Asymmetric Self-Renewal and Commitment
of Satellite Stem Cells in Muscle
Shihuan Kuang,1 Kazuki Kuroda,1 Fabien Le Grand,1 and Michael A. Rudnicki1,2,*
1
The Sprott Center for Stem Cell Research, Ottawa Health Research Institute, Molecular Medicine Program, 501 Smyth Road,
Ottawa, ON K1H 8L6, Canada
2
University of Ottawa, Department of Medicine, 501 Smyth Road, Ottawa, ON K1H 8L6, Canada
*Correspondence: mrudnicki@ohri.ca
DOI 10.1016/j.cell.2007.03.044
SUMMARY
Satellite cells play a central role in mediating the
growth and regeneration of skeletal muscle.
However, whether satellite cells are stem cells,
committed progenitors, or dedifferentiated
myoblasts has remained unclear. Using Myf5-
Cre and ROSA26-YFP Cre-reporter alleles,
we observed that in vivo 10% of sublaminar
Pax7-expressing satellite cells have never ex-
pressed Myf5. Moreover, we found that Pax7+/
Myf5 satellite cells gave rise to Pax7+/Myf5+
satellite cells through apical-basal oriented
divisions that asymmetrically generated a basal
Pax7+/Myf5 and an apical Pax7+/Myf5+ cells.
Prospective isolation and transplantation into
muscle revealed that whereas Pax7+/Myf5+
cells exhibited precocious differentiation, Pax7+/
Myf5 cells extensively contributed to the satel-
lite cell reservoir throughout the injected mus-
cle. Therefore, we conclude that satellite cells
are a heterogeneous population composed of
stem cells and committed progenitors. These
results provide critical insights into satellite
cell biology and open new avenues for thera-
peutic treatment of neuromuscular diseases.
INTRODUCTION
Satellite cells reside in a niche beneath the basal lamina
closely juxtaposed to the muscle fiber, and are responsi-
ble for the growth, maintenance, and repair of postnatal
skeletal muscle. Satellite cells are normally mitotically qui-
escent but are activated (i.e., enter the cell cycle) in re-
sponse to stress induced by weight bearing or by trauma
such as injury. The descendants of activated satellite cells,
called myogenic precursor cells, undergo multiple rounds
of division prior to terminal differentiation and fusion to
form multinucleated myofibers. Activated satellite cells
also generate progeny that restore the pool of quiescent
satellite cells. However, the molecular mechanisms regu-
lating satellite cell self-renewal and differentiation remain
poorly understood (McKinnell et al., 2005).
Early experiments using quail-chick chimeras sug-
gested that satellite cells were derived from the somite
(Armand et al., 1983). Recent experiments support
this work and indicate that a significant proportion of
the progenitors of satellite cells originate in embryonic
somites as Pax3/Pax7-expressing cells (Ben-Yair and
Kalcheim, 2005; Gros et al., 2005; Kassar-Duchossoy
et al., 2005; Relaix et al., 2005; Schienda et al., 2006). In
addition, satellite cells may also be derived from cells
associated with the embryonic vasculature including
the dorsal aorta (De Angelis et al., 1999) and from other
adult stem cells during regeneration (Asakura et al.,
2002; LaBarge and Blau, 2002; Polesskaya et al., 2003).
However, whether satellite cells are stem cells, committed
progenitors, or dedifferentiated myoblasts remains
unresolved.
Satellite cells are widely considered to be a homoge-
neous population of committed muscle progenitors (Bis-
choff, 1994). However, several studies have raised the
possibility that the satellite cell compartment may be a het-
erogeneous population. Radioisotope labeling of growing
rat muscle revealed that satellite cells are a mixture of
80% fast-cycling cells and 20% slow-cycling ‘‘reserve
cells’’ (Schultz, 1996). Examination of the expression of
satellite cell markers CD34, M-cadherin, and Myf5-nLacZ
in freshly prepared myofibers suggested that a subpopula-
tion of satellite cells exhibits heterogeneous expression of
these markers (Beauchamp et al., 2000). However, the
molecular identity of any potential subpopulations has
not been defined, and the prospective isolation and char-
acterization of these cells has not been achieved.
Transplantation of the cultured primary myoblasts into
regenerating muscle typically results in extensive loss of
the transplanted cells, terminal differentiation of the
surviving cells, and virtually no contribution to the satellite
cell compartment (Beauchamp et al., 1999; El Fahime
et al., 2003; Fan et al., 1996; Hodgetts et al., 2000; Qu
et al., 1998; Rando and Blau, 1994). By contrast, experi-
ments involving transplant of intact fibers carrying satellite
cells (Collins et al., 2005) or fresh isolated satellite cells
(Montarras et al., 2005) have suggested that at least
some portion of satellite cells have the capacity to
Cell 129, 999–1010, June 1, 2007 ª2007 Elsevier Inc. 999
repopulate the satellite cell compartment as well as exten-
sively contribute to regenerating muscle.
In the present study, we report the existence of hierar-
chical subpopulations of satellite cells that are distinct in
phenotype and function. We document that sublaminar
satellite cells expressing Pax7 are heterogeneous based
on Myf5 expression. Satellite cells that express Pax7 but
not Myf5 give rise to Myf5-expressing cells through subla-
minar cell divisions in a basal-apical orientation. Finally,
we observe that Pax7+/Myf5 satellite cells are capable
of efficiently contributing to the satellite cell reservoir
following prospective isolation and transplantation into
Pax7 / muscle. Together, these experiments demon-
strate that satellite cells are a heterogeneous population
composed of stem cells and committed myogenic
progenitors.

RESULTS

Satellite Cells Are Heterogeneous

by Myf5 Expression
Satellite cells uniformly express Pax7 (Seale et al., 2000)
and have been suggested to express the Myf5-nLacZ
knockin allele in the quiescent sublaminar state (Beau-
champ et al., 2000). We hypothesized that Myf5 transcrip-
tion occurs in satellite cells that had undergone commit-
ment to the myogenic lineage. We reasoned that if we
could detect satellite cells that had not expressed Myf5,
these would represent a candidate stem cell of the satellite
cell compartment.
We first examined whether all satellite cells expressing
Pax7 also express Myf5 in single myofiber preparations
fixed immediately following isolation from extensor digito-
rum longus (EDL) muscles of Myf5-nLacZ mice. Immuno-
histological analysis revealed that the majority of satellite
cells contained readily detectable levels of Pax7 and
b-Gal proteins. Notably, 13 ± 4% (n = 3 mice, > 100 cells/
mouse) of Pax7+ satellite cells did not contain detectable
levels of b-Gal, indicating that Myf5 was not uniformly
expressed (Figure 1A).
To investigate whether the Pax7+/b-Gal satellite cells
reflect a transient downregulation of Myf5 or represent
a distinct population that never expressed Myf5, we em-
ployed the Cre-LoxP system to genetically mark cells
that have expressed Myf5. We generated heterozygous Figure 1. Satellite Cells Are a Heterogeneous Population
mice carrying a Myf5-Cre allele (Tallquist et al., 2000) Based on Myf5 Expression
and a Cre-dependent YFP reporter knocked into the ubiq- (A) 87% of Pax7+ satellite cells expressed b-Gal (left column), and 13%
uitously expressed ROSA26 locus (Srinivas et al., 2001; of Pax7+ cells were b-Gal (right column, n = 3 mice) on single fibers
isolated from Myf5-nLacZ mice.
Figure S1A). In the Myf5-Cre/ROSA-YFP mice, any satel-
(B) 90% of Pax7+ cells expressed YFP (left column), and 10% of Pax7+
lite cells that have once expressed Myf5-Cre should cells were YFP (right column, n = 18 mice) on single fibers isolated
express YFP irreversibly. Conversely, any cells that are from Myf5-Cre/ROSA-YFP mice.
YFP should have never expressed Myf5-Cre in the past. (C and D) Total number of Pax7+ satellite cells per EDL myofiber and
Analysis of myofibers isolated from Myf5-Cre/ROSA- percentage of Pax7+/YFP cells at different ages (n = 3, 4, 6, and 4
YFP EDL muscle demonstrated that 90 ± 2% of satellite mice for 0.5-, 1-, 2-, and 6-month-old mice, respectively). Error bars
indicate standard error of mean (SEM).
cells expressing Pax7 also expressed YFP. Importantly,
(E) Sublaminar localization of Pax7+/YFP (left) and Pax7+/YFP+ (right)
10 ± 2% (n = 18 mice, >100 cells/mouse) of Pax7+ satellite cells.
cells did not contain detectable levels of YFP, confirming Scale bars are 10 mm in (A) and (B) and 25 mm in (E).
that these cells never expressed Myf5 (Figure 1B). The

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relative proportion of Pax7+/YFP satellite cells persisted
in muscle in mice up to 6 months old (Figures 1C and 1D).
Similar proportions of b-Gal-positive and -negative
satellite cells were observed when Myf5-Cre/ROSA26R3
reporter mice that express b-Gal only in the presence of
Myf5-Cre were examined (Figure S1B). By contrast, all
Pax7+ satellite cells were also YFP+ in Pax3-Cre/ROSA-
YFP mice (n = 4 mice, data not shown), confirming the
efficacy of the Cre-LoxP system and the notion that
satellite cells are derived from embryonic Pax3+/Pax7+
progenitors.
To ask whether the Pax7+/Myf5 represent newborn
satellite cells that have never undergone activation, we
treated muscles with cardiotoxin (CTX) to induce the acti-
vation of satellite cells and muscle regeneration. We ob-
served substantial numbers of both Pax7+/YFP and
Pax7+/b-Gal cells in regenerating Myf5-Cre/ROSA-YFP
and Myf5-nLacZ muscles, respectively (Figure S2). Many
of the Pax7+/b-Gal cells had incorporated BrdU, indicat-
ing they were undergoing DNA synthesis and hence were
progressing through the cell cycle (Figure S2B). Therefore,
the lack of Myf5 expression in satellite cells does not
equate to quiescence.
To confirm the satellite cell identity of the Pax7+/YFP
cells, we triple labeled Myf5-Cre/ROSA-YFP muscle sec-
tions with antibodies to Pax7, YFP, and laminin. All Pax7+/
YFP cells were located beneath the basal lamina in a sat-
ellite cell position (Figure 1E). Furthermore, Pax7+/YFP
and Pax7+/b-Gal cells were found to express typical sat-
ellite cell markers including CD34, M-Cad, Syn4, and
NCAM (Figure S3). These data confirm that sublaminar
satellite cells are a heterogeneous population on the basis
of Myf5 expression.
Prospective Isolation of Pax7+/YFP and Pax7+/YFP+
Satellite Cells
To further characterize Pax7+/YFP and Pax7+/YFP+ sat-
ellite cells, we developed a method to prospectively iso-
late the different satellite cell subpopulations based on
the previously reported approach using fluorescence-
activated cell sorting (FACS) with antibody reactive with
the cell-surface protein a7-integrin (int; Blanco-Bose
et al., 2001). Immunostaining of isolated myofibers con-
firmed that a7- and its dimerization partner b1-int were
expressed on both YFP and YFP+ satellite cells (Figures
S4A and S4B).
Initially, we employed positive selection for a7-int, to-
gether with negative selection (Lin , for Sca1, CD31,
CD45). FACS analysis of mononuclear cells from Myf5-
Cre/ROSA-YFP muscle revealed that the a7-int+ fraction
contained both YFP+ and YFP cells (Figure S4B, right
column). RT-PCR analysis indicated that mRNA for
Pax7, Myf5, and Cre were only present in the a7-int+ frac-
tion (Figure S4C), suggesting that the a7-int+ fraction con-
tained all the Pax7+ satellite cells. However, only 20 ± 5%
(n = 4) FACS-isolated a7-int+Lin YFP cells were Pax7+;
therefore, we introduced b1-int as a second positive-
selection marker and further narrowed the gating based
on the side-scatter (SSC) range of satellite cells.
Next, we isolated the YFP+/a7b1-int+/Lin and the
YFP /a7b1-int+/Lin (termed YFP+ and YFP hereafter)
cells by FACS (Figure 2A). Immunostaining of freshly
sorted cells revealed that 90 ± 1% (n = 3) of the YFP+ cells
expressed detectable levels of Pax7, whereas 67 ± 9%
(n = 3) of YFP cells expressed Pax7 (Figure 2B). There-
fore, we established an approach for the prospective iso-
lation of the different satellite cell subpopulations with
a high degree of purity.
RT-PCR analysis of YFP+ cells confirmed expression of
Pax7, Myf5, Cre, and YFP mRNAs. By contrast, YFP cells
expressed similar levels of Pax7 but did not express Myf5,
Cre, or YFP (Figure 2C). Importantly, Myf5 protein was
readily detectable in the FACS-purified YFP+ but not in
the YFP cells (Figure 2D). Together, these data directly
confirmed that the YFP cells did not express Myf5 at
either mRNA or protein level.
Pax7+/Myf5 Cells Give Rise to Pax7+/Myf5+ Cells
To investigate the developmental origin and relationship
between the Pax7+/Myf5 and Pax7+/Myf5+ cells, we ex-
amined the satellite cell progenitors in the somites and
limb buds of E11.5 embryos. Interestingly, about 10% of
Pax7-expressing cells in the dermamyotome and migrat-
ing progenitors near the limb bud were YFP among the
large majority of YFP+ progenitors (Figure S5A and S5B).
To analyze the clonal growth of satellite cells on isolated
myofibers in vitro, individual myofibers were isolated from
Myf5-Cre/ROSA-YFP EDL muscle and cultured in sus-
pension under growth conditions for 1–3 days. Typically,
satellite cells undergo their first cell division at around
24 hr and form 4–8 cell aggregates within 3 days. Live-
imaging analysis confirmed that these aggregates were
of clonal origin (not shown). After 3 days of culture almost
all clones uniformly expressed YFP, but the levels of Pax7
within each clone were variable (Figure 3A). Importantly,
nearly 10% of the clones contained both Pax7+/YFP
and Pax7+/YFP+ cells at day 3 (Figure 3B) and day 2
(Figure 3C; 6 out of 70 clones from 4 experiments). Since a
colony of mixed YFP+ and YFP cells can only be derived
from a common YFP progenitor, these results indicate
that Pax7+/Myf5 cells can form additional Pax7+/Myf5
satellite cells as well as give rise to Pax7+/Myf5+ satellite
cells.
To confirm that Pax7+/Myf5 cells give rise to Pax7+/
Myf5+ cells, we conducted time-lapse imaging analysis
of satellite cell division on cultured Myf5-Cre/ROSA-YFP
myofibers. We observed that single YFP satellite cells di-
rectly gave rise to one YFP and one YFP+ cell (Figure 4;
Movie S1). Furthermore, we examined expression of
MyoD, an established marker for myogenic commitment
and differentiation. Notably, Pax7+/YFP cells did not ex-
press MyoD (Figure S5C), consistent with the hypothesis
that Pax7+/YFP cells have yet to undergo myogenic
commitment.
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Figure 2. Isolation of Pax7+/YFP and Pax7+/YFP+ Cells from Myf5-Cre/ROSA-YFP Muscle
(A) Top panels: FACS profile of cells from control mice with no staining. Bottom panels: FACS profile of Myf5-Cre/ROSA-YFP cells stained with PE
conjugated to Sca-1, CD31, CD45 (PE-Lin), a7-integrin, and b1-integrin. The gates used to prospectively isolate a7-int+/b1-int+/Lin /YFP and
a7-int+/b1-int+/Lin /YFP+ cells are shown by boxed or circled areas. The bottom right panel shows the a7-int and YFP profile after gating.
(B) Pax7 expression in FACS-isolated YFP+ (upper panels) and YFP (lower panels) cells.
(C) RT-PCR analysis of sorted cells showing the absence of Myf5, Cre, and YFP expression in the YFP cells. Myoblasts indicates cultured primary
myoblasts isolated from Myf5-Cre/ROSA-YFP mice. NTC indicates no template control.
(D) Absence of Myf5 protein in FACS-isolated YFP cells.
Scale bars are 25 mm.

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To examine satellite cell divisions within their natural
niche, we developed an approach to analyze satellite
cell proliferation in vivo. Typically, induction of muscle
regeneration with CTX results in death of myofibers and
massive immunoinfiltration, making it technically difficult
to isolate viable myofibers. Therefore, we instead isolated
fibers from the adjacent EDL muscle 4–5 days following
CTX injection into the tibialis anterior (TA) muscle. We
found that this approach allowed us to isolate viable myo-
fibers containing numerous satellite cells that were under-
going cell division in vivo (Figure 3D).
Numerous doublets of sister cells (double arrows in
Figure 3D) were observed beneath the basal lamina on vi-
able regenerating myofibers, which contained long arrays
of centrally located nuclei (Figure 3D). The close physical
proximity of the cells within each doublet, the expression
of proliferation markers in both cells (Ki67 and phospho-
H3; Figures 3G and 3H), and their sublaminar localization
within the same satellite cell niche (Refer to Figures 5C and
5D) evidence the clonal origin of these doublet sister cells.
The vast majority of the doublets in Myf5-Cre/ROSA-YFP
mice displayed identical expression of Pax7 and YFP
(Figure 3E). Nonetheless, we identified doublets of prolif-
erating cells with one Pax7+/YFP cell and one Pax7+/
YFP+ cell (Figure 3F), presumably derived from a common
Pax7+/YFP progenitor.
Altogether, our in vivo and in vitro analyses support
the notion that Pax7+/Myf5 cells give rise to both
Figure 3. Clonal Analysis Implies a Devel-
opmental Relationship between Pax7+/
Myf5 and Pax7+/Myf5+ Satellite Cells
(A) Satellite-cell-derived clonal clusters from
Myf5-Cre/ROSA-YFP reporter mice after 3
days in suspended culture. Shown is a clonal
cluster of cells that uniformly express YFP.
(B) Shown is a clonal cluster containing both
Pax7+/YFP+ (arrow) and Pax7+/YFP (arrow-
head) cells.
(C) A couplet of sister cells with one Pax7+/
YFP cell (arrow head) and one Pax7+/YFP+
cell (arrow) after 2 days in culture.
(D) Extensive in vivo proliferation of satellite
cells on a regenerating EDL myofiber 4 days
post CTX injection. The majority of satellite
cells have undergone mitosis as indicated by
the presence of two adjacent Pax7+ nuclei
(double arrows) within a single satellite cell
niche.
(E) Couplet of identical sister cells that were
both Pax7+/YFP+.
(F) Couplet of sister cells with one Pax7+/YFP
cell (arrowhead) and one Pax7+/YFP+ cell (ar-
row).
(G) Satellite cell couplets expressed the meta-
phase mitosis marker phosphorylated his-
tone-H3 (H3-P).
(H) Satellite cell couplets expressed the prolif-
eration marker Ki67. b-Gal labeling denotes
Myf5-nLacZ expression.
Scale bars are 20 mm in (A), (B), and (D) and
10 mm in (C), (E), (F), (G), and (H).
Pax7+/Myf5 and Pax7+/Myf5+ cells and therefore repre-
sent a reservoir that maintains a hierarchical composition
of the satellite cell compartment.
Apical-Basal Location Determines Self-Renewal
versus Differentiation
We next investigated whether the fate of daughter cells is
determined by their relative orientation within the satellite
cell compartment. Theoretically, sister cells can either be
in a planar orientation where both cells remain in direct
contact with the basal lamina and host myofiber (Fig-
ure 5A) or in an apical-basal orientation where one daugh-
ter cell is pushed toward the basal lamina and the other
cell apically toward the host myofiber (Figure 5B).
Examination of myofibers isolated from regenerating
EDL muscle revealed doublets of sister satellite cells be-
neath the basal lamina in both planar and apical-basal ori-
entations (Figures 5C and 5D). Doublets of cells within the
satellite cell niche were predominantly planar in orienta-
tion (91%, n = 248 pairs). Planar divisions were observed
to give rise almost exclusively (92%, n = 89 pairs) to iden-
tical daughter cells that were either both Pax7+/Myf5 or
both Pax7+/Myf5+, as reported by Myf5-nLacZ and
ROSA-YFP reporter mice (Figures 5A, 5E, and 5G).
Strikingly, we observed apical-basal oriented doublets
where the sister cells assumed asymmetric cell fates
(82%, n = 38 pairs; Figure 5B). The majority of apical-basal
oriented doublets contained a Pax7+/Myf5 cell against
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Figure 4. Pax7+/Myf5 Satellite Cells Give Rise to Pax7+/
Myf5+ Satellite Cells
Time-lapse image analysis showing a YFP satellite cell (arrowhead at
T = 0) give rise to YFP+ (arrows) and YFP (arrowheads) daughter cells.
EDL myofibers isolated from Myf5-Cre/ROSA-YFP reporter mouse
were plated on Matrigel, and satellite cell growth was monitored with
a live imaging system. Cell division occurred at approximately 0 min
(around 36 hr in real time), and YFP expression became apparent in
the lower daughter cell at 240 min. Scale bar is 25 mm.

the basal surface and a Pax7+/Myf5+ cell located on the

apical side against the muscle fiber (Figures 5F, 5H, and
S6–S7). In addition, we observed rare events where the
basal cell expressed Pax7 and Myf5 and the apical cell
had downregulated Pax7 and was presumably undergo-
ing terminal differentiation (Figure S7).
Taken together, these results demonstrate that satellite
cell niche plays an important role in the maintenance of
stem cell identity of newly divided daughter cells. The
daughter cell attached to the basal lamina remains
Myf5 , whereas the daughter that loses contact with the
basal lamina upregulates Myf5 and becomes a committed
myogenic cell.
Notch Regulation of Asymmetric Self-Renewal
To further explore the signaling mechanism underlying the
apical-basal asymmetric self-renewal of satellite stem
cells, we conducted gene-expression analysis of FACS-
purified YFP+ and YFP satellite cells. Importantly, YFP+
cells expressed the Notch ligand Delta-1 whereas YFP
cells did not (Figure 6A). Moreover, YFP cells but not
YFP+ cells expressed high levels of Notch-3 (Figure 6A).
By contrast, Notch-1 and Notch-2 receptors and the
Notch signaling inhibitor Numb were equally expressed
in both cell types.
Immunostaining of isolated myofibers revealed that
Delta-1 was expressed at high levels in Pax7+/Myf5+ sat-
ellite cells but low levels in Pax7+/Myf5 cells (Figure 6B).
In cultured satellite cells, Delta-1 expression was upregu-
lated in Myf5+ but not in Myf5 cells (Figure 6C). In newly
Figure 5. Orientation of Cell Division within the Satellite Cell
Niche Determines Cell Fate
Regenerating EDL myofibers were isolated from Myf5-Cre/ROSA-YFP
(C–F) or Myf5-nLacZ (G–H) reporter mice and fixed immediately for
labeling of Pax7 and YFP or b-Gal. Myofibers were horizontally ori-
ented in all panels.
(A and B) Schematics of planar (A) and apical-basal (B) oriented sister
cells and the corresponding frequency of symmetric (Sym) versus
asymmetric (Asym) expression of Myf5 (based on YFP or b-Gal). Sat-
ellite cells (red and green cells) are located beneath the basal lamina
(green) and are adjacent to the myofiber (brown).
(C and D) Newly formed sister cells (double arrows) were both located
under the basal lamina (Lam) but were separated from myofiber by
M-cadherin (M-Cad) regardless of their relative orientation (C, planar;
D, apical-basal).
(E and F) Examples of planar (double arrows) and apical-basal (arrow
and arrowhead)-oriented Pax7+ sister cells that displayed symmetric
and asymmetric YFP expression, respectively.
(G and H) Examples of planar (double arrows in G) and apical-basal
(arrow and arrowhead in H)-oriented Pax7+ sister cells that displayed
symmetric and asymmetric b-Gal expression, respectively. Centrally
located myonuclei indicated the regenerating status of the myofibers.
Scale bar is 12.5 mm.

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divided cells, high-level Delta-1 expression was observed
specifically in Myf5-expressing cells (Figures 6C–6D).
To directly test the role of Notch signaling in satellite cell
self-renewal, we treated proliferating satellite cells on
freshly isolated myofibers with DAPT, an inhibitor of
g-secretase that is necessary for Notch signaling. DAPT
treatment significantly reduced the total number of cells
after 3 days of culture. Importantly, Pax7+/MyoD satellite
cells were markedly reduced in number following DAPT
treatment. By contrast, the percentage of Pax7 /MyoD+
cells was significantly increased (Figure 6E). Together,
these results strongly suggest that Notch signaling plays
a critical role in the maintenance of Pax7+/YFP satellite
cells in an undifferentiated stem cell state.
Transplanted Pax7+/Myf5 Cells Extensively
Contribute to the Satellite Cell Compartment
Our experiments provided compelling evidence that satel-
lite cells are a hierarchal population composed of Pax7+/
Myf5 and Pax7+/Myf5+ satellite cells. To examine the
biological function of these two subpopulations of satellite
cells, we transplanted FACS-purified YFP+ and YFP sat-
ellite cells from Myf5-Cre/ROSA-YFP mice into Pax7 /
muscle and assessed: (1) their ability to terminally dif-
ferentiate and (2) their contribution to the satellite cell
compartment. Notably, Pax7 / mice lack functional sat-
ellite cells (Kuang et al., 2006; Seale et al., 2000). There-
Figure 6. A Role for Notch Signaling in
Self-Renewal of Pax7+/Myf5 Satellite
Cells
(A) RT-PCR analysis of FACS-purified YFP
and YFP+ satellite cells from Myf5-Cre/ROSA-
YFP muscles.
(B) Fresh isolated Myf5-nLacZ myofiber show-
ing Pax7+/b-Gal (arrowhead) and Pax7+/
b-Gal+ (arrow) satellite cells (B1) and their cor-
responding expressions of Delta-1 (B2).
(C) Satellite-cell-derived cultures showing dif-
ferential expression of Delta-1 in Pax7+/Myf5
(arrowhead) and Pax7+/Myf5+ (arrows) cells.
(D) High levels of Delta-1 were observed in
Pax7+/Myf5+ cells and low levels of Delta-1 in
Pax7+/Myf5 cells in newly formed sister dou-
blets.
(E) Abrogation of satellite cell self-renewal by
the g-secretase inhibitor DAPT. (E1–E2) Ex-
pression of the self-renewing marker Pax7
and myogenic differentiation marker MyoD in
colonies of satellite cell progenies in control
(E1) and DAPT (E2)-treated (3 day) myofiber
cultures. Notice the lack of Pax7 expression
in DAPT-treated cells. (E3–E5) Quantification
of cell number (E3) and the relative proportion
of Pax7+ (E4) and MyoD+ (E5) cells. Numbers
in the graphs indicate n. Error bars indicate
SEM.
Scale bar is 15 mm.
fore, the transplanted cells can readily colonize the unoc-
cupied satellite cell niche.
Three weeks after transplantation (900–10,000 cells/TA
muscle), we observed donor-derived cells as indicated by
the expression of YFP or Pax7 (n = 6 animals in each
group). Both YFP+ and YFP donor cells were capable
of undergoing terminal differentiation as revealed by the
presence YFP+ myofibers (Figures 7A and 7B). Transplan-
tation of YFP+ cells typically gave rise to clusters of 20–60
YFP+ myofibers at the injection site, whereas transplanted
YFP cells formed 0–22 YFP+ fibers. Furthermore, we ob-
served in half of the transplanted muscles (n = 6) a massive
accumulation of cells (DAPI nuclei in Figures 7A and 7E)
surrounding the newly formed YFP+ myofibers, suggest-
ing a host immune reaction against donor cells. Little or
no immune reaction was observed in muscle transplanted
with YFP cells (Figures 7B, 7D, and 7F). These data indi-
cate that YFP+ donor cells readily differentiated following
transplantation.
Transplanted YFP cells efficiently gave rise to both
Pax7+/YFP and Pax7+/YFP+ satellite cells (94 ± 32 cells/
section/10000 Pax7+ cells injected or 13 ± 3 cells/cross-
section, n = 6 mice). Many YFP satellite cells were
observed to have migrated large distances into the host
muscle tissue, and were located in sublaminar positions
(Figures 7D and 7F). By contrast, transplanted YFP+ cells
only occasionally gave rise to Pax7+/YFP+ satellite cells
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Figure 7. Transplanted Pax7+/Myf5 Cells Extensively Con-
tribute to the Satellite Cell Compartment
TA muscles of Pax7 / mice were engrafted with freshly sorted YFP+
and YFP cells from Myf5-Cre/ROSA-YFP mice. Three weeks later,
transplanted muscles were examined for the expression of YFP and
Pax7.
(A and B) Transplanted YFP+ (A) and YFP (B) satellite cells both differ-
entiated into YFP+ myofibers; however, YFP satellite cells also gave
rise to several Pax7+/YFP cells associated with host (YFP ) myo-
fibers.
(C) Transplanted YFP+ cells gave rise to a satellite cell (arrowhead) that
was associated with a YFP+ fiber.
(D) Transplanted YFP satellite cells gave rise to several Pax7+/YFP
(arrows) satellite cells that migrated onto host fibers (YFP ) and lo-
cated beneath the basal lamina (white staining). Notice that one
Pax7+/YFP+ (arrowhead) cell closely associated with a YFP+ myofiber.
(E) TA muscle transplanted with YFP+ satellite cells showing numerous
nuclei surrounding a YFP+ myofiber and two interstitial Pax7+/YFP+
cells (arrowheads) indicative of host immune rejection.
(F) TA muscle transplanted with YFP satellite cells showing several
Pax7+/YFP cells (arrows) and two YFP+ cells (arrowheads) associ-
ated with host (YFP ) myofibers and minimal immune rejection.
(G and H) Number of donor-derived satellite cells/TA cross-section af-
ter transplantation of YFP+ (G) and YFP (H) satellite cells (n = 6 mice).
Error bars indicate SEM.
Scale bars are 25 mm in (A) and (B) and 12.5 mm in (C)–(F).
(12 ± 7 cells/section/10000 Pax7+ cells injected or 4 ± 2
cells/cross-section, n = 6 mice), and these were virtually
all associated with the new YFP+ myofibers formed at
the injection site (Figure 7C). Rare mononuclear YFP+ cells
were also observed in interstitial spaces between fibers
(Figure 7E).
1006 Cell 129, 999–1010, June 1, 2007 ª2007 Elsevier Inc.
Finally, we assessed the relative repopulating efficiency
of YFP and YFP+ cells after engrafting myofibers isolated
from Myf5-Cre/ROSA-YFP mice into Pax7 / recipients.
We hypothesized that the relative ratio of reconstituted
YFP and YFP+ satellite cells should remain unchanged
or decline if both populations repopulate equally well
(given that some YFP can turn into YFP+ cells). Ten
freshly isolated EDL myofibers containing 7% YFP and
93% YFP+ satellite cells were grafted into the TA muscle
of Pax7 / mice. Four weeks later, the satellite cells de-
rived from the grafted fibers differentiated into bundles
of 20–100 YFP+ myofibers and also gave rise to numerous
Pax7+ satellite cells (14.7 ± 2.5 cells/cross-section, three
mice; Figure S8). Importantly, 24 ± 8% of engrafted
Pax7+ satellite cells remained YFP . Most of these YFP
satellite cells had migrated away from the injection site
and infiltrated the surrounding muscle tissue to repopulate
the host muscle. By contrast, almost all of the YFP+ satel-
lite cells remained at the injection site (Figure S8). Con-
sistent with the notion that Pax7+/Myf5 satellite cells
represent a stem cell population within the satellite cell
compartment, YFP satellite cells rapidly expanded in
number as they repopulated the vacant satellite cell niche
in Pax7 / muscle.
Together, these data indicate that transplanted Pax7+/
Myf5+ satellite cells preferentially undergo terminal differ-
entiation, whereas transplanted Pax7+/Myf5 satellite
cells extensively contribute to the satellite compartment
and give rise to both classes of satellite cells. Therefore,
we conclude that the Pax7+/Myf5 satellite cells represent
a novel population of stem cells that actively maintain the
homeostatic composition of stem cells and committed
progenitors within the satellite cell niche.
DISCUSSION
Previously, our laboratory found that satellite cells uni-
formly express the transcription factor Pax7 (Seale et al.,
2000). Using a Myf5-Cre knockin allele and a ROSA-YFP
Cre reporter, we observed that in vivo about 10% of satel-
lite cells express Pax7 but have never expressed Myf5.
Moreover, we found that Pax7+/Myf5 satellite cells give
rise to Pax7+/Myf5+ satellite cells through basal-apical
oriented divisions within the satellite cell niche. Prospec-
tive isolation followed by transplantation confirmed that
Pax7+/Myf5+ satellite cells preferentially differentiate,
whereas Pax7+/Myf5 satellite cells extensively contrib-
ute to the satellite cell compartment. Therefore, anatomi-
cal and functional experiments provide compelling evi-
dence that the satellite cell population is composed of
hierarchal subpopulations of stem cells (Pax7+/Myf5 )
and committed myogenic progenitors (Pax7+/Myf5+).
This discovery provides a new paradigm that redefines
satellite cell biology and opens new doors for therapeutic
intervention for the treatment of devastating neuromus-
cular diseases such as Duchenne muscular dystrophy
(DMD).
 Transplantation experiments have raised the possibility
that only a portion of satellite cells exhibit repopulating ac-
tivity. Unlike cultured primary myoblasts (Collins et al.,
2005; Cousins et al., 2004; Heslop et al., 2001), some por-
tion of freshly isolated satellite cells or single myofibers
carrying satellite cells give rise to satellite cells after trans-
plantation (Collins et al., 2005; Montarras et al., 2005).
These results suggest that satellite cells in vivo contain
a subpopulation of repopulating cells that are lost in
culture. Our findings are consistent with this body of
work and demonstrate that repopulating cells represent
a pool of premyogenic cell as evidenced by lack of Myf5
expression.
Recent advances have provided important insights into
the role played by the microenvironment in regulating
stem cell identity and the asymmetric generation of com-
mitted daughter cells (Fuchs et al., 2004; Knoblich, 2001;
Moore and Lemischka, 2006). In several other systems,
stem cell polarity and spindle orientation relative to the
basal lamina determines the fate of daughter cells. Stem
cell divisions in a planar orientation are symmetric and
generate identical daughter stem cells. By contrast,
stem cell divisions in an apical-basal orientation are asym-
metric and give rise to a stem cell at the basal surface and
a committed daughter cell destined for differentiation on
the apical surface (Fuchs et al., 2004; Knoblich, 2001;
Lechler and Fuchs, 2005). Therefore, the discovery of
the stem cell niche provides an anatomical means to es-
tablish the identity of a candidate stem cell population
within a particular tissue.
Muscle satellite cells occupy a niche featuring a struc-
tural foundation for asymmetric self-renewal. The basal
side of satellite cells is covered by the basal lamina en-
sheathing the muscle fiber, while the apical side of satellite
cells is adjacent to the myofiber. The laminin receptor
a7b1 integrin is specifically expressed in satellite cells
on the basal surface, whereas the cell-adhesion molecule
M-cadherin is specifically expressed on the apical surface
toward the muscle fiber (Bornemann and Schmalbruch,
1994; Burkin and Kaufman, 1999; Collo et al., 1993;
Irintchev et al., 1994). Our data document the asymmetric
generation of Pax7+/Myf5 stem cells and Pax7+/Myf5+
committed cells upon apical-basal oriented cell divisions
as well as symmetric generation of daughters upon planar
cell divisions. Notably, we observed that Pax7+/Myf5
cells remained at the basal surface, whereas Pax7+/Myf5+
cells were localized to the apical surface against the mus-
cle fiber. Presumably the apical cell became committed
due to loss of contact with the basal lamina and the extra-
cellular matrix. These results suggest that the satellite cell
niche plays an important role in regulating both the main-
tenance of stem cell identity and the generation of com-
mitted daughter cells through asymmetric cell divisions.
Within the stem cell niche, signaling pathways such as
Wnt, Notch, BMP/TGFb, and STAT and proteins such as
Numb, PAR, PKCz, LGL, and NUMA have been implicated
in the regulation of asymmetric cell division in model sys-
tems (Betschinger and Knoblich, 2004; Betschinger et al.,
2003; Fuchs et al., 2004; Knoblich, 2001; Knoblich et al.,
1995; Moore and Lemischka, 2006; Rhyu et al., 1994).
We observed that sublaminar satellite stem cells dis-
played elevated expression of Notch-3, whereas com-
mitted progenitors exhibited upregulation of Delta-1. We
further demonstrated that in newly divided primary myo-
blasts the level of Delta-1 is high in Myf5+ and low in
Myf5 sister cells. Moreover, we observed that inhibition
of Notch signaling resulted in the loss of the Pax7+/
Myf5 population. Therefore, activation of the Notch sig-
naling pathway, probably stimulated by Delta-1 ligand
and Notch-3 receptor interactions, plays a positive role
in the self-renewal of satellite stem cells.
Several lines of evidence implicate Notch signaling in
the regulation of satellite cell function. Delta-1 inhibits
myogenic differentiation in cell culture and during embry-
onic myogenesis (Delfini et al., 2000; Kuroda et al., 1999).
Cosegregation of template DNA strand and asymmetric
distribution of Numb to one daughter cell were observed
during satellite cell division (Conboy et al., 2005; Shinin
et al., 2006). Furthermore, asymmetric distribution of
Numb to the basal side of dividing cells occurs in the der-
mamyotome during embryonic myogenesis (Holowacz
et al., 2006; Venters and Ordahl, 2005). Finally, Inscute-
able and Numb are segregated to the apical and basal sib-
ling cells, respectively, during muscle differentiation in
Drosophila, and perturbation of this segregation pattern
leads to the failure of muscle development (Carmena
et al., 1998). One of the challenges for future study is to
pinpoint the intrinsic and extrinsic molecules and signaling
pathways that control the asymmetric distribution of Delta
and Numb in satellite cells and how this asymmetry leads
to cell fate segregation.
BrdU-labeling experiments in freshly isolated satellite
cells have suggested that template DNA cosegregates
with Numb in label-retaining cells that express the self-re-
newal marker Pax7 (Shinin et al., 2006). The relationship
between Pax7+/Myf5 satellite cells and label-retaining
cells thus represents an interesting question. In prelimi-
nary short-term label-retention experiments, we applied
a short pulse of BrdU to label the newly synthesized
DNA strand in proliferating satellite cells, which was
followed by a 3 day chase. From cross-sections, we ob-
served that 14% (n = 71) of Pax7+/Myf5 cells were
BrdU+, whereas 41% (n = 309) of Pax7+/Myf5+ cells
were BrdU+. From single myofibers, we observed cose-
gregation of template DNA to Pax7+/Myf5 cells in 38%
(5 out of 13) of newly divided sister cells. By contrast,
only 9% (2 out of 23) of newly divided Pax7+/Myf5+ cells
displayed cosegregation of template DNA to one sister
cell. These results are consistent with the observations
of Shinin and coworkers (Shinin et al., 2006).
In studies of mdx mice, a mouse model of human DMD,
it has been noted that a radiation-resistant subpopulation
of satellite cells is depleted relative to wild-type muscle
(Heslop et al., 2000). Moreover, satellite-cell-derived myo-
blasts display accelerated differentiation kinetics when
isolated from mdx mice (Yablonka-Reuveni and Anderson,
Cell 129, 999–1010, June 1, 2007 ª2007 Elsevier Inc. 1007
2006). These data suggest that under pathological condi-
tions, the equilibrium between stem cells and committed
progenitors within the satellite cell compartment is per-
turbed. It is interesting to speculate that this phenomenon
contributes to the progression and severity of DMD.
It is worth mentioning that symmetric expansion of sat-
ellite stem cells and myogenic cells may represent a pri-
mary means of cell division during CTX-induced muscle
regeneration, as manifested by the focal enrichment of
Pax7+/YFP cells (Figure S2). We suspect that symmetric
expansion of stem cells represents the primary mecha-
nism of self-renewal under certain devastating pathologi-
cal conditions, whereas asymmetric self-renewal occurs
as the primary mechanism for the maintenance of the ho-
meostasis of satellite cells under normal physiological
conditions.
The identification of a satellite stem cell represents an
important advance in our understanding of satellite cell bi-
ology and opens new avenues to explore for the treatment
of degenerative neuromuscular diseases. Satellite stem
cells could be used for direct transplantation into diseased
muscle. Molecular characterization of satellite stem cells
should lead to identification of markers for their prospec-
tive isolation from human muscle tissue. Alternatively, un-
derstanding the molecular regulation of satellite stem cell
symmetric versus asymmetric cell division will lead to
identification of biologics or small drugs that specifically
target the relevant pathway leading to satellite stem cell
expansion. Future experiments will investigate both the
utility of satellite stem cells for cell transplantation and
the molecular control of cell fate determination.
EXPERIMENTAL PROCEDURES
Mice and Animal Care
ROSA26-LacZ (ROSA26R3) reporter mice, mdx mice carrying homol-
ogous mutated dystrophin gene, and Pax3-Cre mice strains were
purchased from JaxMice (Jackson Laboratories, MI). Heterozygous
Myf5-nLacZ (Tajbakhsh et al., 1996) mice were directly used, whereas
Myf5-Cre (Tallquist et al., 2000) heterozygous mice were bred with
ROSA26-YFP (Srinivas et al., 2001), also referred to as ‘‘ROSA-YFP’’
in the text, or ROSA26R3 reporter mice. Pax7 mutant mice carrying
the lacZ knockin mutation were maintained in an inbred 129SV/J back-
ground (Kuang et al., 2006). All mice are maintained inside a barrier
facility, and experiments were performed in accordance with the
University of Ottawa regulations for animal care and handling.
Myofiber Isolation and Immunohistochemistry
Single myofibers were isolated from the EDL muscles as previously de-
scribed (Rosenblatt et al., 1995). To induce activation of satellite cells,
6- to 8-week-old Myf5-nLacz or Myf5-Cre/ROSA-YFP mice were
injected with 25 ml CTX (10 mM, Latoxan, France) into the TA muscles
and examined 4 days later. For analysis of satellite cell proliferation
in vitro, single myofibers were either plated on Matrigel-coated cham-
ber slides or cultured in suspension in 60 mm Petri dishes coated with
horse serum to prevent fiber attachment (Kuang et al., 2006). Primary
myoblasts were isolated from hindlimb muscles and cultured as previ-
ously reported (Megeney et al., 1996).
Histological processing and immunochemical labeling of cryo-
sections, single myofibers, and cells were performed as previously
reported (Kuang et al., 2006). M.O.M. blocking kit (BMK-2202, Vector
Lab) was used in staining of muscle sections. Primary antibodies are
1008 Cell 129, 999–1010, June 1, 2007 ª2007 Elsevier Inc.
listed in Table S1. Secondary antibodies used were Alexa488,
Alexa568, and Alexa647 conjugated to specific IgG types (Invitrogen
Molecular Probes) that matched the primary antibodies (Invitrogen
Molecular Probes). Zenon tricolor anti-Rabbit IgG labeling kit (Invitro-
gen Molecular Probes) and Avidin-conjugated fluorophores were
also used where applicable. Nuclei were counterstained with DAPI.
Cell Sorting, PCR Analysis, and Transplantation
Cells were isolated from hindlimb muscle of 6- to 8-week-old mice as
previously described (Megeney et al., 1996). Erythrocytes were re-
moved with the Red Blood Cell Lysing Buffer Hybri-Max (Sigma).
Mononuclear cells were blocked with goat serum for 10 min and incu-
bated with primary antibodies in DMEM with 2% FBS at 1–3 3 107 cell/ml
for 15 min at 4 C. Cells were briefly washed and incubated with
appropriate secondary antibodies (1: 1000) at 4 C for 15 min. After
staining, cells were washed, passed through 30 mm filters (Miltenyi
Biotec) and suspended at a concentration of 1 3 107 cells/ml. Cells
were separated on a MoFlo cytometer (DakoCytomation) equipped
with three lasers. Sorting gates were strictly defined based on single
antibody-stained control cells as well as the forward and SSC patterns
of satellite cells based on preliminary tests. Total RNA was prepared
from 2 3 104 of sorted cell populations by TRIzol (GIBCO) or MicroRNA
kit (Qiagen). cDNA synthesis and PCR were performed as previously
described (Minoguchi et al., 1997). Primers are listed in Table S2.
Equal numbers of FACS-purified YFP and YFP+ cells were injected
with 0.5 cc insulin syringes into the left and right TA muscles of the
same Pax7 / mouse. The number of cells used for each injection
varied from 900–10,000 based on availability of FACS-purified cells.
Injected TA muscles were harvested 3 weeks later for analysis. For
myofiber graft, 10 EDL myofibers were grafted together with 10 ml
DMEM into the TA muscles of Pax7 / mice with custom-made glass
needles controlled by a microsyringe. Grafted muscles were collected
4 weeks later for analysis.
Microscopy and Live Imaging Analysis
Samples were visualized with a Zeiss Axioplan 2 microscope, and im-
ages were acquired using a Zeiss AxioCam camera and the Axioview
3.1 software as previously described (Kuang et al., 2006). For clonal
tracing of satellite cells, single myofiber cultures were monitored using
a Zeiss Axiovert 200 microscope equipped with an incubator (XL-3,
Zeiss), a CO2 controller, and a heating unit that cooperate to maintain
37 C and 5% CO2 within the incubator. Fluorescent and phase-
contrast images were recorded at one frame every 5 min using a
203 objective (LD Plan-Neofluar N.A. 0.4) with a Zeiss AxioCam and
the AxioView 4.5 software.
Supplemental Data
Supplemental Data include eight figures, two tables, and one movie
and can be found with this article online at http://www.cell.com/cgi/
content/full/129/5/999/DC1/.
ACKNOWLEDGMENTS
We thank Caroline Vergette and Vanessa Seale for expert technical
assistance and Iain McKinnell and Mark Gillespie for careful reading
of the manuscript. We thank Drs. P. Soriano for the Myf5-Cre mice,
F. Costantini for the ROSA-YFP mice, and S. Tajbakhsh for the
Myf5-nLacZ mice. M.A.R. holds the Canada Research Chair in Molec-
ular Genetics and is an International Research Scholar of the Howard
Hughes Medical Institute. This work was supported by grants to
M.A.R. from the Canadian Institutes of Health Research, the National
Institutes of Health, Muscular Dystrophy Association, the Howard
Hughes Medical Institute, and the Canada Research Chair Program.
S.K. was supported by an NSERC Postdoctoral Fellowship.
The authors declare no conflict of interests.
Received: June 14, 2006
Revised: February 7, 2007
Accepted: March 7, 2007
Published: May 31, 2007
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