RNA from the myoﬁber (see Supplemental Information for

details).

RNA Extraction and Quality Control

RNA was extracted using the RNeasy Kit (QIAGEN) according

to the manufacturer’s instructions. The quality and concentration

of the RNA was assessed using a Bioanalyzer (Agilent 2100, Agilent)

or a Nanodrop (Thermo Fisher).

Microarrays

Sample labeling and microarray hybridizations were carried

out according to Agilent’s One-Colour Microarray-Based Gene

Expression Analysis Protocol, v.6.6 (manual part number

G4140-90040). Further details can be found in the Supplemental

Experimental Procedures. Raw microarray data are available from

Figure 7. Schematic Representation of

the Role of p53 in SC-Mediated Myo-

genesis

In uninjured muscle (red myoﬁber on the

left), SCs (oval yellow cell on the my-

oﬁber) are quiescent, in a reversible G0

phase of the cell cycle. Upon injury (bolt)

SCs become activated and re-enter the

cell cycle in G1 (yellow/green, star-shaped

cell). At each division cycle the two

daughter cells (myoblasts, green rounded

cells) choose between three main fates:

divide again (and complete the cell cycle),

or exit the cell cycle and then either

differentiate (red elongated cells), or

become quiescent (again oval yellow cell).

Although a transient increase in p53 levels

(upward blue arrow) allows cell-cycle exit,

p53 must return to basal levels in order

for differentiation to proceed (downward

blue arrow). In contrast, a further and sustained increase in p53 levels (larger upward blue arrow) leads to differentiation

inhibition and promotion of self-renewal (return to a quiescent, undifferentiated state).

980 Stem Cell Reports jVol. 10 j970–983 jMarch 13, 2018

the GEO public depository under the accession number: GEO:

GSE109052.

Microarray Analysis

Raw Agilent text ﬁles were read using the read. Agilent function in

the marray R package (Yang, Paquet, and Dudoit, Exploratory anal-

ysis for two-color spotted microarray data. R package v.1.50.0.

http://www.maths.usyd.edu.au/u/jeany/). Data were background

corrected and loess normalized (using the normalizeWithinArrays

function within the Limma package, followed by a quantile

normalization using the normalizeBetweenArrays function)

against their respective 48-hr data point to allow for a direct

comparison between the two culture systems at both time points.

Data were then summarized at the gene level by ﬁrst averaging

identical probes and then selecting a representative probe per

gene name that showed the highest average intensity across the

dataset. Genes with a median intensity level <5 across all arrays

were removed. Differentially expressed genes were identiﬁed

using the samr package (Tusher et al., 2001) for each of the three

comparisons: (1) 72 versus 48 hr myoﬁber-associated myoblasts

(Figure S2A), (2) 72 versus 48 hr dispersed myoblasts (Figure S2B),

and (3) 72 hr/48 hr dispersed versus 72 hr/48 hr myoﬁber-associ-

ated myoblasts (Figure S2C). Genes signiﬁcant at 1% FDR in each

comparison were extracted and used for further analysis.

Manual Annotation of Gene Families Involved in

Myogenesis

Gene families involved in myogenesis were identiﬁed manually via

screening of literature published in PubMed between 01/01/1986

and 01/01/2016. A gene family was annotated as being involved

in myogenesis when at least one member of the family was

described as involved in myogenesis in at least one peer-reviewed

publication. This list included but was not limited to: myogenesis

regulatory factors, Pax genes, skeletal muscle sarcomeric proteins,

various extracellular matrix components, calcitonin receptor,

Wnt pathway, Notch pathway, receptor tyrosine kinase pathways

(ﬁbroblast growth factor, hepathocyte growth factor, epidermal

growth factor, insulin growth factor, etc.), transforming growth

factor bfamily pathways, interleukins, cytokines, chemokines,

mitogen-activated protein kinases, p53 family, cyclins, cyclin-

dependent kinases, etc. The comparative analysis ﬁltered through

this manually curated list is reported in full in Table S3.

Transfection

Myoﬁber and C2C12 cell cultures were transfected as described

previously (Ghadiali et al., 2016). Further details can be found in

Supplemental Experimental Procedures.

Western Blotting

Primary and C2C12 myoblasts were lysed in modiﬁed RIPA buffer

(50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% IGEPAL, 0.5% SDS,

and 0.1% sodium deoxycholate) and western blotting performed

as described previously (Arecco et al., 2016). A list of primary and

secondary antibodies and concentrations used can be found in

the Supplemental Experimental Procedures. Quantitative analysis

of western blots was performed using the ‘‘Analyze Gel’’ function

of ImageJ.

Statistical Analysis of Cell and Biochemistry

Experiments

Experiments shown in Figures 3,4,5, and 6were statistically

analyzed using a t test as the data distributed normally. A p value <

0.05 was considered signiﬁcant.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental

Procedures, seven ﬁgures, and three tables and can be found

with this article online at https://doi.org/10.1016/j.stemcr.2018.

01.007.

AUTHOR CONTRIBUTIONS

V.F. designed and performed experiments shown in Figures 1,2,

and S2. R.S.G. designed and performed experiments shown in Fig-

ures 4 and S3. P.A. performed the microarray analysis and the CR

analysis. A.R. performed experiments shown in Figure 5. J.E.T.

contributed to experimental design. A.P. contributed to experi-

mental design and execution throughout the study and drafted

the manuscript. All authors contributed to manuscript preparation

by providing critical feedback.

ACKNOWLEDGMENTS

This work was funded by a Marie Curie IEF (grant no. 302113) and

a Wellcome Trust ISSF to A.P., a BBSRC-DTP PhD studentship to

R.S.G., and Natural Environment Research Council grant to P.A.

(NE/M01939X/1). We wish to thank Prof. Dave Fernig for the

FGF2, Prof. Brad Olwin for the anti-SDC3 antibody, and Ms. Lisa

Olohan and Ms. Pia Koldkjaer, University of Liverpool, for their

help with the microarrays. We also wish to thank the staff of the

Biomedical Services Unit at the University of Liverpool for the su-

perb care they take of the mouse colonies.

Received: October 11, 2016

Revised: January 11, 2018

Accepted: January 12, 2018

Published: February 8, 2018

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Stem Cell Reports jVol. 10 j970–983 jMarch 13, 2018 983

Stem Cell Reports, Volume 10

Supplemental Information

The Satellite Cell Niche Regulates the Balance between Myoblast Differ-

entiation and Self-Renewal via p53

Valentina Flamini, Rachel S. Ghadiali, Philipp Antczak, Amy Rothwell, Jeremy E.

Turnbull, and Addolorata Pisconti

The satellite cell niche regulates the balance between myoblast

differentiation and self-renewal via p53

Valentina Flamini, Rachel S. Ghadiali, Philipp Antczak, Amy Rothwell, Jeremy E.

Turnbull and Addolorata Pisconti

Supplementary Information

SUPPLEMENTARY METHODS

Myofibre cultures

Mice were culled under a CO2 raising gradient and the gastrocnemius muscles

dissected, incubated with 400 U/mL collagenase type I (Worthington) for 90 min then

individual myofibres were picked using a glass Pasteur pipette whose tip had been

flame-polished and flushed with fresh primary myoblast growth medium (F12 + 0.4

mM CaCl2 + 15% horse serum + 1% penicillin + 1% streptomycin + 2 mM

GlutaMax) in a cell culture incubator (humidified 37 °C, atmospheric O2 and 5%

CO2). After 3 subsequent passages in fresh primary myoblast growth medium to

remove debris and contaminant fibroblasts, FGF2 was added at the final concentration

of 2 nM in the last passage. 2nM FGF2 was added again at 24 h and then the medium

was changed once at 48 hours supplementing again with 2nM FGF2. Although it is

still debated whether SCs derived from different myofibre types are also different and

retain these different properties once cultured ex vivo, we chose to use the

gastrocnemius muscle for myofibre preparations, rather than the most commonly used

extensor digitorum longus (EDL) muscle, because the gastrocnemius muscle contains

all myofibre types and therefore a myofibre-associated myoblast preparation obtained

from the gastrocnemius muscle is more representative of all possible “types” of SCs

than if the preparation was obtained from the EDL muscle, which contains almost

exclusively fast twitch myofibres. This was especially important because the

dispersed myoblast preparations were obtained from whole limb muscles and

therefore they too contained all “types” of SCs. The same batch of horse serum was

used throughout the work presented in this manuscript.

Myofibre-associated myoblast RNA quality control

We ran preliminary experiments to test for the presence of RNA from myofibre debris

in our myofibre-associated myoblast preparations. We re-suspended the final pellet

obtained after myoblast stripping into primary myoblast growth medium, plated this

cell suspension on gelatin-coated dishes and the day after we extracted RNA from

both the adherent cells and the supernatant. If significant amounts of contaminant

RNA from the myofibres had been present in the final myoblast pellet, we should

have been able to extract it and detect it from the supernatant of the plated myoblasts.

Instead we only detected RNA from the adherent cell population not from the

supernatant, which, once analysed by Bioanalyzer, showed only non-RNA

contaminants (Fig. S7D-E), indicating that the final pellet obtained from myoblast

isolation from myofibres is highly pure and does not contain myofibre RNA.

Preparation of dispersed myoblasts

Mice were culled under a CO2 raising gradient and all the hind-limb muscles

dissected, finely minced with scissors and then incubated with 400 U/mL collagenase

type I for 60 minutes at 37 °C. After centrifuging at low speed (30 xg) for 3 minutes

to pellet larger tissue debris, the supernatant containing mono-nucleated cells was

collected, diluted with two volumes of primary myoblast growth medium (F12 + 0.4

mM CaCl2 + 15% horse serum + 1% penicillin + 1% streptomycin + 2 mM

GlutaMax) and then centrifuged again at 500 xg to pellet the mono-nucleated cells.

Fibroblasts were removed via two subsequent pre-plating steps (2h + 1h, each one on

gelatin-coated dishes, at humidified 37 °C, atmospheric O2 and 5% CO2). After the

second pre-plating the floating cells were plated on gelatin-coated dishes in primary

myoblast growth medium, supplemented with 2 nM FGF2 and incubated for 48 or 72

hours, with one medium change at 48 hours. This protocol yielded a preparation of

great purity (over 95% MYF5+/PAX7+ cells). For the microarray experiments, at 48

and 72 h after plating, cells where washed with phosphate buffered saline (PBS) and

lysed with RLT buffer (Qiagen) prior to RNA extraction. For the reserve cell assays,

48 h after plating cells were washed twice with F12C (F12 + 0.4 mM CaCl2 ) and then

switched to primary myoblast low serum medium (F12 + 0.4 mM CaCl2 + 3% horse

serum + 1% penicillin/streptomycin + 2 mM GlutaMax) for 3 days. After 3 days, cells

were either fixed and immunostained or “washed” with F12C and incubated again in

primary myoblast growth medium (containing 15% horse serum) for 2 days, prior to

being fixed and immunostained. The same batch of horse serum was used throughout

the work presented in this manuscript.

C2C12 myoblast cultures

C2C12 myoblasts (Yaffe and Saxel, 1977) were cultured in C2C12 growth medium

(DMEM + 10% foetal bovine serum + 2 mM L-glutamine + 1% penicillin + 1%

streptomycin) up to 90% confluence then washed twice with DMEM and switched to

C2C12 low serum medium (DMEM + 3% horse serum + 2 mM L-glutamine + 1%

penicillin + 1% streptomycin) to induce differentiation and reserve cell generation

(Yoshida et al., 1998) in the presence/absence of either 20 µM Nutlin-3 (Sigma

Aldrich) or an equal volume of vehicle (DMSO, Sigma Aldrich) for an additional 3

days. After 3 days, cells were either immediately fixed and immunostained or

“washed” with DMEM and re-incubated in C2C12 growth medium (containing 10%

foetal bovine serum) for an additional 2 days prior to being fixed and immunostained.

The same batch of foetal bovine serum was used throughout the work presented in

this manuscript.

Antibodies

For immunofluorescence, primary antibodies used were: mouse anti-Pax7 (DSHB)

1:100, mouse anti-p53 (Cell Signaling, Cat No. 2524) 1:100, rabbit anti-myogenin

(SCBT, Cat. No. sc-576) 1:500, rabbit anti-SDC3 (kindly donated by Prof Brad

Olwin, University of Colorado (Cornelison et al., 2001) 1:500, rabbit anti-KI67

(Abcam, Cat. No. ab15580) 1:400, mouse anti-myogenin (DSHB, clone F5D) 1:100,

mouse anti-myosin heavy chain (DSHB, clone MF20) 1:100, rabbit anti-MYF5

(SCBT, Cat. No. sc-302) 1:500, rabbit anti-MYOD (SCBT, Cat. No. sc-760) 1:600.

Secondary antibodies conjugated to Alexa488, Alexa555 or Alexa647 (Invitrogen)

were used at 1:500. DAPI (Invitrogen) was used at 2 µM in PBS.

For western blotting, primary antibodies used were: mouse anti-p53 (Cell Signaling,

Cat No. 2524) 1:1,000; mouse anti-GAPDH (Sigma Aldrich, Cat. No. G8795)

1:3,000; mouse anti-myogenin (DSHB, clone F5D) at 1:1,000 and mouse anti-

sarcomeric myosin (DSHB, clone MF20) at 1:3,000. Secondary antibodies were from

Santa Cruz Biotechnology and used at 1:10,000. Chemiluminescence was detected on

a LAS 4,000 (GE) image doc system.

Microscopy

Microscope images were acquired on an EVOS FL imaging system (Life

Technologies) with a 10X, a 20X or a 40X objective and consistent imaging

parameters.

Microarrays

Fluorescently labelled amplified complementary RNA (cRNA) was generated using a

Low Input Quick Amp Labelling kit, One-Colour (Agilent). The method utilises an

oligo (dT) primer bearing a T7 promoter, and MMLV-RT, producing double stranded

cDNA from mRNA; the cDNA then serves as template for in vitro transcription with

T7 RNA Polymerase, which linearly amplifies target material whilst simultaneously

incorporating cyanine 3-labelled CTP. For each labelling reaction, 70 ng of total RNA

was used as input, along with appropriately diluted Spike Mix from the One-Colour

RNA Spike-In kit (Agilent). cRNA was purified using an RNeasy Mini Kit (Qiagen)

and quantified on a NanoDrop ND-1000 Spectrophotometer version 3.3.0. For array

hybridisation, 600 ng of each cyanine 3-labelled cRNA, was combined with 5 µl of

10X Gene Expression Blocking Agent and 1 µl of 25X Fragmentation Buffer (both

from the Agilent Gene Expression Hybridisation kit) in a total volume of 25 µl.

Target mixtures were then incubated at 60 °C for 30 min to fragment the RNA to

approximately 150 nucleotides. Fragmentation was terminated by cooling on ice for 1

min followed by addition of 25 µl of 2X Hi-RPM Hybridisation Buffer (Agilent).

Agilent SurePrint 8X 60K Mouse Gene Expression microarrays (Design ID 028005)

were loaded and hybridised using Agilent hardware, namely: gasket slides, SureHyb

chambers and hybridisation oven. Hybridisation was carried out at 65 °C with 10

rotations per minute for 17 h. After this time, microarrays were washed using an

Agilent Gene Expression Wash Buffer Kit according to the manufacturer’s

instructions. Arrays were scanned immediately at a resolution of 3 µm using an

Agilent DNA Microarray Scanner to generate 20 bit tiff images. Data was extracted

and QC reports generated using Agilent Feature Extraction version 11.0.1.1. Array

quality was assessed by visual inspection of each tiff and analysis of the associated

QC report, which indicates the dynamic range of the experiment, hybridisation and

background uniformity, as well as an evaluation of metrics associated with the RNA

spike-ins added to the labelling reactions.

Transfection

C2C12 myoblast cultures that had reached 90% confluence or myofibre cultures were

washed twice with PBS and switched to OptiMem before receiving a 1:3,

volume:volume mixture of siRNA (diluted in OptiMem for a final 30 or 60

nM):Lipofectamine2000. After addition of the siRNA:Lipofectamine2000 mixture,

plates where incubated for three hours in a cell culture incubator (humidified 37 °C,

atmospheric O2 and 5% CO2), then the medium was replaced with low serum medium

that contained either 20 µM Nutlin-3 or an equal volume of DMSO and incubated for

another day (for western blot analysis of C2C12 cells) or three days (for

immunofluorescence analysis of C2C12 cells) or 2 days (for immunofluorescence

analysis of myofibre cultures). siRNAs were either control (scrambled) siRNA

(Sigma Aldrich) or anti-p53 siRNA (Sigma Aldrich, SASI_Mm02_00310137).

SUPPLEMENTARY FIGURES

Supplementary Figure S1 – supplementing Fig. 1A-B: Dispersed myoblasts

cultured on gelatin in the presence of high serum and FGF2 undergo

spontaneous differentiation. Primary SC-derived myoblasts were cultured on

gelatin-coated plates in primary myoblast growth medium supplemented with 2 nM

FGF2 for 4 days. During the first 2-3 days cells mostly proliferated, around the third

day they underwent spontaneous differentiation such that myotubes were visible at

day 4 (black arrows).

Supplementary Figure S2 – supplementing Fig. 1E: Replicate reproducibility

was excellent in both dispersed and myofibre-associated myoblast cultures,

which appeared very different in their transcriptome signature during myogenic

progression. A-B) Hierarchical clustering of the Top 200 differentially expressed

genes between 48 and 72 hours in culture in myofibre-associated (A) and dispersed

(B) myoblasts. For each condition the biological replicates clustered together

suggesting high reproducibility and high quality of the data. Moreover, as visually

shown by the heatmaps, the transcriptomic signature at 48 hours is very different from

the transcriptomic signature at 72 hours for both culture conditions. C) Gene

expression regulation between 48 and 72 hours in myofibre-associated myoblasts and

in dispersed myoblasts are different.

Supplementary Figure S3 – supplementing Fig. 3: p53 protein levels do not

significantly increase over time in primary dispersed myoblast cultures. A-B)

Primary dispersed myoblasts were cultured for up to 96 hours post-isolation, lysed at

three time points: 48, 72 and 96 hours, then total proteins extracted and analysed by

western blot to detect p53 levels. Ponceau staining was used as loading control. A

representative western blot image is shown in (A) while (B) is the quantification of

three independent experiments.

Supplementary Figure S4 – supplementing Fig. 4: siRNA-mediated p53

knockdown is highly efficient. C2C12 myoblast were cultured in growth medium

until they reached 90% confluence then transfected with either control siRNA (30

nM, lane 1; 60 nM, lane 3) or anti-p53 siRNA (30 nM, lane 2; 60 nM, lane 4), 3 h

later switched to low serum medium supplemented with either DMSO or 20 µM

Nutlin-3 and cultured for an additional 24 h prior to being lysed and analysed by blot

to reveal p53 (top gel) and GAPDH (bottom gel) abundance. The intensity of the p53

bands normalised to the intensity of the GAPDH bands are reported below the gel

images as percentage of the control band for each one of the two siRNA

concentrations tested. Since the level of knockdown was comparable between 30 nM

and 60 nM siRNA, all subsequent experiments were carried out using 30 nM siRNA.

Supplementary Figure S5 – supplementing Fig. 5: Nutlin-3 treatment of primary

dispersed myoblasts inhibits proliferation without increasing cell death. A)

Primary dispersed myoblasts were expanded for two days prior to being detached and

re-plated at clonal density for an additional three days. The day after plating (time 0

treatment) culture medium was supplemented with either DMSO or Nutlin-3 at 3

different concentrations: 5 µM, 10 µM or 20 µM. Samples were fixed either the day

after (24 h treatment) or 2 days later (48 h treatment) and the numbers of cells/clone

scored and plotted as average across 3 independent experiments ± S.E.M. At 24 h,

only 20 µM Nutlin-3 significantly reduces proliferation (* = p < 0.05), at 48 h both 10

µM and 20 µM Nutlin-3 significantly reduce proliferation (** = p < 0.01). B) Primary

dispersed myoblasts were plated and expanded for one day before being treated with

either DMSO or 20 µM Nutlin-3, then fixed either the day after (24 h treatment) or 2

days later (48 h treatment), processed for TUNEL assay (green) and counter-stained

with DAPI to detect DNA (blue). At each time point, a non-treated control was also

fixed and treated with DNAse-I before TUNEL assay to induce DNA breakage – this

is labelled as DNAse-I/positive control. Although in the positive control all cells are

TUNEL+ indicating that the TUNEL assay worked, in both the DMSO and Nutlin-3-

treated samples, at both time points, only very few TUNEL+ spots are detected,

which do not co-localise with DAPI staining (hence the lack of quantification) and

might therefore be an artifact or might be late apoptotic cells whose DNA was so

extremely fragmented that no DAPI staining was detectable any more. In any case,

there appear to be no difference between the DMSO-treated and the Nutlin-3-treated

samples.

Supplementary Figure S6 – supplementing Fig. 5 and Fig. 6: A sustained

increase in p53 levels reduces C2C12 myoblast differentiation. A-C) C2C12

myoblast cultures were grown to 90% confluence then switched to low serum

medium (DMEM + 3% horse serum + 1% pen/strep + 2 mM L-glutamine) either in

the presence of 20 µM Nutlin-3 or in the presence of an equal volume of DMSO and

lysed at the following time points: Pr (= proliferating) is the day before reaching 90%

confluence; 0 is the day when 90% confluence was reached; 1, 2 and 3 are the days

subsequent to medium switch. At each time point cells were lysed and then all time

points loaded onto a 10% SDS-PAGE for western blot analysis to reveal the

abundance of myosin heavy chain (MYHC), myogenin (MYOG), p53 and GAPDH.

Representative gels from three independent experiments are shown in (A) where to

top GAPDH gel is related to the p53 gel, while the bottom GAPDH gel is related to

the MYHC and MYOG gel. In (B-C) is the quantification of three independent

experiments carried out as in (A) where the average band intensity for p53 (B) or

MYHC (C) normalised to the band intensity for its related GAPDH at each time point

is plotted. Error bars are S.E.M. across the three independent experiments. D-E)

Immunofluorescence analysis of C2C12 myoblasts switched to low serum medium

when 90% confluence was reached and treated with either 20 µM Nutlin-3 or DMSO

for 3 days prior to fixation and immunostaining to detect MYHC (green) and DNA

(DAPI, blue) reveals that differentiation is reduced by Nutlin-3 treatment. In (D) one

representative of several (> 10) independent experiments is shown. In (D) quantitation

of three independent experiments across 10 technical replicates for each one of three

biological replicates (N = 30) as the percentage of MYHC+ cells over the total

numbers of DAPI+ cells is plotted. Error bars are S.E.M. ** = p < 0.01.

Supplementary Figure S7 – cited in Experimental Procedures: Validation of the

protocol to isolate myofibre-associated myoblasts. A) Myofibre-associated

myoblasts were stripped off myofibres using trypsin as described in the Experimental

Procedures section then cytospun and immunostained to detect the SC/myoblast

markers PAX7 and MYF5: 100% of the cells cytspun were positive for at least one of

these two markers. B-C) Isolated myofibre-associated myoblasts were tested for

viability and functionality by plating them on gelatin-coated dishes in primary

myoblast growth medium. Three days later isolated myofibre-stripped myoblasts had

extensively proliferated in culture (B) and after switching them to low serum medium

they underwent differentiation into myotubes (C). D-E) Bioanalyzer analysis of the

RNA extracted from: negative control (water, lane 2); adherent cells (lane 3);

supernatant of isolated myofibre-associated myoblasts plated on gelatin-coated dishes

immediately after isolation (lane 4). Please not the massive differences in the Y-axis

scales in the chromatograms shown in (E). This analysis reveals that no RNA was

present in the supernatant indicating that no RNA was carried over from myofibre

debris into the RNA preparations that were used for microarray analysis. F)

Bioanlyzer analysis of the RNA extracted from all the biological replicates of

myofibre-associated myoblasts at 48 hours and 72 hours post-myofibre isolation.

Only for one of the 4 replicates of myofibre-associated myoblasts at 48 h, two distinct

preparations were pooled to produce one biological replicate. In all other cases each

biological replicate corresponded to one independent preparation. G) Table reporting,

for each RNA sample used in the microarray experiment: sample name, type of

myoblast prep, system for preparation, method of quantification, concentration, RIN

where applicable and 260/280 ratio where applicable. All RNA samples were eluted

in the same volume (14 µL) therefore the concentrations reported are a direct measure

of the total amount of RNA extracted.

REFERENCES

Cornelison, D.D., Filla, M.S., Stanley, H.M., Rapraeger, A.C., Olwin, B.B., 2001.

Syndecan-3 and syndecan-4 specifically mark skeletal muscle satellite cells and

are implicated in satellite cell maintenance and muscle regeneration. Dev Biol

239, 79–94. doi:10.1006/dbio.2001.0416

Yaffe, D., Saxel, O., 1977. Serial passaging and differentiation of myogenic cells

isolated from dystrophic mouse muscle. Nature 270, 725–727.

Yoshida, N., Yoshida, S., Koishi, K., Masuda, K., Nabeshima, Y., 1998. Cell

heterogeneity upon myogenic differentiation: down-regulation of MyoD and

Myf-5 generates “reserve cells.” J Cell Sci 111 ( Pt 6), 769–779.

Supplementary Table 1 – Cited in Results, section 4: Top three GO terms that are

most significantly enriched in genes with CR > 2.

Term

p-value

Genes

Fold

enrichment

Negative regulation

of transport

0.00002

Lif, Il6, Nos1, Snph, Edn1, Pkia,

Adora1, Htr2a

8.99

Striated muscle

development

0.00004

Musk, Actc1, Myl2, Tnc, Myog, Ttn,

Neurl2

10.48

Taxis

0.00005

Ccr8, Ccl2, Cxcl3, Cxcl2, Ecscr, Amot,

Ccl5, Ccl7, Slit2

6.68

Supplementary Table 2 – Cited in Results, section 4: Top three GO terms that are

most significantly enriched in genes with CR < 0.5.

Term

p-value

Genes

Fold

enrichment

Cell adhesion

0.0004

Col18a1, Selp, Pcdhb7, Pcdhb6, Pcdhb3,

Pcdhb4, Itgb4, Cdhr5, Vtn, Pcdh17,

Megf10, Dchs1, Cldn15, Aplp1, Wnt7b,

Lamb3, Itga6, Lama5, Tro, Otog, Msln

2.42

Cell morphogenesis

involved in

differentiation

0.0016

Col18a1, Ablim1, Sema6a, Notch1,

Slc1a3, Rtn4rl1, Cxcr4, Sema3a, Gas1,

Ngfr, Cxcl12

3.35

Cell fate commitment

0.0019

Notch3, Fgfr4, Notch1, Tbx2, Pax7,

Pparg, Cyp26b1, Gas1, Sox8

3.96

Supplementary Table 3 – Cited in Experimental Procedures and in Results, section

4: Comparative analysis of genes that change between 48 and 72 hours according to

the formula: [(72h/48h)dispersed]/[(72h/48h)myofibre-associated] filtered through a manually

curated list of genes involved in myogenesis.

Gene$Name$

Fold$Change$

q-value$

Diras2'

0.054013395(

0.0000000(

Mc4r'

0.093310186(

0.0000000(

Aqp5'

0.096597149(

0.0000000(

Ngfr'

0.105205253(

0.0000000(

Cd5l'

0.179134772(

0.0000000(

Sema6b'

0.228863483(

0.0000000(

Trpc3'

0.233711819(

0.0235839(

Fzd6'

0.235680061(

0.0000000(

Fgfr4'

0.236169718(

0.0000000(

Epha1'

0.250161594(

0.0000000(

Itgb4'

0.25121833(

0.0000000(

Pitx3'

0.255210552(

0.0000000(

Itga6'

0.263171022(

0.0000000(

Notch1'

0.277726352(

0.0000000(

Col18a1'

0.278990429(

0.0000000(

Tmem119'

0.290233444(

0.0000000(

Notch3'

0.291949448(

0.0000000(

Col10a1'

0.300396372(

0.0000000(

Cxcr4'

0.315090189(

0.0000000(

Cdkn2a'

0.316454481(

0.0000000(

Ly6a'

0.316960439(

0.0000000(

Sox8'

0.323270186(

0.0000000(

Tmem44'

0.328784523(

0.0000000(

Megf10'

0.335582833(

0.0000000(

Cdc42ep1'

0.337906165(

0.0000000(

Hes6'

0.338134183(

0.0000000(

Pax7'

0.344352698(

0.0000000(

Hey1'

0.345761151(

0.0000000(

Cxcl12'

0.348607325(

0.0000000(

Sema6a'

0.349215321(

0.0000000(

Rnf152'

0.350092944(

0.0000000(

Tmem191c'

0.350341532(

0.0235839(

Pparg'

0.361863203(

0.0000000(

Rnf144a'

0.400662004(

0.0000000(

Cdkn2b'

0.401434376(

0.0000000(

Lama5'

0.412932128(

0.0000000(

Sema3a'

0.41859219(

0.0000000(

Tcf7'

0.423415284(

0.0000000(

Pdgfc'

0.427927325(

0.0235839(

Tmem176a'

0.428260942(

0.0235839(

Jam2'

0.43211217(

0.0000000(

Pdgfrl'

0.436975917(

0.0000000(

Sdc3'

0.437947492(

0.0000000(

Timp4'

0.442403723(

0.0000000(

Cnnm2'

0.442786114(

0.0000000(

Mmp15'

0.449408686(

0.0000000(

Rnf125'

0.454254155(

0.0000000(

Lamb3'

0.455321565(

0.0000000(

Rnf208'

0.463874638(

0.0000000(

Tnfaip8l1'

0.465211468(

0.0000000(

Tmem30b'

0.471209484(

0.0235839(

Cd200'

0.472625308(

0.0000000(

Fzd2'

0.481798531(

0.0000000(

Ace'

0.485118212(

0.0000000(

Lrp12'

0.487980149(

0.0000000(

Wnt7b'

0.491717209(

0.0000000(

Gper'

0.493160426(

0.0000000(

Creb3l1'

0.50032382(

0.0000000(

Tcf4'

0.502701894(

0.0000000(

Tnfrsf25'

0.511265133(

0.0235839(

Trim62'

0.511388518(

0.0000000(

Megf9'

0.512288348(

0.0235839(

Tmem176b'

0.512579672(

0.0335022(

Chrna1'

0.512895398(

0.0000000(

Ephb4'

0.513271937(

0.0000000(

Cdh15'

0.513597528(

0.0000000(

Igfbp4'

0.518688547(

0.0000000(

Megf6'

0.520352228(

0.0335022(

Tmem195'

0.524521151(

0.0000000(

Cd276'

0.526542761(

0.0000000(

Traf5'

0.534764828(

0.0235839(

Map3k11'

0.541734759(

0.0000000(

Cd2ap'

0.550591348(

0.0000000(

Tmem121'

0.56017007(

0.0235839(

Smad6'

0.56064096(

0.0000000(

Tmem9'

0.562422561(

0.0000000(

Il20rb'

0.563193529(

0.0000000(

Tmem229b'

0.571979361(

0.0000000(

Wnt6'

0.579395389(

0.0000000(

Ctnnb1'

0.58218307(

0.0235839(

Pth1r'

0.583389597(

0.0000000(

Hspa12b'

0.58409558(

0.0000000(

Il16'

0.587001345(

0.0235839(

Cdc42ep4'

0.591780882(

0.0000000(

Cd97'

0.595509301(

0.0000000(

Jak3'

0.600945954(

0.0000000(

Cd38'

0.603198555(

0.0235839(

Tmem86a'

0.606710437(

0.0000000(

Il18rap'

0.608983054(

0.0335022(

Mmp11'

0.61109568(

0.0000000(

F2r'

0.618226226(

0.0000000(

Cdk19'

0.618693237(

0.0000000(

Il34'

0.619225504(

0.0000000(

Mmp17'

0.62185378(

0.0235839(

Map4k2'

0.623623769(

0.0000000(

Cxcr7'

0.624860007(

0.0000000(

Hmgb3'

0.624875968(

0.0000000(

Tmem173'

0.630723339(

0.0000000(

Six2'

0.631694902(

0.0335022(

Ctnnbip1'

0.63742738(

0.0335022(

Fzd3'

0.639107577(

0.0335022(

Socs2'

0.64750778(

0.0335022(

Hmgn5'

0.648216068(

0.0235839(

Stat2'

0.658367534(

0.0235839(

Cdk2ap2'

0.659850247(

0.0000000(

Met'

0.665855282(

0.0000000(

Sdc4'

0.667108623(

0.0235839(

Tcf3'

0.668449518(

0.0000000(

Trim12'

0.671382361(

0.0335022(

Ilkap'

0.675550964(

0.0335022(

Ccna2'

0.675932308(

0.0235839(

Mapk3'

0.686093157(

0.0000000(

Itga3'

0.688256775(

0.0000000(

Ilf2'

0.689515315(

0.0000000(

Grb10'

0.691704377(

0.0000000(

Tmem110'

0.694182038(

0.0335022(

Itm2c'

0.701841327(

0.0335022(

Cd3eap'

0.704417076(

0.0335022(

Rnf38'

0.704939689(

0.0335022(

Nfkbil2'

0.705436177(

0.0335022(

Map3k12'

0.711453353(

0.0235839(

Mybl2'

0.712538267(

0.0235839(

Cdh8'

0.733437491(

0.0235839(

Cdkn2c'

0.733704766(

0.0235839(

Cd151'

0.734003656(

0.0235839(

Casp2'

0.740118071(

0.0335022(

Cdk10'

0.742337344(

0.0235839(

Atoh8'

0.743759899(

0.0235839(

Cdk2ap1'

0.759884675(

0.0335022(

Tmem179b'

0.790983217(

0.0335022(

Tmem50a'

0.791326965(

0.0335022(

Col9a2'

0.805013649(

0.0235839(

Nos2'

0.808038002(

0.0235839(

Rnf160'

1.182986633(

0.0335022(

Ppara'

1.226111384(

0.0235839(

Rnf103'

1.265119029(

0.0000000(

Abl2'

1.28007608(

0.0235839(

Rnf181'

1.28600617(

0.0335022(

Sod2'

1.288438354(

0.0235839(

Tmem126a'

1.297795278(

0.0335022(

Large'

1.304309246(

0.0335022(

Tmem9b'

1.31584281(

0.0335022(

Rnf170'

1.32832226(

0.0235839(

Lamc1'

1.338170117(

0.0335022(

Crybg3'

1.352691383(

0.0235839(

Rnf19b'

1.377284374(

0.0335022(

Nfix'

1.428716223(

0.0335022(

Cd300lg'

1.487985697(

0.0335022(

Itga2b'

1.518339592(

0.0335022(

Cdc42ep3'

1.520005509(

0.0335022(

Trim23'

1.53151972(

0.0335022(

Cdkn1c'

1.568324588(

0.0235839(

Ccng1'

1.608196047(

0.0335022(

Tmem143'

1.681525317(

0.0000000(

Ar'

1.706092125(

0.0235839(

Mef2d'

1.71779398(

0.0000000(

Tmem51'

1.720317928(

0.0335022(

Myc'

1.727137888(

0.0235839(

Tmem14a'

1.741341997(

0.0000000(

Rnf113a2'

1.748848333(

0.0000000(

Traf3'

1.749643353(

0.0000000(

Rnf128'

1.758049346(

0.0000000(

Col4a5'

1.764058974(

0.0235839(

Hes2'

1.807606777(

0.0335022(

Rnf123'

1.822805482(

0.0335022(

Itga5'

1.824026319(

0.0335022(

Tmem65'

1.844739221(

0.0000000(

Mapk14'

1.877026513(

0.0000000(

Tmem25'

1.877475243(

0.0000000(

Tmem170'

1.890353302(

0.0335022(

Myh7'

1.904240751(

0.0335022(

Hdac9'

2.039262001(

0.0335022(

Cd93'

2.077909983(

0.0335022(

Ang2'

2.119929417(

0.0000000(

Fgf10'

2.136513435(

0.0335022(

Rnf39'

2.142887692(

0.0235839(

Rnf115'

2.185271495(

0.0335022(

Tmem74'

2.189673882(

0.0235839(

Wnt5a'

2.225643565(

0.0000000(

Tgfbr3'

2.243237532(

0.0335022(

Nos1'

2.244337119(

0.0000000(

Tnfaip3'

2.370856078(

0.0335022(

Tmem22'

2.382163203(

0.0335022(

Timp3'

2.389738255(

0.0000000(

Grb14'

2.450073063(

0.0335022(

Icam1'

2.480875001(

0.0000000(

Cd34'

2.502741332(

0.0235839(

Fgf13'

2.522457048(

0.0335022(

Tnfrsf26'

2.526705886(

0.0335022(

Cxcl1'

2.571387986(

0.0335022(

Rnf150'

2.613735241(

0.0000000(

Cxcr6'

2.666414714(

0.0235839(

Hspb1'

2.671942324(

0.0335022(

Igf2bp1'

2.685073278(

0.0335022(

Lama4'

2.694176556(

0.0000000(

Col11a1'

2.81007029(

0.0000000(

Il1rl1'

2.883004106(

0.0235839(

Angpt2'

2.889406621(

0.0000000(

Ctgf'

2.917124574(

0.0000000(

Trim55'

2.943127311(

0.0235839(

Sema3c'

2.962051241(

0.0000000(

Itga11'

3.097928862(

0.0335022(

Smad3'

3.143103306(

0.0335022(

Itgb7'

3.180213711(

0.0000000(

Des'

3.276458094(

0.0335022(

Wnt9a'

3.473096788(

0.0000000(

Tmem132e'

3.637282601(

0.0000000(

Tnfaip6'

3.705210584(

0.0000000(

Crym'

3.715182105(

0.0335022(

Pdgfra'

3.799874363(

0.0000000(

Tmem56'

3.824028259(

0.0000000(

Stac3'

4.096755699(

0.0000000(

Itga2'

4.151801886(

0.0000000(

Myl2'

4.478819312(

0.0000000(

F3'

4.53209333(

0.0000000(

Itgb1bp2'

5.052382919(

0.0000000(

Cxcl2'

5.21740209(

0.0000000(

Il6'

5.883417321(

0.0000000(

Cxcl5'

7.072889179(

0.0335022(

Mmp10'

8.091003904(

0.0000000(

Mmp3'

9.798800323(

0.0000000(

Csf2'

10.41366572(

0.0000000(

Postn'

13.13746879(

0.0000000(

Fgf21'

13.98192167(

0.0000000(

Cxcl3'

15.22060208(

0.0000000(

Myl3'

18.83977572(

0.0000000(

Myog'

20.03261426(

0.0000000(

Mmp13'

28.20644517(

0.0000000(

mmc2.p

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Conditional Loss of Pten in Myogenic Progenitors Leads to Postnatal Skeletal Muscle Hypertrophy but Age-Dependent Exhaustion of Satellite Cells

Article

Full-text available

Nov 2016

Skeletal muscle stem cells (satellite cells [SCs]) are normally maintained in a quiescent (G0) state. Muscle injury not only activates SCs locally, but also alerts SCs in distant uninjured muscles via circulating factors. The resulting GAlert SCs are adapted to regenerative cues and regenerate injured muscles more efficiently, but whether they provide any long-term benefits to SCs is unknown. Here, we report that embryonic myogenic progenitors lacking the phosphatase and tensin homolog (Pten) exhibit enhanced proliferation and differentiation, resulting in muscle hypertrophy but fewer SCs in adult muscles. Interestingly, Pten null SCs are predominantly in the GAlert state, even in the absence of an injury. The GAlert SCs are deficient in self-renewal and subjected to accelerated depletion during regeneration and aging and fail to repair muscle injury in old mice. Our findings demonstrate a key requirement of Pten in G0 entry of SCs and provide functional evidence that prolonged GAlert leads to stem cell depletion and regenerative failure.

Loss of niche-satellite cell interactions in syndecan-3 null mice alters muscle progenitor cell, improving muscle regeneration

Article

Full-text available

Oct 2016

Background The skeletal muscle stem cell niche provides an environment that maintains quiescent satellite cells, required for skeletal muscle homeostasis and regeneration. Syndecan-3, a transmembrane proteoglycan expressed in satellite cells, supports communication with the niche, providing cell interactions and signals to maintain quiescent satellite cells. Results Syndecan-3 ablation unexpectedly improves regeneration in repeatedly injured muscle and in dystrophic mice, accompanied by the persistence of sublaminar and interstitial, proliferating myoblasts. Additionally, muscle aging is improved in syndecan-3 null mice. Since syndecan-3 null myofiber-associated satellite cells downregulate Pax7 and migrate away from the niche more readily than wild type cells, syxndecan-3 appears to regulate satellite cell homeostasis and satellite cell homing to the niche. Conclusions Manipulating syndecan-3 provides a promising target for development of therapies to enhance muscle regeneration in muscular dystrophies and in aged muscle. Electronic supplementary material The online version of this article (doi:10.1186/s13395-016-0104-8) contains supplementary material, which is available to authorized users.

Dynamic changes in heparan sulfate during muscle differentiation and ageing are associated with regulation of myoblast cell fate and FGF2 signalling

Article

Full-text available

Aug 2016

Satellite cells (SCs) are skeletal muscle stem cells residing quiescent around healthy muscle fibres. In response to injury or disease SCs activate, proliferate and eventually differentiate and fuse to one another to form new muscle fibres, or to existing damaged fibres to repair them. The sulfated polysaccharide heparan sulfate (HS) is a highly variable biomolecule known to play key roles in the regulation of cell fate decisions, though the changes that muscle HS undergoes during SC differentiation are unknown. Here we show that the sulfation levels of HS increase during SC differentiation; more specifically, we observe an increase in 6-O and 2-O-sulfation in N-acetylated disaccharides. Interestingly, a specific increase in 6-O sulfation is also observed in the heparanome of ageing muscle, which we show leads to promotion of FGF2 signalling and satellite cell proliferation, suggesting a role for the heparanome dynamics in age-associated loss of quiescence. Addition of HS mimetics to differentiating SC cultures results in differential effects: an oversulfated HS mimetic increases differentiation and inhibits FGF2 signalling, a known major promoter of SC proliferation and inhibitor of differentiation. In contrast, FGF2 signalling is promoted by an N-acetylated HS mimetic, which inhibits differentiation and promotes SC expansion. We conclude that the heparanome of SCs is dynamically regulated during muscle differentiation and ageing, and that such changes might account for some of the phenotypes and signalling events that are associated with these processes.

Elastase levels and activity are increased in dystrophic muscle and impair myoblast cell survival, proliferation and differentiation

Article

Full-text available

May 2016

In Duchenne muscular dystrophy, progressive loss of muscle tissue is accompanied by fibrosis, chronic inflammation and reduced muscle regenerative capacity. Although much is known about the development of fibrosis and chronic inflammation in muscular dystrophy, less is known about how they are mechanistically linked to loss of muscle regenerative capacity. We have developed a proteomics method to discover dystrophy-associated changes in the muscle progenitor cell niche, which identified serine proteases, and especially neutrophil elastase, as candidates. We show that elastase activity is increased in dystrophic (mdx4cv) muscle and impairs myoblast survival in culture. While the effect of elastase on C2C12 cell survival correlates with the kinetics of elastase-mediated degradation of the substrate to which the cells adhere, the effect of elastase on satellite cell-derived primary myoblast growth and differentiation is substrate-independent and even more dramatic than the effect on C2C12 cells, suggesting a detrimental role for elastase on myogenesis in vivo. Additionally, elastase impairs differentiation of both primary and C2C12 myoblasts into myotubes. Our findings evidence the importance of neutrophil-mediated inflammation in muscular dystrophy and indicate elastase-mediated regulation of myoblast behaviour as a potential mechanism underlying loss of regenerative capacity in dystrophic muscle.

Hypoxia inhibits myogenic differentiation through p53-dependent induction of Bhlhe40

Article

Full-text available

Oct 2015

Satellite cells are muscle-resident stem cells capable of self-renewal and differentiation to repair injured muscles. However, muscle injury often leads to an ischemic hypoxia environment that impedes satellite cell differentiation and reduces the efficiency of muscle regeneration. Here we performed microarray analyses and identified the basic helix-loop-helix family transcription factor Bhlhe40 as a candidate mediator of the myogenic inhibitory effect of hypoxia. Bhlhe40 is strongly induced by hypoxia in satellite cell-derived primary myoblasts. Overexpression of Bhlhe40 inhibits Myog expression and mimics the effect of hypoxia on myogenesis. Inhibition of Bhlhe40, conversely, up-regulates Myog expression and promotes myogenic differentiation. Importantly, Bhlhe40 knockdown rescues myogenic differentiation under hypoxia. Mechanistically, Bhlhe40 binds to the proximal E-boxes of the Myog promoter and reduces the binding affinity and transcriptional activity of MyoD on Myog. Interestingly, hypoxia induces Bhlhe40 expression independent of HIF1α but through a novel p53-dependent signaling pathway. Our study establishes a crucial role of Bhlhe40 in mediating the repressive effect of hypoxia on myogenic differentiation and suggests that inhibition of Bhlhe40 or p53 may facilitate muscle regeneration after ischemic injuries.

Intrinsic and extrinsic mechanisms regulating satellite cell function

Article

Full-text available

May 2015
DEVELOPMENT

Muscle stem cells, termed satellite cells, are crucial for skeletal muscle growth and regeneration. In healthy adult muscle, satellite cells are quiescent but poised for activation. During muscle regeneration, activated satellite cells transiently re-enter the cell cycle to proliferate and subsequently exit the cell cycle to differentiate or self-renew. Recent studies have demonstrated that satellite cells are heterogeneous and that subpopulations of satellite stem cells are able to perform asymmetric divisions to generate myogenic progenitors or symmetric divisions to expand the satellite cell pool. Thus, a complex balance between extrinsic cues and intrinsic regulatory mechanisms is needed to tightly control satellite cell cycle progression and cell fate determination. Defects in satellite cell regulation or in their niche, as observed in degenerative conditions such as aging, can impair muscle regeneration. Here, we review recent discoveries of the intrinsic and extrinsic factors that regulate satellite cell behaviour in regenerating and degenerating muscles. © 2015. Published by The Company of Biologists Ltd.

p53 Suppresses muscle differentiation at the myogenin step in response to genotoxic stress

Article

Full-text available

Dec 2014
CELL DEATH DIFFER

Acute muscle injury and physiological stress from chronic muscle diseases and aging lead to impairment of skeletal muscle function. This raises the question of whether p53, a cellular stress sensor, regulates muscle tissue repair under stress conditions. By investigating muscle differentiation in the presence of genotoxic stress, we discovered that p53 binds directly to the myogenin promoter and represses transcription of myogenin, a member of the MyoD family of transcription factors that plays a critical role in driving terminal muscle differentiation. This reduction of myogenin protein is observed in G1-arrested cells and leads to decreased expression of late but not early differentiation markers. In response to acute genotoxic stress, p53-mediated repression of myogenin reduces post-mitotic nuclear abnormalities in terminally differentiated cells. This study reveals a mechanistic link previously unknown between p53 and muscle differentiation, and suggests new avenues for managing p53-mediated stress responses in chronic muscle diseases or during muscle aging.Cell Death and Differentiation advance online publication, 12 December 2014; doi:10.1038/cdd.2014.189.

The Muscle Stem Cell Niche in Health and Disease

Chapter

Jan 2017
Curr Top Dev Biol

The regulation of stem cells that maintain and regenerate postnatal tissues depends on extrinsic signals originating from their microenvironment, commonly referred to as the stem cell niche. Complex higher-order regulatory interrelationships with the tissue and factors in the systemic circulation are integrated and propagated to the stem cells through the niche. The stem cell niche in skeletal muscle tissue is both a paradigm for a structurally and functionally relatively static niche that maintains stem cell quiescence during tissue homeostasis, and a highly dynamic regenerative niche that is subject to extensive structural remodeling and a flux of different support cell populations. Conditions ranging from aging to chronically degenerative skeletal muscle diseases affect the composition of the niche and thereby impair the regenerative potential of muscle stem cells. A holistic and integrative understanding of the extrinsic mechanisms regulating muscle stem cells in health and disease in a broad systemic context will be imperative for the identification of regulatory hubs in the niche interactome that can be targeted to maintain, restore, or enhance the regenerative capacity of muscle tissue. Here, we review the microenvironmental regulation of muscle stem cells, summarize how niche dysfunction can contribute to disease, and discuss emerging therapeutic implications.

Role of AhR in positive regulation of cell proliferation and survival

Article

Aug 2016
CELL PROLIFERAT

The aryl hydrocarbon receptor (AhR) is an important nuclear transcription factor that is best known for mediating toxic responses by adjusting numbers of metabolism-related enzymes, including CYP1A1 and CYP1B1. Previous findings have revealed that, in addition to negatively regulating cell proliferation and survival, AhR may also positively regulate these pathways. Here, we review these findings and summarize distinct mechanisms by which AhR promotes cell proliferation and survival, including modulation of receptor expression, growth factor signalling and apoptosis, regulating the cell cycle and promoting cytokine expression. This review will aid better understanding the role of AhR in positive regulation of cell proliferation and survival.

Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle

Article

Jan 1977

The Satellite Cell Niche Regulates the Balance between Myoblast Differentiation and Self-Renewal via p53

Article

February 2018

Valentina Flamini · Rachel S. Ghadiali · Philipp Antczak · Amy Rothwell · Addolorata Pisconti

Document S2. Article plus Supplemental Information

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