Published October 14, 2002

JCB
Article

Myogenic specification of side population cells

in skeletal muscle
Atsushi Asakura,1 Patrick Seale,1,2 Adele Girgis-Gabardo,2 and Michael A. Rudnicki1,2
1
Molecular Medicine Program, Ottawa Health Research Institute, Ottawa, Ontario, K1H 8L6 Canada
2
Department of Biology, McMaster University, Hamilton, Ontario, L8S 4K1 Canada

S
keletal muscle contains myogenic progenitors called to myocytes but did not express Sca-1 or CD45 and never
satellite cells and muscle-derived stem cells that have formed hematopoietic colonies. Importantly, muscle SP
been suggested to be pluripotent. We further investigated cells exhibited the potential to give rise to both myocytes
the differentiation potential of muscle-derived stem cells and satellite cells after intramuscular transplantation. In
and satellite cells to elucidate relationships between these addition, muscle SP cells underwent myogenic specification
two populations of cells. FACS® analysis of muscle side after co-culture with myoblasts. Co-culture with myoblasts
population (SP) cells, a fraction of muscle-derived stem or forced expression of MyoD also induced muscle differ-

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cells, revealed expression of hematopoietic stem cell entiation of muscle SP cells prepared from mice lacking
marker Sca-1 but did not reveal expression of any satellite Pax7 gene, an essential gene for satellite cell development.
cell markers. Muscle SP cells were greatly enriched for Therefore, these data document that satellite cells and
cells competent to form hematopoietic colonies. Moreover, muscle-derived stem cells represent distinct populations
muscle SP cells with hematopoietic potential were CD45 and demonstrate that muscle-derived stem cells have the
positive. However, muscle SP cells did not differentiate potential to give rise to myogenic cells via a myocyte-
into myocytes in vitro. By contrast, satellite cells gave rise mediated inductive interaction.

Introduction
Myogenic satellite cells reside beneath the basal lamina of from their daughter myoblasts by both biochemical and bio-
adult skeletal muscle closely juxtaposed against skeletal muscle logical criteria (Seale and Rudnicki, 2000).
fibers and account for 2–5% of sublaminal nuclei in adult Satellite cells have long been considered monopotential
muscle. Satellite cells are normally mitotically quiescent, but stem cells with the ability to only give rise to cells of the
are activated (initiate proliferation) to mediate the postnatal myogenic lineage. However, recent experiments have identified
growth and regeneration of muscle (Seale and Rudnicki, the existence of adult stem cells present in most (if not all)
2000). The progeny of activated satellite cells, termed myo- tissues that appear to exhibit the ability to differentiate into
genic precursor cells, undergo multiple rounds of cell division many different cell types after reintroduction in vivo. This
before terminal differentiation. The number of quiescent work has raised important questions regarding the develop-
satellite cells in adult muscle remains relatively constant over mental potential of stem cells derived from diverse tissues
multiple cycles of degeneration and regeneration, suggesting including muscle, bone marrow, and brain (Seale et al.,
a capacity for self-renewal within the satellite cell compartment. 2001). For example, hematopoietic stem cells (HSCs),* in
Satellite cells express specific genes such as M-cadherin, addition to their ability to produce all blood cell lineages,
CD34, and Pax7, and after activation give rise to a large also exhibit developmental plasticity when introduced into
number of daughter myoblasts in addition to repopulating different tissues. HSCs can differentiate into hepatic cells
the satellite cell pool (Beauchamp et al., 1999, 2000; Seale et (Lagasse et al., 2000), cardiac muscle and vascular endothelium
al., 2000). Therefore, satellite cells represent a unique popu- (Jackson et al., 2001), and several epithelial cell types
lation of committed myogenic progenitors that are distinct (Krause et al., 2001) after transplantation. Therefore, many
or all tissues appear to contain a population of adult stem
Address correspondence to Michael A. Rudnicki, Molecular Medicine
Program, Ottawa Health Research Institute, 501 Smyth Rd., Ottawa,
*Abbreviations used in this paper: CMV, cytomegalovirus; HSC, hemato-
Ontario, K1H 8L6 Canada. Tel.: (613) 739-6740. Fax: (613) 737-8803. poietic stem cell; IL, interleukin; LTR, long-term repeat; MHC, myosin
E-mail: [email protected] heavy chain; MP, main population; SP, side population; TA, tibialis
Key words: stem cell; satellite cell; Pax7; hematopoiesis; Sca-1 anterior; WP, whole population.

 The Rockefeller University Press, 0021-9525/2002/10/123/12 $5.00

The Journal of Cell Biology, Volume 159, Number 1, October 14, 2002 123–134
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124 The Journal of Cell Biology | Volume 159, Number 1, 2002

cells that differentiate in a context-specific manner, presum-

ably in response to growth factors and signals provided by
their host tissues (Seale et al., 2001).
Satellite cells are believed to be the committed stem cell of
the myogenic cells responsible for the postnatal growth and
regeneration of muscle (Seale et al., 2001). The notion that
satellite cells exclusively accomplish the regeneration of adult
muscle has been challenged by the demonstration that muscle
also contains a population of adult stem cells, called muscle-
derived stem cells. Muscle-derived stem cells exhibit the ca-
pacity to reconstitute the entire hematopoietic repertoire after
intravenous injection into lethally irradiated mice (Jackson et
al., 1999; Kawada and Ogawa, 2001). Muscle-derived stem
cells isolated by FACS® of side population (SP) cells, on the
basis of Hoechst dye exclusion, exhibit the capacity to give
rise to hematopoietic cells after intravenous injection into le-
thally irradiated mice. In addition, transplanted SP cells iso-
lated from bone marrow or muscle actively participate in the Figure 1. Adult muscle contains hematopoietic progenitors.
(A, B, and D–F) Muscle-derived cells formed myeloid hematopoietic
formation of skeletal myotubes during regeneration (Gussoni colonies in Methocult M3434. Both large tight colonies 1 mm (A)
et al., 1999). Importantly, Pax7-deficient muscle entirely and small colonies (B) were formed in cultures of muscle-derived
lacks myogenic satellite cells, yet contains muscle SP cells that cells. (C) Muscle-derived cells also formed pre-B cell colonies in

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exhibit a high level of hematopoietic progenitor activity Methocult M3630. May-Grunwald’s Giemsa staining (D) and
(Seale et al., 2000). Together, these data demonstrate that immunohistochemistry against Mac1 (E) and Gr1 (F). Arrows and
arrowheads indicate Mac-1/Gr-1–positive monocytes containing
muscle-derived stem cells represent a distinct cell population some vacuoles and granulocytes with polymorphic nuclei, respec-
from satellite cells. These studies suggested the hypothesis tively. (G) B220-positive pre-B cells were contained in colonies
that induction of Pax7 in muscle-derived stem cells induces derived from muscle cells in Methocult M3630. Bars: (A and B) 200
satellite cell specification by restricting alternate developmen- m; (C) 100 m; and (D–G) 25 m.
tal programs. However, direct proof that muscle-derived stem
cells represent the progenitors of satellite cells and any under- emsa staining revealed that large colonies contained a vari-
standing of the mechanisms that regulate this developmental ety of hematopoietic cells including Mac-1– and Gr-1–
step remains lacking. positive granulocytes and monocytes (Fig. 1, D–F). The
To investigate the relationship between muscle-derived incidence of hematopoietic colonies was 18.8/104 muscle-
stem cells and myogenic satellite cells, we used a variety of derived cells plated versus 81.2/104 bone marrow cells
cell culture assays as well as in vivo intramuscular transplan- plated (Fig. 2 E), which is consistent with previously re-
tation to explore their developmental potential. Our experi- ported works (Kawada and Ogawa, 2001; McKinney-Free-
ments demonstrate that the ability of muscle-derived stem man et al., 2002). By contrast, no hematopoietic colonies
cells to undergo myogenic specification is a regulated process were formed after plating of 104 nucleated peripheral
that appears to involve cell-mediated inductive interactions. blood cells (Fig. 2 E), ruling out the possibility that he-
matopoietic progenitors were derived from circulating pe-
ripheral blood cells contaminating the muscle preparation.
Results Similar results were also reported previously (Kawada and
Adult muscle is a rich source Ogawa, 2001).
of hematopoietic progenitors To test whether muscle-derived stem cells can differenti-
Adult skeletal muscle contains muscle-derived stem cells ate into lymphoid cells in vitro, muscle-derived cells were
that efficiently reconstitute the entire hematopoietic reper- cultured in methylcellulose medium (Methocult M3630)
toire after intravenous injection into lethally irradiated containing IL-7, permitting the clonal growth of pre-B cell
mice (Gussoni et al., 1999; Jackson et al., 1999; Kawada lineage. By 10 days, cultures of muscle-derived cells gave rise
and Ogawa, 2001; McKinney-Freeman et al., 2002). to hematopoietic colonies consisting of small pre-B cells as
Therefore, a hallmark property of muscle-derived stem confirmed by immunohistochemistry with B220 antibodies
cells is the ability to undergo hematopoietic differentia- (Fig. 1, C and G). Therefore, these results indicate that adult
tion. To elucidate whether muscle-derived stem cells can skeletal muscle contains a rich source of hematopoietic pro-
differentiate into hematopoietic cells in vitro, we prepared genitors for both myeloid and lymphoid lineages.
dissociated muscle–derived cells by enzymatic digestion
and cultured the cells in methylcellulose medium (Metho- Satellite cells do not exhibit hematopoietic potential
cult M3434) containing stem cell factor, interleukin (IL)-3, Satellite cells have long been considered monopotential stem
IL-6, and erythropoietin, permitting the clonal growth of cells, with the ability to only give rise to cells of the myogenic
myeloid-type hematopoietic cells. By 14 days, cultures of lineage. However, recent experiments have indicated that
muscle-derived cells efficiently gave rise to high numbers satellite cells readily differentiate into different cell types such
of hematopoietic colonies consisting of large colonies (Fig. as osteocytes and adipocytes (Asakura et al., 2001). To inves-
1 A) and small colonies (Fig. 1 B). May-Grunwald’s Gi- tigate whether satellite cells have the potential to differentiate
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Myogenic specification of muscle SP cells | Asakura et al. 125

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Figure 2. Satellite cells do not exhibit hematopoietic potential.
Isolated single muscle fibers prepared from Myf5-nlacZ transgenic
mice were cultured in Methocult M3434. (A) Satellite cells expressing
Myf5-nlacZ (arrowheads) were detected on freshly isolated muscle
fibers (day 0). (B) Migrating satellite cells derived from muscle fibers
were detected after 4 d of culture (arrowheads). (C) By day 10, the
migrating cells formed colonies containing multinucleated myotubes
that expressed Myf5-nlacZ (arrows). (D) By 14 days, differentiated
myotubes (arrow) were detected adjacent the original muscle fiber
(indicated by F). (E) Single muscle fibers (total fibers; n  180 for
three independent experiments) or 1  104 cells prepared from
whole muscle, bone marrow, or peripheral blood were cultured
in Methocult M3434. By day 14, hematopoietic colonies were
counted. The error bars represent the SEM from three independent
experiments. Bar: (A–C) 50 m; and (D) 100 m.

into hematopoietic cells, we cultured single muscle fibers Figure 3. Muscle SP cells express Sca-1 and CD45 but do not
from heterozygous Myf5-nlacZ mice in Methocult M3434. express Myf5-nlacZ or MD6.0-lacZ. (A) FACS® fractionation of SP
The expression of Myf5-nlacZ recapitulates the expression of cells from bone marrow and muscle was used for the isolation of
the endogenous Myf5 mRNA both during embryogenesis adult stem cells by Hoechst dye exclusion. Both muscle and bone
marrow SP cells stained with Hoechst dye were sensitive to verapamil.
(Tajbakhsh et al., 1996) and in adult muscle (Beauchamp et (B) x- and y-axes indicate intensity of CD45-FITC and Sca-1-PE,
al., 2000). Isolated muscle fibers maintained in culture for respectively. The percentage of cells in each quadrant is indicated
14 d gave rise to colonies uniformly composed of Myf5- in the panel. (C) Unsorted (WP) or sorted cells prepared from
nlacZ–positive multinucleated, contractile myotubes (Fig. 2, Myf5-nlacZ or MD6.0-lacZ transgenic mouse muscle were stained
A–D). Importantly, hematopoietic colonies were never de- with X-gal. The error bars represent the SEM from three independent
experiments. At least a total of 1,000 cells were counted in
tected in these long-term cultures of isolated muscle fibers each fraction.
(Fig. 2 E; n  180 fibers). By contrast, 10–30 hematopoietic
colonies were formed after plating of 1  104 unfractionated
muscle cell suspension. The inability of satellite cells to un- Muscle SP cells in vitro undergo hematopoietic
dergo hematopoietic differentiation and the preferential dif- but not myogenic differentiation
ferentiation of Myf5-nlacZ–expressing cells into muscle (Fig. The expression of Myf5-nlacZ was readily detected in the
2 C) strongly support the hypothesis that satellite cells are re- nuclei of satellite cells on freshly isolated muscle fibers (Fig.
stricted in their developmental potential within the mesen- 2 A; Beauchamp et al., 2000). A similar expression profile of
chymal range of cell lineages. lacZ within myonuclei and satellite cell nuclei was also ob-
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126 The Journal of Cell Biology | Volume 159, Number 1, 2002

Figure 4. Muscle SP cells efficiently

form hematopoietic colonies in vitro.
(A) 1  104 unsorted (WP) or sorted MP
or SP cells prepared from muscle and
bone marrow were cultured in Methocult
M3434 (left). 1  103 nucleated sorted
muscle SP subfractions (CD45:Sca-1,
CD45:Sca-1, CD45:Sca-1, and
CD45:Sca-1) were cultured in Meth-
ocult M3434 (right). By day 14, hemato-
poietic colonies were counted. (B) 1 
104 unsorted (WP) or sorted MP or SP
cells prepared from muscle and bone
marrow were cultured in Methocult
M3434 for 14 days (left) and in myoblast
growth medium by 10 days (right).
Muscle colonies were counted after
cultures. (C). In myoblast growth
medium, the muscle MP fraction
contained Myf5-nlacZ–expressing myo-
blasts (arrows), whereas the muscle SP
fraction did not contain any lacZ-
expressing cells. The error bars represent
the SEM from three independent experi-
ments. Bar: 50 m.

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served in single muscle fiber cultures prepared from MD6.0- 16% of muscle SP cells expressed CD45 consisting of both
lacZ transgenic mice carrying myoD upstream–driving lacZ Sca-1–positive (9.2%) and –negative (6.8%) fractions (Fig.
gene (Asakura et al., 1995; unpublished data). Therefore, if 3 B). Next, sorted cells (MP and SP) and unfractionated
purified muscle SP cells (a fraction of muscle-derived stem whole population (WP) cells prepared from Myf5-nlacZ or
cells) contained a fraction of satellite cells, Myf5-nlacZ– or MD6.0-lacZ mouse muscle were stained with X-gal. Both
MD6.0-lacZ–expressing cells should be detected within the WP and MP cells contained a similar proportion of lacZ-
SP compartment. To test this possibility, we used the expressing cells (Fig. 3 C). By contrast, the SP fraction did
FACS®/Hoechst method (5 g/ml Hoechst 33342) to frac- not contain any cells that expressed Myf5-nlacZ or MD6.0-
tionate muscle cell suspensions into SP and main population lacZ. Desmin and Pax7 are also known to be expressed in
(MP). Compared to bone marrow–derived SP cells (a frac- both quiescent satellite cells and derivative myoblasts in
tion of HSCs), increased numbers of SP cells were detected adult muscle (Seale et al., 2000). Importantly, no desmin or
in muscle (0.2 vs. 2.3% of total cells; Fig. 3 A). Similar re- Pax7 expression was detected in cells from the muscle SP
sults were obtained when skeletal muscle cells were stained fraction (unpublished data). Therefore, satellite cells and
with 12.5 g/ml Hoechst 33342 (unpublished data) as de- their daughter myoblasts strictly reside within the MP frac-
scribed previously (Gussoni et al., 1999). Both bone marrow tion of cells by FACS®/Hoechst analysis.
and muscle SP cells stained with Hoechst dye were sensitive We have demonstrated that putative muscle-derived
to verapamil, which is consistent with reported results (Jack- stem cells differentiate into hematopoietic cells in vitro.
son et al., 1999). Recent studies have demonstrated stem However, to confirm that these hematopoietic progenitors
cells derived from skeletal muscle that exhibit hematopoietic were derived from the SP compartment, cells prepared
potential express the hematopoietic marker CD45 and Sca-1 from muscle and bone marrow were fractionated by
(Kawada and Ogawa, 2001; McKinney-Freeman et al., FACS® into SP and MP populations. The purified frac-
2002). To investigate the origin of the hematopoietic pro- tions were cultured in Methocult M3434 or in myoblast
genitors in muscle SP cells, FACS® analysis was used to sub- growth medium to score hematopoietic (Fig. 4 A) and
fractionate muscle SP cells with CD45 and Sca-1 antibodies. myogenic (Fig. 4, B and C) colony formation. In Methoc-
Most of the muscle SP cells (92%) expressed Sca-1, which is ult M3434, both bone marrow and muscle SP cells effi-
consistent with previous observation (Gussoni et al., 1999). ciently formed numerous hematopoietic colonies. By con-
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Myogenic specification of muscle SP cells | Asakura et al. 127

trast, WP or MP fractions gave rise to markedly fewer

hematopoietic colonies (Fig. 4 A). Importantly, CD45-
positive muscle SP cells exclusively gave rise to hematopoi-
etic colonies; CD45:Sca-1 muscle SP cells efficiently
gave rise to hematopoietic colonies, whereas CD45:Sca-
1 muscle SP cells gave rise to a lower number of hemato-
poietic colonies. Therefore, these results indicate that mus-
cle SP cells that exhibit hematopoietic potential are CD45
positive. In addition, most of the SP population was nega-
tive for hematopoietic lineage markers such as B220, CD3,
Gr-1, MAC1, and Terr119 (Gussoni et al., 1999; unpub-
lished data), suggesting that muscle SP cells contain primi-
tive hematopoietic progenitors.
Importantly, by day 10 after being plated, muscle SP cells
did not form differentiated muscle colonies, and instead
formed colonies that displayed fibroblastic morphology (Fig.
4 C). By contrast, the WP and MP cell fractions prepared
from muscle gave rise to 8–30 differentiated muscle colonies
per plate under both culture conditions (Fig. 4, B and C).
None of the cells derived from bone marrow differentiated
into muscle colonies under these conditions. Importantly,

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most bone marrow SP cells, in contrast to muscle SP cells,
did not attach to the plate and failed to survive in myoblast
growth medium. Therefore, muscle SP cells undergo prefer-
ential hematopoietic differentiation in vitro and do not ap-
pear capable of forming myogenic progenitors under the
culture conditions tested.

Sca-1–positive cells are located outside

of muscle fibers
To further confirm that muscle SP cells are distinct cell
population from satellite cells, we first examined detection
of HSC markers CD34, Sca-1, and CD45, in satellite cells
on freshly isolated muscle fibers. Immunohistochemical
detection clearly demonstrated that CD34 was expressed
in satellite cells on muscle fibers (Fig. 5 A). About three
to four CD34-expressing satellite cells were normally
detected per muscle fiber (unpublished data), which is
consistent with previous observation (Beauchamp et al., Figure 5. Sca-1–positive cells are located outside of muscle fibers
2000). By contrast, satellite cells on muscle fibers were and are associated with vasculature. (A) Satellite cells on freshly
never observed to express Sca-1 or CD45 (n  20 fibers; isolated muscle fibers expressed CD34 (arrow) but not Sca-1 or
Fig. 5 A). Previously, it has been reported that the major- CD45. The satellite cell nuclei and myonuclei were visualized by
ity of muscle SP cells express Sca-1, an important cell sur- counterstaining with DAPI. These fibers were one example out of 20
face marker for HSCs (Gussoni et al., 1999; Jackson et al., muscle fibers stained with antibodies. (B) Left panels show double
immunostaining with anti–Sca-1 and laminin, and anti–Sca-1 and
1999). To examine where Sca-1–expressing cells are lo- PECAM antibodies for cross sections of tibialis anterior (TA) muscle.
cated within muscle, double immunostaining was per- Right panels show light field with DAPI nucleus staining for each
formed. Clearly, Sca-1–positive cells were located outside dark field at the left panel. Sca–1–positive cells (red) indicated by
of the muscle fiber basal lamina as revealed by staining arrows were detected between muscle fibers. Laminin was
with anti–laminin antibodies (Fig. 5 B). In addition, Sca- expressed in basal lamina of muscle fibers (green). Sca-1–positive
cells (red) were also detected in endothelium (arrowheads) and
1–expressing cells were frequently detected closely juxta- outer layer (arrows) of blood vessels indicated by black circles in
posed to blood vessels as well as endothelium that ex- the right panel. PECAM-positive endothelial cells (green) were
pressed PECAM (Fig. 5 B). Similar results were reported detected in blood vessels. Bars: (A) 50 m; and (B) 25 m.
in previous works in which Sca-1–positive cells were de-
tected in endothelium and outer layer of blood vessels, but
were not detected beneath the basal lamina of skeletal Myogenic specification of muscle SP cells
muscle fibers (Zammit and Beauchamp, 2001). These re- after intramuscular injection
sults further confirm that muscle-derived stem cells are Culture of purified muscle SP cells in Methocult M3434 or
distinct cell population from satellite cells and may be as- myoblast growth medium did not give rise to any SP-
sociated with the vasculature within muscle. derived myoblasts or myocytes (Fig. 4, B and C). To exam-
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128 The Journal of Cell Biology | Volume 159, Number 1, 2002

Figure 6. Muscle SP cells undergo myogenic

specification and satellite cell differentiation in
vivo after intramuscular injection. Myf5-nlacZ
muscle SP cells were injected into regenerating
tibialis anterior (TA) muscle of scid/bg mice. (A) By
day 14, whole-mount staining of the TA muscle
showed clusters of lacZ-positive nuclei. (B and C)
By day 14, cross-sections showed that muscle SP
cell–derived myocytes that expressed lacZ in the
nucleus were integrated into desmin (B) or MHC-
positive (C) regenerating muscle fibers (arrows). By
1 month, TA muscles were used for histochemical
(D–K) and culture (L) experiments to detect muscle
SP cell–derived satellite cells. (D–F) The lacZ-
expressing satellite cell was detected between the
basal lamina (laminin; red) and sarcolemma
(dystrophin; green) of muscle fiber (arrows). The
lacZ-expressing satellite cells were positive for
Pax7 (G–I) in the nucleus and desmin (J and K) in
the cytoplasm (arrows). (E, F, I, and K) DAPI staining
indicates nuclei (blue). Arrowheads indicate myo-
nuclei. Dissociated cells prepared from the TA
muscle contained a lacZ-positive myoblast cluster
(L). Bar: (A) 50 m; (B and C) 10 m; (D–F) 7 m;
(G–K) 5 m; and (F) 40 m.

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ine whether muscle SP cells have a potential to undergo positive nuclei are within a satellite cell. By contrast, desmin
myogenic specification in vivo, we intramuscularly in- protein was not located surrounding myonuclei (Fig. 6, J and
jected FACS®/Hoechst-purified muscle SP cells derived K). Next, we cultured dissociated cells prepared from TA
from Myf5-nlacZ or ROSA26 mice into regenerating tibialis muscle 1 mo after injection of cells. After 4 d of culture,
anterior (TA) muscle of adult scid/bg immunodeficient mice. Myf5-nlacZ–expressing myoblast clusters were occasionally
In brief, cardiotoxin was injected into TA muscles to induce detected (Fig. 6, F and L). These results strongly suggest that
muscle damage, cells were injected after a further 24 h, and intramuscularly transplanted muscle SP cells are competent
the muscle was allowed to heal for an additional 2 wk–1 mo. to give rise to satellite cells within the recipient muscle fibers.
By day 14, whole-mount staining of the TA muscle injected In addition, these SP cell–derived satellite cells have a normal
with Myf5-nlacZ muscle SP cells showed clusters of lacZ- proliferative potential in vitro. Together, these data indicate
positive nuclei (Fig. 6 A). Examination of frozen sections re- that muscle SP cells have the capability to undergo myogenic
vealed the presence of lacZ-expressing myocytes as con- specification and suggest that muscle-derived stem cells un-
firmed by immunohistochemistry with antibodies reactive dergo myogenic specification via a myocyte-mediated induc-
with desmin (Fig. 6 B) or myosin heavy chain (MHC; Fig. 6 tive interaction. In addition, the in vivo potential of muscle
C). The total number of Myf5-nlacZ–expressing nuclei SP cells to give rise to satellite cells supports our hypothesis
counted in serial sections was 1.4% (n  3) of the total mus- that at least some portion of satellite cells originates from
cle SP cells injected. The efficiency of engraftment is similar muscle-derived stem cells.
to previous results after intramuscular cell injections into re-
generating muscle (Beauchamp et al., 1999; Galli et al., Muscle SP cells undergo myogenic specification
2000). Desmin/lacZ double-positive regenerating muscle fi- after co-culture with primary myoblasts
bers were also detected in cross sections prepared from TA Intramuscular transplantation experiments suggest that mus-
muscle after injection of ROSA26 muscle SP cells (unpub- cle-derived stem cells undergo myogenic specification via a
lished data). myocyte-mediated inductive interaction. To investigate
By 1 mo after injection of My5-nlacZ muscle SP cells, a whether cell-mediated interactions could influence the de-
few lacZ-positive nuclei were detected between the basal lam- velopmental fate of muscle SP cells in vitro, we co-cultured
ina (laminin) and sarcolemma (dystrophin) of TA muscle FACS®/Hoechst-purified muscle SP cells from ROSA26
fibers (Fig. 6, D–F). The lacZ-positive nuclei were also posi- mice, expressing lacZ protein ubiquitously (Zambrowicz et
tive for Pax7 (Fig. 6, G–I), a satellite cell maker in resting al., 1997), with equal numbers of myoblasts or fibroblasts.
muscle (Zammit and Beauchamp, 2001). Cytoplasm sur- Immunohistochemistry for desmin was performed to con-
rounding the lacZ-positive nuclear-expressed desmin and the firm the differentiation of lacZ-expressing ROSA26 cells
plasma membrane was also evident, indicating that the lacZ- into myocytes. Culture of muscle SP cells alone resulted in
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Myogenic specification of muscle SP cells | Asakura et al. 129

Figure 7. Myogenic specification of muscle SP

cells after co-culture with myoblasts. (A) Cultures
of ROSA26 muscle SP cells alone or together with
equal numbers of primary myoblasts were performed
followed by staining with X-gal to mark all input
ROSA26 cells and antidesmin to identify myogenic
cells. Muscle SP cells co-cultured with myoblasts
gave rise to X-gal/desmin-positive multinucleated
myotubes (black arrows). (B) GFP-expressing muscle
SP cells prepared from GFP mice were cultured
alone or together with equal numbers of primary
myoblasts. Muscle SP cells co-cultured with myo-
blasts formed GFP/MHC-positive myotubes (white
arrows). White arrowheads indicate GFP-negative
and MHC-positive myoblast–derived myotubes.
(C) Myf5-nlacZ muscle SP cells were cultured
alone or together with equal numbers of primary
myoblasts. Muscle SP cells co-cultured with
primary myoblasts underwent Myf5-nlacZ–
expressing multinucleated myotubes (black
arrows) and mononuclear myocytes (arrowheads).
Bars: 25 m.

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the formation of any lacZ-expressing myogenic cells (Fig. 7 muscle SP cells isolated from Myf5-nlacZ mice with primary
A). By contrast, co-culture of muscle SP cells with primary myoblasts resulted in the formation of Myf5-nlacZ–express-
myoblasts resulted in the formation of multinucleated myo- ing mononuclear myoblasts and multinucleated myotubes
tubes that coexpressed lacZ and desmin. The number of nu- (Fig. 7 C), indicating that muscle SP cells did not simply
clei of myogenic cells that coexpressed desmin and lacZ was form heterokaryon myotubes fused with primary myoblasts.
3.8% (n  4) of the total lacZ-expressing ROSA26 cell nu- By contrast, we failed to detect any muscle differentiation of
clei. We also observed myogenic conversion in co-cultures of bone marrow SP cells after co-culture with primary myo-
muscle SP cells with C2C12 myoblasts but not with blasts (unpublished data), suggesting that there is a biologi-
C3H10T1/2 fibroblasts or conditioned medium from pri- cal difference between muscle and bone marrow SP cells.
mary myoblast cultures (unpublished data). We also co-cul- Together, these data unequivocally demonstrate that some
tured 1  104 FACS®/Hoechst-purified muscle SP cells portion of muscle SP undergoes myogenic specification after
from TgN(GFPU)5-Nagy (GFP) mice, expressing a GFP co-culture with primary myoblasts.
ubiquitously (Hadjantonakis et al., 1998), with equal To investigate the myogenic potential of CD45-negative
numbers of primary myoblasts. Immunohistochemistry for (CD45 mSP) and CD45-positive (CD45 mSP) muscle
MHC was performed to confirm the differentiation of GFP- SP cells, preparations from GFP mice were co-cultured with
expressing muscle SP cells into muscle cells after co-culture primary myoblasts. Immunostaining with anti-MHC anti-
with primary myoblasts (Fig. 7 B). Finally, co-culture of body revealed the presence of GFP-expressing multinucle-
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130 The Journal of Cell Biology | Volume 159, Number 1, 2002

tures (Fig. 9). The number of nuclei of myogenic cells that

coexpressed MHC and GFP was 7.6% (Pax7/; n  3)
and 4.7% (Pax7/; n  3) of the total GFP-positive cell
nuclei. These data demonstrate that some portion of mus-
cle SP cells undergoes terminal myogenic differentiation af-
ter co-culture with primary myoblasts and that Pax7 is not
required for this step.
The muscle-specific transcription factor MyoD readily in-
duces the myogenic program when ectopically expressed in
many nonmuscle cell types (Weintraub et al., 1991). To in-
vestigate whether MyoD expression was sufficient to induce
SP myogenic conversion, purified SP cells from wild-type
and Pax7/ muscle were infected with retrovirus express-
ing an LTR/CMV promoter–driven MyoD gene. Notably,
SP cells from wild-type and Pax7/ muscle differentiated
into MyoD/desmin-positive myoblasts (Fig. 10, D, F, J, and
Figure 8. CD45-positive and -negative muscle SP cells exhibit L). The efficiencies of retrovirus infection and myogenic
myogenic potential. Sorted CD45-negative (CD45 mSP) and conversions in all cultures were 50% of the total cells
CD45-positive muscle SP cells (CD45 mSP) prepared from GFP based on the expression of MyoD and desmin (Fig. 10, D
mice were cultured together with equal numbers of primary myo- and J). Importantly, Pax7 protein could not be detected in
blasts. Both CD45 mSP and CD45 mSP gave rise to GFP/MHC- the MyoD-induced myoblasts derived from wild-type mus-
positive myotubes (arrows). Bar: 25 m.

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cle SP cells (Fig. 10 E). Therefore, MyoD-induced myo-
genic differentiation of muscle SP cells is Pax7 independent.
ated myotubes in both cultures (Fig. 8). The number of nu- In addition, the lack of Pax7 induction in MyoD-expressing
clei of myogenic cells that coexpressed MHC and GFP was myoblasts suggests that Pax7 is not regulated by MyoD and
5.1% (CD45 mSP; n  3) and 8.8% (CD45 mSP; n  is therefore likely upstream of MyoD during satellite cell de-
3) of the total GFP-positive cell nuclei. These data demon- velopment.
strate that some portion of both CD45 mSP and CD45
mSP undergo myogenic differentiation after co-culture with
primary myoblasts. Discussion
Satellite cells are committed myogenic progenitors that me-
Myogenic differentiation of muscle SP cells diate muscle growth during the postnatal period as well as
is Pax7 independent regeneration after injury (Seale and Rudnicki, 2000). The
Recently, we demonstrated that mice lacking the Pax7 gene observation that muscle SP cells introduced intravenously
display complete absence of satellite cells (Seale et al., participate in the formation of new myofibers and poten-
2000). To test whether muscle differentiation of muscle SP tially give rise to satellite cells (Gussoni et al., 1999) raised
cells requires Pax7, we co-cultured Pax7/ derived mus- the possibility that muscle-derived stem cells were in fact sat-
cle SP cells with myoblasts. To distinguish muscle SP cells ellite cells (Jackson et al., 1999). However, our data reported
from myoblasts, GFP retrovirus containing virus long term here that muscle-derived stem cells and satellite cells dis-
repeat (LTR) and cytomegalovirus (CMV) promoter-driv- played distinct developmental potentials and were not copu-
ing enhanced GFP gene was used to infect purified muscle rified during FACS®/Hoechst isolation confirms that they
SP cells before initiating co-culture with myoblasts. Immu- are distinct cell populations. Consistent with these data, mu-
nostaining with anti-MHC antibody revealed the presence tant mice lacking Pax7 display a complete absence of satel-
of GFP-expressing multinucleated myotubes in both cul- lite cells, yet contain a normal proportion of SP cells in mus-

Figure 9. Myogenic differentiation of muscle SP

cells after co-culture with myoblasts is Pax7.
Muscle SP cells were prepared from Pax7/
and Pax7/ mice, followed by infection with
GFP retrovirus. Muscle SP cells were cultured
alone or together with equal numbers of primary
myoblasts. Both Pax7/ and Pax7/ muscle
SP cells formed GFP/MHC-positive myotubes
(arrows) after co-culture with myoblasts. Arrow-
heads indicate GFP-negative and MHC-positive
myoblast–derived myotubes. Bar: 25 mm.
Published October 14, 2002

Myogenic specification of muscle SP cells | Asakura et al. 131

Figure 10. MyoD-induced myogenic differentia-

tion of muscle SP cells is Pax7 independent. Muscle
SP cells prepared from Pax7/ (A–F) and Pax7/
(G–L) mice were infected with control retrovirus
vector (A–C and G–I) or with MyoD retrovirus
vector (D–F and J–L). Muscle SP cells after infection
with control retrovirus did not form any desmin-
positive myoblasts (C and I). Infection with MyoD
retrovirus induced desmin-positive myoblast differ-
entiation of both muscle SP cells (F and L). MyoD
expression was detected in both muscle SP cells
after infection with MyoD retrovirus (D and J).
Pax7 protein was not detected in MyoD-induced
myoblasts derived from Pax7/ muscle SP cells (E).
Bar: 25 m.

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cle (Seale et al., 2000). Interestingly, isolated muscle SP cells et al., 2002). By contrast, we have observed that CD45 mSP
efficiently formed hematopoietic colonies in culture but cells have the potential to give rise to adipocytes and osteo-
never gave rise to muscle cells. These muscle SP cells with cytes after induction (unpublished data). Therefore, it is in-
hematopoietic potential were observed to express the he- teresting to hypothesize that the CD45 mSP fraction of cells
matopoietic marker CD45. By contrast, satellite cells from contains progenitor cells that are similar to multipotential
cultured myofibers were capable of differentiation into myo- mesenchymal stem cells (Pittenger et al., 1999).
cytes, adipocytes, and osteocytes (Asakura et al., 2001), but Interestingly, muscle SP prepared from Pax7/ mice,
not into hematopoietic cells. Gussoni et al. (1999) suggested when co-cultured with primary myoblasts, also resulted in
that muscle SP cells converted into myoblasts after 2 wk in the formation of multinucleated myotubes. In addition,
culture, but did not document this observation. It remains forced expression of MyoD could induce myogenic differen-
possible that muscle SP cells undergo spontaneous myoblast tiation of Pax7/ muscle SP cells. Furthermore, Pax7
differentiation after a long-term culture. However, in nu- could not be induced by MyoD in myoblasts derived from
merous experiments, we have never noted the presence wild-type muscle SP cells. Therefore, these data suggest that
of SP-derived myogenic progenitors. Together, these data terminal myogenic differentiation of muscle-derived stem
strongly support the notion that muscle-derived stem cells cells is a Pax7-independent process and that Pax7 functions
and satellite cells represent distinct cellular populations upstream of MyoD during satellite cell development.
within adult skeletal muscle. Muscle SP cells differentiated into myocytes and formed
Importantly, myogenic specification of muscle SP cells and new myofibers after intramuscular injection into regenerating
the formation of mononuclear myoblasts were observed to muscle. Therefore, the observed Pax7-independent myogenic
occur in vitro after co-culture with primary myoblasts. In ad- differentiation of muscle SP cells suggests that Pax7 is re-
dition, muscle SP cells contain at least two distinct fractions quired for the specification of satellite cells, but not for termi-
with myogenic potential as CD45 mSP and CD45 mSP. nal myogenic differentiation. Together, these data indicate
Both exhibit the potential to give rise to myogenic cells after that muscle-derived stem cells are competent for induction of
co-culture with primary myoblasts. Recent work demon- myogenic specification as well as for direct myogenic differ-
strated that both intramuscular and intravenous transplanta- entiation. The inability of muscle SP cells to undergo myo-
tion of bone marrow and intravenous transplantation of bone genic specification except in the presence of myogenic cells
marrow SP cells contributed to skeletal muscle fiber forma- suggests that the process is subject to regulation via a mecha-
tion (Ferrari et al., 1998; Gussoni et al., 1999; Fukada et al., nism that involves cell-mediated inductive interactions.
2002). More recently, it has been demonstrated that muscle- Primary myoblasts derived from neonatal mice readily give
derived CD45-positive cells integrate into regenerating mus- rise to satellite cells after intramuscular injection (Heslop et
cle fibers after intramuscular injection (McKinney-Freeman al., 2001). Muscle SP cells have been suggested to differenti-
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132 The Journal of Cell Biology | Volume 159, Number 1, 2002

ate into satellite cells based on their location after intrave- CD45-positive (CD45 mSP). Bone marrow HSCs cannot
nous injection into irradiated mice (Gussoni et al., 1999). be cultured under the conditions used in this work as re-
Furthermore, fetal liver and bone marrow cells have a poten- ported previously (Traycoff et al., 1996). By contrast, mus-
tial to differentiate into satellite cells based on the location cle cells with hematopoietic potential are readily cultured in
after injection into busulphan-treated neonatal mice (Fukada myoblast medium and have been suggested to maintain their
et al., 2002). Here, we demonstrate that muscle SP cells have hematopoietic potential for at least 5 d in vitro (Jackson et
the potential to differentiate into satellite cells after intra- al., 1999). HSCs express several surface markers such as
muscular injection into regenerating muscle. These satellite Sca-1 and c-kit (Goodell et al., 1997; Gussoni et al., 1999).
cells express satellite cell markers Myf5-nlacZ, Pax7, and Moreover, although freshly isolated muscle SP cells express
desmin, and are proliferative in vitro, suggesting that muscle Sca-1, they do not express c-kit (Gussoni et al., 1999). Fur-
SP cells can differentiate into functional satellite cells. The in thermore, we failed to detect any muscle differentiation of
vivo potential of muscle SP cells that give rise to satellite cells bone marrow SP cells after co-culture with primary myo-
supports our hypothesis that at least some portion of satellite blasts. Therefore, marker expression and biological assays
cells originates from muscle-derived stem cells. However, it suggest some important differences between HSCs and mus-
remains unclear whether both CD45 mSP and CD45 cle-derived cells with hematopoietic potential. In any case,
mSP have the potential to give rise to satellite cells after in- these results strongly suggest that skeletal muscle is an adult
tramuscular transplantation. In addition, it remains to be organ containing a resident population of adult stem cells
elucidated whether Pax7 is required for muscle SP cells to with hematopoietic potential. Recent bone marrow trans-
differentiate into satellite cells after intramuscular transplan- plantation experiments have been interpreted to suggest that
tation. We are currently investigating these questions. hematopoietic progenitors in muscle have a bone marrow
An important outstanding question concerns the location origin (Kawada and Ogawa, 2001). However, a clear under-

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of muscle-derived stem cells within skeletal muscle. Exami- standing of the identity and relationship between HSCs in
nation of regeneration in dystrophic muscle has revealed bone marrow and hematopoietic potential cells within mus-
that recruitment of cells via the circulatory system does not cle remains to be elucidated. For example, it is interesting to
provide a substantial source of myoblasts for muscle repair speculate that muscle-derived hematopoietic potential cells
after radiation of mouse limbs (Heslop et al., 2000). It represent the progenitors for hematopoietic cells such as
is conceivable that the vascular-associated progenitors of macrophages and polymorphonuclear lymphocytes, which
myogenic satellite cells in the embryo persist in close associ- are mobilized in high numbers in regenerating muscle tissue
ation with the vasculature as adult stem cells in skeletal (Seale and Rudnicki, 2000).
muscle (De Angelis et al., 1999). Indeed, muscle SP cells Direct proof that a single stem cell within the muscle SP
express Sca-1, and importantly, Sca-1–positive cells reside fraction of cells is indeed capable to undergo hematopoietic,
between muscle fibers prominently associated with blood myogenic, or other differentiation remains lacking. It is en-
vessels. By contrast, satellite cells on freshly isolated muscle tirely possible that the muscle SP fraction of cells contains
fibers express CD34 but not Sca-1. In addition, Sca-1– multiple types of progenitors each with a restricted range of
expressing cells lines isolated from muscle exhibit a high de- potential. It has been suggested recently that a single pluri-
gree of plasticity (Torrente et al., 2001; Qu-Petersen et al., potent HSC can give rise to many different cell types in a
2002; Tamaki et al., 2002). Together, these observations wide range of tissues in mice (Krause et al., 2001). Similar
suggest that muscle-derived stem cells located as vascular- experiments using muscle SP cells should establish the po-
associated cells in skeletal muscle are a progenitor for sat- tentiality of muscle-derived stem cells. Nevertheless, our
ellite cells or muscle precursor cells. According to this data strongly support the existence of a tier of stem cells in
hypothesis, muscle-derived stem cells and satellite cell pop- addition to satellite cells in skeletal muscle with the potential
ulations would co-exist as distinct stem cell tiers in some state to undergo myogenic specification.
of equilibrium within adult muscle. Soon after initiation of
muscle regeneration, the number of myogenic precursor cells
thought to be derived from satellite cells are much greater Materials and methods
than that of the resident satellite cells (Grounds et al., 1992). Transgenic mice
Myf5-nlacZ (Tajbakhsh et al., 1996) and MD6.0-lacZ (Asakura et al., 1995)
Therefore, one explanation for this apparent paradox is that transgenic mice were provided from Drs. Shahragim Tajbakhsh (Pasteur In-
muscle-derived stem cells give rise to the myogenic precursor stitute, Paris, France) and Stephen J. Tapscott (Fred Hutchinson Research
cells. Additional experiments are required to deduce whether Center, Seattle, WA), respectively. ROSA26 mice and TgN(GFPU)5-Nagy
the flux between muscle-derived stem cells and satellite cell mice were purchased from Jackson ImmunoResearch Laboratories (Zam-
browicz et al., 1997; Hadjantonakis et al., 1998). Heterozygous mice lack-
tiers is an ongoing process or is normally limited to a re- ing Pax7 gene, provided by Dr. Peter Gruss (Max-Planck Institute for Bio-
stricted period during development. physical Chemistry, Gottingen, Germany), were maintained by breeding
Muscle SP cells clearly possess the ability to form multiple with C57BL/6.
hematopoietic colonies in vitro as well as hematopoietic dif-
FACS®
ferentiation in vivo after intravenous injection into irradi- Hoechst staining and FACS® analysis was performed as described previ-
ated mice (Gussoni et al., 1999). Recently, two groups dem- ously (Jackson et al., 1999). In brief, hindlimb skeletal muscle was digested
onstrated that CD45-positive cells within skeletal muscle with collagenase type B and dispase II (Roche). About 2  107 cells were
normally obtained from four adult mice (1- to 2-mo old). Bone marrow
contain HSC activity (Kawada and Ogawa, 2001; McKin- was prepared as described previously (Goodell et al., 1997). FACS® analy-
ney-Freeman et al., 2002). We also demonstrated that cells sis was performed on a cell sorter (MoFlo®; Cytomation, Inc.) equipped
from muscle SP with in vitro hematopoietic potential are with dual lasers. Hoechst staining was performed in DME supplemented
Published October 14, 2002

Myogenic specification of muscle SP cells | Asakura et al. 133

with 2% FCS, 10 mM Hepes, 5 g/ml Hoechst 33342 (Sigma-Aldrich), and experiments as described previously (Sabourin et al., 1999). Cultures were
with or without 50 M verapamil (Sigma-Aldrich) at 37C for 90 min. After maintained for 4 d in myoblast growth medium on collagen-coated dishes.
Hoechst staining, immunostaining was performed by using antibodies re-
active to Sca-1 or CD45 conjugated with phycoerythrin or FITC (PharMin- Hematopoietic colony assays
gen), respectively. Hoechst dye was excited at 351 nm by UV laser and its Cells prepared from muscle, bone marrow, peripheral blood, and isolated
fluorescence was detected at two wavelengths using 424/44 (Hoechst muscle fibers were cultured in Methocult M3434 or M3630 (StemCell
blue) and 675/LP (Hoechst red) filters. FITC or phycoerythrin was excited Technologies) for 10–14 d. The number of colonies consisting of 50 cells
at 488 nm by argon laser and its fluorescence was detected at FL1 (530/40) were scored using an inverted microscope (Axiovert 25; Carl Zeiss Micro-
or FL2 (580/30) filter, respectively. Dead cells and debris were excluded Imaging, Inc.). Hematopoietic cells were identified by May-Grunwald’s
from the plots based on propidium iodide staining (2 g/ml; Sigma- Giemsa (Sigma-Aldrich) and by immunohistochemistry with Mac1 and
Aldrich). Sorted cells were characterized by X-gal staining or immunohis- Gr1 antibodies (PharMingen) for detection of granulocyte/monocyte or
tochemistry on slide glass. B220 antibodies (PharMingen) for detection of pre-B cells followed by
Vectastain ABC kit (Vector Laboratories).
Cell culture
Single muscle fibers and dissociated muscle cells (muscle-derived cells)
We thank Stephen J. Tapscott and Shahragim Tajbakhsh for providing
were isolated from hindlimb skeletal muscles prepared from 2-month-old
MD6.0-lacZ and Myf5-nlacZ strains of mice, respectively. We thank J. Ish-
heterozygous Myf5-nlacZ or MD6.0-lacZ transgenic mice by digestion in
ibashi for making retrovirus. We also thank Sophie Charge for critical read-
0.4% collagenase type A (Roche) as described previously (Beauchamp et
ing of the manuscript.
al., 2000). Isolated muscle fibers were immediately fixed or cultured in
This work was supported by grants to M.A. Rudnicki from the National
Methocult M3434 (StemCell Technologies Inc.). 5  103–104 FACS® frac-
Institutes of Health, the Canadian Institutes of Health Research, the Muscu-
tionated TgN(GFPU)5-Nagy (GFP)–, ROSA26-, or Myf5-nlacZ–muscle SP
lar Dystrophy Association, and the Canada Research Chair Program. A.
cells were cultured or co-cultured with equal numbers of either primary
Asakura is supported by a development grant from the Muscular Dystro-
myoblasts (Sabourin et al., 1999), C2C12 myoblasts, or C3H10T1/2 fibro-
phy Association. P. Seale holds a pre-doctoral fellowship from the Cana-
blasts. Conditioned medium was obtained from growing primary myo-
dian Institutes of Health Research. M.A. Rudnicki holds the Canada Re-
blasts cultured for 4 d. Muscle SP cells were also purified from 2-week-old
search Chair in molecular genetics and is an international scholar of the
Pax7/ and Pax7/ mice. pHAN-puro retrovirus vector was used as a
Howard Hughes Medical Institute.
control retrovirus vector, and retroviruses were prepared as previously de-

Downloaded from jcb.rupress.org on March 15, 2010

scribed (Soneoka et al., 1995). pHAN-eGFP and pHAN-MyoD contain vi- Submitted: 20 February 2002
rus LTR and CMV promoter–driving enhanced GFP gene and MyoD gene, Revised: 30 July 2002
respectively. 1  104 FACS®-fractionated muscle SP cells were cultured in Accepted: 28 August 2002
myoblast growth media on collagen-coated dishes for 24 h, followed by
infection with retrovirus vectors for 3 h with polybrene. 24 h after retrovi-
rus infection, cultures of muscle SP cells alone or together with equal num-
bers of primary myoblasts were performed. Cultures were maintained for References
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