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DEVELOPMENTAL DYNAMICS 208:505–515 (1997)
Chemotaxis of Skeletal Muscle Satellite Cells
RICHARD BISCHOFF*
Department of Anatomy and Neurobiology, Washington University School of Medicine, Saint Louis, Missouri
ABSTRACT
Migration of myogenic cells occurs extensively during both embryogenesis and
regeneration of skeletal muscle and is important
in myoblast gene therapy, but little is known
about factors that promote chemotaxis of these
cells. We have used satellite cells from adult rats
purified by Percoll density gradient centrifugation to test growth factors and wound fluids for
chemotactic activity in blind-well Boyden chambers. Of a variety of growth factors tested only
hepatocyte growth factor (HGF) and transforming growth factor-beta (TGF-b) exhibited significant chemotactic activity. The dose-response
curves for both of these factors was bell-shaped
with maximum activity in the 1–10 ng/ml range.
Checkerboard analysis of TGF-b showed that
chemotaxis occurred only in response to a positive concentration gradient. An extract of rat
platelets also exhibited chemotactic activity for
satellite cells. Half-maximal activity of this material was about 3 mg/ml, and there was no evidence
of inhibition of migration at high concentrations.
Checkerboard analysis of platelet extract exhibited evidence of both chemotaxis and chemokinesis, or increase in random motility of cells. Inhibition experiments showed that most, but not all, of
the chemotactic activity in platelet extract could
be blocked with a neutralizing antibody to TGF-b.
A saline extract of crushed muscle was found to
contain both mitogenic and motogenic factors for
satellite cells. The two activities were present in
different fractions after heparin affinity chromatography. We propose that the proliferation and
migration of satellite cells during regeneration is
regulated by overlapping gradients of several
effector molecules released at the site of muscle
injury. These molecules may also be useful for
enhancing the dispersion of injected myoblasts
during gene therapy. Dev. Dyn. 208:505–515,
1997. r 1997 Wiley-Liss, Inc.
Key words: skeletal muscle; satellite cell; regeneration; platelet; TGF-b; HGF
INTRODUCTION
Skeletal muscle precursor cells are capable of migration throughout life. Early in development, myogenic
cells emigrate from the somites into the limb buds
where they proliferate and fuse to form the primary
myotubes of the appendicular muscles (Chevallier et
al., 1977; Christ et al., 1977). This migration is induced
r 1997 WILEY-LISS, INC.
by a signal arising from the mesoderm of the lateral
body wall (Hayashi and Ozawa, 1995) and is dependent
upon attachment of the cells to fibronectin present in
the extracellular matrix that forms the substratum for
migration (Brand-Saberi et al., 1993).
Once muscle differentiation is underway, most myogenic cells become enclosed by the myotube basal
lamina; however, these satellite cells are capable of
limited migration between myotubes. During the early
postnatal period in rat development, proliferating satellite cells labeled in situ with a retrovirus subsequently
give rise to progeny, some of which move across basal
laminae and fuse with neighboring myofibers (Hughes
and Blau, 1990). The myofiber basal lamina becomes
thicker and stronger with age (Kovanen et al., 1988),
and most satellite cells become quiescent, so it is likely
that little migration occurs in the adult under normal
conditions.
After muscle trauma, however, there is abundant
evidence for the directed migration of satellite cells in
response to injury. During the free grafting of larger
muscles, satellite cells migrate from the central necrotic area toward the periphery (Schultz et al., 1988),
and later, during revascularization, they migrate back
toward the center where they undergo myogenesis
(Phillips et al., 1987). Satellite cells also migrate from
the viable half to the killed half of a longitudinally split
muscle autograft (Phillips et al., 1990). Localized muscle
trauma apparently produces factors that stimulate
chemotaxis of satellite cells from distant sites (Watt et
al., 1994). Focal crush injury at one end of a muscle
results in the activation and movement of satellite cells
beneath the basal lamina from distant uninjured tissue
toward the crush site (Schultz et al., 1985). If the injury
stimulus is perpendicular to the direction of the myofibers, some cells are able to cross the basal lamina of
uninjured myofibers and migrate transversely through
the muscle toward the injury (Klein-Ogus and Harris,
1983).
In addition to migrating within the muscle, satellite
cells are even capable of moving between adjacent
muscles under certain conditions. Experiments using
genetic variants of metabolic enzymes to identify donor
and host tissue in adult mice demonstrated that grafted
regenerating muscle becomes invaded by myogenic
cells migrating from neighboring muscles (Watt et al.,
*Correspondence to: Richard Bischoff, Ph.D., Department of Anatomy
and Neurobiology, Box 8108, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110.
Received 4 October 1996; Accepted 27 December 1996
506
BISCHOFF
1987). Similar experiments carried out in adult rats,
however, failed to show satellite cell migration from
adjacent muscles (Ghins et al., 1984), and it was
suggested that the thicker epimysium of rat muscle
may block invasion (Partridge, 1991). This was confirmed by the demonstration that regenerating rat
muscle would be invaded by satellite cells from an
adjacent muscle only if a ‘‘bridge’’ of tissue was created
between the two muscles by damaging the epimysium
(Schultz et al., 1986). There is evidence that migration
of myogenic cells between muscles depends upon the
presence of soluble chemotactic factors, perhaps released during muscle regeneration or inflammation
(Moens et al., 1996).
Taken together, these studies demonstrate the extensive migratory capacity of satellite cells, but little is
known of the chemotactic signals involved. Venkatasubramanian and Solursh (1984) reported that quail limb
mesoderm cells respond chemotactically to plateletderived growth factor (PDGF), but the cells were tested
after their migration from the somites and were not a
pure population. More recently, Daston et al. (1996)
have shown that the transcription factor Pax-3 is
necessary for emigration of myogenic cells from the
somites in mouse embryos. This factor has been implicated in regulating the expression of the c-met-encoded
receptor tyrosine kinase, also essential for myogenic
cell emigration (Bladt et al., 1995). Mice bearing null
mutations of this allele are devoid of limb muscles
owing to failure of migration of muscle precursor cells
from the somites. The ligand for the c-met receptor is
hepatocyte growth factor (HGF; Bottaro et al., 1991),
and the forced expression of HGF in the nervous system
induces ectopic formation of skeletal muscle (Takayama
et al., 1996). These studies indicate that early migration of myogenic cells, probably in response to a chemoattractant, plays an essential role in the origin of appendicular muscles. This chemotactic responsiveness may
persist in satellite cells of adult muscle, the descendants of embryonic myoblasts.
The present study was undertaken to test several
defined growth factors and components of wound fluid
for chemotactic activity towards muscle satellite cells.
Results show that HGF and TGF-b are potent chemoattractants. Chemotactic activity is also present in extracts of platelets and crushed muscle.
RESULTS
Preliminary experiments were carried out to determine the optimum incubation time (Fig. 1A) and satellite cell concentration (Fig. 1B) for chemotaxis. A saline
extract of crushed skeletal muscle from adult rats was
used as the chemoattractant (Robertson et al., 1993; see
below). Based upon these results, chemotaxis chambers
were loaded with 1,500 satellite cells/mm2 filter surface
and incubated for 7 hr.
Because growth factors are released during muscle
regeneration and may serve as chemoattractants, we
Fig. 1. Effect of cell density and incubation time on chemotactic
response of satellite cells to 0.5 mg/ml crushed muscle extract. A:
Chemotactic chambers were incubated for 1–7 hr before fixation and
staining. Cells were counted on both sides of the filter. B: Various
concentrations of satellite cells were placed in the upper well and
incubated for 6 hr. Cells were scraped from the upper surface of the filter
before fixation, and only migrated cells were counted.
tested the response of satellite cells to a range of
concentration of purified growth factors (Table 1). Several isoforms of PDGF were tested because this has
been reported to be a chemoattractant for chick embryo
cells (Venkatasubramanian and Solursh, 1984) and
mouse myoblasts (Robertson et al., 1993). Although
basic fibroblast growth factor (bFGF) and PDGF are
mitogens for satellite cells (DiMario and Strohman,
1988; Allen and Boxhorn, 1989; Allen and Rankin,
1990; Yablonka-Reuveni et al., 1990; Jin et al., 1991),
they exhibited, as did epidermal growth factor (EGF),
only weak chemotactic activity. PDGF-AB at the highest concentration (2,000 ng/ml) induced migration to
only twice the level of the negative control value, as
compared with the almost sevenfold stimulation of
embryo extract, used as a positive control. HGF and
TGF-b, however, displayed significant chemotactic activity, amounting to 5–6 times that of the negative
control wells. Both factors displayed evidence of bell-
CHEMOTAXIS OF SATELLITE CELLS
507
TABLE 1. Migration of Satellite Cells in Response
to Various Growth Factors
Attractant
None
5% chick EE
PDGF-AA
PDGF-AA
PDGF-AA
PDGF-BB
PDGF-BB
PDGF-BB
PDGF-AB
PDGF-AB
PDGF-AB
bFGF
bFGF
bFGF
EGF
EGF
EGF
HGF
HGF
HGF
TGF-b
TGF-b
TGF-b
Concentration
(ng/ml)
—
—
12
25
100
12
25
100
125
500
2,000
1
10
100
10
100
1,000
1
10
100
2.5
5.0
25.0
Cells crossing
filter (%)a
6.7 6 1.7
45.0 6 2.6*
3.5 6 0.8
8.7 6 2.8
10.6 6 2.4
4.9 6 1.8
4.7 6 1.0
7.0 6 2.1
4.6 6 0.9
3.4 6 1.6
12.1 6 2.9
8.8 6 2.2
5.2 6 0.8
5.6 6 2.9
9.0 6 2.3
9.6 6 1.7
5.8 6 1.5
23.0 6 2.4*
36.4 6 1.2*
3.2 6 0.8
18.2 6 2.6*
29.6 6 2.3*
8.2 6 1.2
aThese
results are representative of at least two experiments
with similar results. The results are expressed as the mean 6
standard error of the mean.
*Values significantly different from the negative control
(P , .05).
Fig. 2. Chemotactic response of satellite cells to increasing concentrations of TGF-b.
shaped dose-response curves, and this was confirmed
by testing a wider range of TGF-b concentrations (Fig.
2). Maximum stimulation of migration occurred at 5
ng/ml for TGF-b, and chemotaxis was suppressed to
control level by 50 ng/ml. A bell-shaped dose-response
curve is characteristic of various purified chemoattractants and cell types (Falk et al., 1980; Lucas et al.,
1988; Adelman-Grill et al., 1990; Phillips et al., 1991).
To examine the response of satellite cells to TGF-b in
more detail, a checkerboard analysis was carried out
(Zigmond and Hirsch, 1973). In this procedure, the
concentration of chemoattractant in wells is varied both
Fig. 3. Checkerboard analysis of chemotaxis for TGF-b. The concentration of TGF-b was varied in both the upper (vertical columns) and lower
(horizontal rows) chambers. The diagonal column of gray boxes represents increasing levels of identical concentrations in both chambers.
Values in the boxes are the percent cells migrating across the filter. The
SEM was less than 20% in all cases; the individual error values were
omitted for clarity.
above and below the filter to establish positive, negative, and null concentration gradients. Chemotaxis is
observed in response to a positive concentration gradient, whereas migration in the presence of a negative or
zero gradient of test substance indicates increased
random motility of cells, or chemokinesis. This analysis
showed that migration occurred only in response to a
positive concentration gradient; there was no evidence
of chemokinesis (Fig. 3). Inhibition of migration in some
instances of high concentration gradient reflects the
bell-shaped dose-response curve observed previously.
Muscle injury leads to the formation of wound fluid
that contains a wide variety of substances released
from the injured tissue, blood, and inflammatory cells.
Previous work has shown that a saline extract of
crushed adult muscle is a potent mitogen for myogenic
cells (Kardami et al., 1985; Bischoff, 1986; Chen and
Quinn, 1992; Haugk et al., 1995), and this material was
also tested for chemotactic activity (Fig. 4). Crude
muscle extract stimulated chemotaxis in a dosedependent manner with about the same half-maximal
activity, 0.4 mg/ml, as that found for the mitogenic
effect (Bischoff, 1986). There was no evidence of inhibition at the highest concentration tested, 1 mg/ml.
Previous work has shown that the satellite cell mitogen
in muscle extract is a heparin-binding growth factor
(Bischoff, 1989; Bischoff, 1990b). When the extract was
partially purified by heparin affinity chromatography,
508
BISCHOFF
reduced, but did not abolish, the activity of platelet
extract (Fig. 8). At the highest concentration tested,
treatment with anti-TGF-b produced 65% inhibition of
chemotaxis as compared with control antibody.
DISCUSSION
Fig. 4. Chemotactic response of satellite cells to increasing concentrations of a crude extract of crushed adult rat muscle.
chemotactic activity was found to reside in the non–
heparin-binding fraction (Fig. 5), whereas all mitogenic
activity for satellite cells is contained in the heparinbinding fraction that elutes from the column with 1 M
NaCl (Bischoff, 1989; Bischoff, 1990b). Although an
extensive dose-response analysis of the non–heparinbinding fraction was not done, the maximum activity
(20 µg/ml) of the several concentrations tested appears
to represent at least a 20-fold purification as compared
with the crude extract.
Another component of wound fluid arises from the
secretion of platelets that are activated during injury
and trigger the clotting mechanism to promote hemostasis. Physical trauma leads to extravasation of blood into
the tissue interstices and is often accompanied by
widespread bruising. To test for chemotactic factors,
platelets were isolated from fresh rat blood and washed,
and secretory products were collected after exposure to
thrombin. The platelet extract exhibited chemotactic
activity for satellite cells with a half-maximal activity
of about 3 µg/ml, and no inhibition of migration was
observed up to 30 µg/ml, the highest concentration
tested (Fig. 6). Checkerboard analysis of platelet extract exhibited evidence of both chemotaxis and chemokinesis (Fig. 7). Increased cell migration occurred whenever there was a positive gradient across the filter, but
migration also occurred in the presence of a negative or
null gradient at high extract concentration. This suggests that platelet extract stimulates both chemotaxis
and chemokinesis of satellite cells in a concentrationdependent manner.
Because platelets are a major source of TGF-b, it is
possible that this factor is responsible for the chemotactic activity of platelet extract. To examine this, we
pretreated platelet extract with an antibody capable of
neutralizing the activity of TGF-b. Both platelet extract
and neutralizing antibody were tested at two concentrations. The antibody treatment completely eliminated
chemotactic activity of purified TGF-b and greatly
Migration of myogenic cells from nearby viable muscle
to a site of injury provides an important means of
augmenting the population of precursor cells during
regeneration. A variety of muscle trauma conditions
stimulate migration including ischemia (Phillips et al.,
1987; Schultz et al., 1988), thermal injury (Morgan et
al., 1987; Phillips et al., 1990), crushing (Schultz et al.,
1985; Watt et al., 1994), and snake venom toxin (KleinOgus and Harris, 1983). Little is known, however,
concerning factors that guide this migration. The findings described here demonstrate that satellite cells are
induced to migrate in response to concentration gradients of several soluble factors known to be released
during muscle injury. These factors may play a role in
the recruitment of satellite cells from distant sites after
injury.
The most interesting chemoattractant identified in
this study is TGF-b, because of extensive prior studies
on the roles of TGF-b in regulating myogenesis. The
TGF-bs comprise a family of at least five closely related
homodimeric proteins first purified from platelets but
also present in many cells and tissues (Roberts and
Sporn, 1991; Massagúe et al., 1992). The effects of TGF-b
on myogenic cells in vitro are complex and depend upon
concentration, medium composition, and cell type. With
satellite cells grown in serum-containing medium, the
most consistent effect observed is suppression of proliferation. Both rat and pig satellite cells exhibit 50–60%
inhibition of proliferation at concentrations of TGF-b in
the 0.1–0.5 ng/ml range (Allen and Boxhorn, 1987;
Pampusch et al., 1990; Cook et al., 1993). Bovine
satellite cells are highly sensitive to TGF-b; maximum
inhibition occurs at 0.001 ng/ml, and mitotic inhibition
diminishes at higher concentrations (Blachowski et al.,
1993). In these studies, the cells were already in the cell
cycle after stimulation by serum mitogens, but TGF-b
is also able to prevent the G0 = G1 transition of
quiescent satellite cells. Bischoff (1990b) reported that
0.05 ng/ml TGF-b blocks proliferation of satellite cells
on single, viable myofibers stimulated with an extract
of crushed muscle. Inhibition of proliferation by TGF-b
may augment its role in chemotactic recruitment of
satellite cells. Because motility is suppressed in mitotic
cells, antiproliferative factors would increase the efficiency of directed migration of satellite cells to the site
of injury.
The response of satellite cells to TGF-b is concentration dependent, but the level of TGF-b after muscle
injury has apparently not been investigated. Cromack
et al. (1987) collected wound fluid from chambers
implanted subcutaneously in rats and found about 1
CHEMOTAXIS OF SATELLITE CELLS
Fig. 5. Chemotactic response to various fractions of crushed muscle
extract. The crude extract was passed through a heparin-Sepharose
affinity column and separated into an unbound fraction and a bound
fraction eluted from the column with 1 M NaCl. Control chambers
509
contained culture medium only, and the extract fractions were tested at a
range of concentrations. Asterisks indicate values significantly different
from the control (P , .05).
Fig. 6. Chemotactic response of satellite cells to increasing concentrations of a saline extract of thrombin-stimulated rat platelets.
ng/ml TGF-b at 3 days, the earliest time measured. The
level reached a peak of about 20 ng/ml at 7 days.
Because the chambers were put in place with minimal
tissue damage, the levels of TGF-b would be higher in
trauma situations involving extensive platelet secretion. TGF-b is also secreted by inflammatory cells, such
as macrophages (Massagúe et al., 1992), and Robertson
et al. (1993) have reported that macrophage secretory
products induce a chemotactic response in muscle
precursor cells. TGF-b may also arise from the injured
muscle tissue itself. Yamazaki et al. (1994) have re-
Fig. 7. Checkerboard analysis of chemotaxis for platelet extract. The
concentration of extract was varied in both the upper (vertical columns)
and lower (horizontal rows) chambers. The diagonal column of gray boxes
represents increasing levels of identical concentrations in both chambers.
Comparison of the 150 µg/ml row and column shows that migration is
stimulated at high concentration of platelet extract with positive, null,
and negative concentration gradients. Values in the boxes are the
percent cells migrating across the filter. The SEM was less than 20% in all
cases.
510
BISCHOFF
Fig. 8. Inhibition of the chemotactic response to TGF-b and platelet
extract by treatment with an antibody to TGF-b. Platelet extract or TGF-b
was incubated 1 hr with neutralizing antibody to TGF-b (5 or 50 µg/ml) or
control IgG (100 µg/ml) before testing in chemotactic chambers. Asterisks
indicate values significantly different from the control antibody for each
chemoattractant (P , .05).
ported that TGF-b is homogeneously localized in necrotic myofibers in Duchenne muscular dystrophy. Production of TGF-b from all sources would lead to a
concentration gradient originating at the site of injury.
In certain injuries, such as blunt trauma, the extensive
hematoma extending some distance from the center of
impact would spread the release of TGF-b from extravasated platelets even further. Because of the bell-shaped
dose-response curve (Fig. 2), satellite cells migrating
centripetally from uninjured areas may reach a point at
which the concentration of TGF-b inhibits further
migration. Concentrations of TGF-b that inhibit chemotaxis are comparable to the maximum levels found in
wound fluid (Cromack et al., 1987).
Satellite cells proliferate at the site of injury during
muscle regeneration, but it is not clear how the antimitotic effects of TGF-b are overcome, thereby allowing
the cells to enter the mitotic cycle. One possibility is
that the anti-mitotic effects of TGF-b are diminished at
higher concentrations, as has been demonstrated for
bovine satellite cells (Blachowski et al., 1993). Alternatively, because the response of myogenic cells to TGF-b
depends upon the presence of other peptide growth
factors (Allen and Boxhorn, 1989; Zentella and Massagúe, 1992; Blachowski et al., 1993; Cook et al., 1993),
the migrating cells may encounter a pro-mitotic environ-
ment as they approach the site of injury. According to
this model (Fig. 9), injury-related mitogens, such as
those present in crushed muscle extract (Bischoff, 1986,
1990a), are localized to the wound site, whereas TGF-b
is more diffuse owing to widespread platelet extravasation. The mitogenic and motogenic behavior of migrating satellite cells may be determined by overlapping
concentration gradients of various effectors.
The present study has also provided the first evidence that HGF, which has been implicated as a
chemoattractant for myogenic cells in the early embryo
(Bladt et al., 1995; Takayama et al., 1996), may also
carry out this function in satellite cells during muscle
regeneration. We found that migration was stimulated
by HGF in the 1–10 ng/ml range and inhibited at higher
concentration. Jennische et al. (1993) have reported
that mRNA for HGF is first expressed in regenerating
muscle 3 days after injury, the earliest time measured,
and declines again to barely detectable levels by a week
after injury. Although concentrations of HGF were not
measured, its location and time of expression are
consistent with its function as a chemoattractant.
HGF may also be involved in other processes during
regeneration. Allen et al. (1995) demonstrated that
HGF acts as a mitogen for rat satellite cells in culture,
primarily by shortening the lag time before quiescent
CHEMOTAXIS OF SATELLITE CELLS
Fig. 9. Hypothetical model for the regulation of satellite cell behavior
after injury by overlapping gradients of mitogenic and motogenic factors.
Injury results in the local release of mitogen, such as crushed muscle
extract (CME), from the damaged myofibers, thereby producing a steep
gradient centered on the injury. Widespread platelet extravasation induced by the inflammatory cascade results in a broad gradient of
motogen, such as TGF-b. Satellite cells distant from the injury migrate up
the TGF-b gradient until high concentration near the injury inhibits further
movement. The high mitogen concentration at the injury site is able to
overcome the inhibition of proliferation by TGF-b.
cells enter the cell cycle after stimulation by serum
factors. This study also showed that quiescent satellite
cells express mRNA for c-met, the receptor for HGF.
Besides stimulating muscle cell movement and growth,
HGF may aid regeneration as an angiogenic factor.
HGF is a potent chemoattractant for endothelial cells in
vitro and promotes neovascularization in the cornea
(Bussolini et al., 1993; Grant et al., 1993).
The present results also suggest an approach for
improving the efficiency of myoblast transfer therapy
for neuromuscular disease. Since the demonstration by
Lipton and Schultz (1979) that implanted satellite cells
expanded in culture become incorporated into the host
myofibers, many studies have used this technique as a
method for introducing normal genes into diseased
muscle to correct the genetic defect. Although encouraging results have been obtained in dystrophic mice
(Karpati et al., 1989; Partridge et al., 1989; Morgan et
al., 1990; Kinoshita et al., 1994), the method has not yet
been effective in humans (Karpati et al., 1993; Law et
al., 1993; Tremblay et al., 1993; Mendell et al., 1995;
Morandi et al., 1995). A significant limitation is the
failure of implanted cells to spread much beyond the
511
injection site (Hughes and Blau, 1990; Satoh et al.,
1993; Rando et al., 1995). Fan et al. (1996) have
reported that implanted mouse myoblasts do not migrate more than 0.1 mm, or approximately the diameter
of two myofibers, and therefore the fraction of host
myofibers that incorporate the donor cells is small.
Morgan et al. (1996) have estimated that 2 3 106
myoblast injections would be required to generate new
muscle in an adult human. Because implanted myoblasts are more readily incorporated into regenerating
muscle (Partridge et al., 1989; Karpati, 1992; Watt et
al., 1994; Huard et al., 1994), attempts have been made
to increase the efficiency of transplant therapy by
injuring the host muscle before or during myoblast
implantation. This has increased migration in some
cases (Morgan et al., 1987; Watt et al., 1994; Huard et
al., 1994; Vilquin et al., 1995), but not others (Satoh et
al., 1993; Rando and Blau, 1994; Fan et al., 1996), and
the reason underlying the different results is not apparent. Injury to host muscle is precluded in human
therapy owing to ethical considerations, but administration of motogenic factors, along with implanted myoblasts, may be used to increase dispersion of the cells.
We have shown that platelet extract contains factors
that greatly increase the random motility of satellite
cells; identification and therapeutic use of these factors
may aid in the dispersion of implanted myoblasts.
EXPERIMENTAL PROCEDURES
Satellite Cells
Satellite cells were isolated from the hindleg muscles,
tibialis anterior, and extensor digitorum longus of 1.5to 2-month-old male Sprague-Dawley rats (Harlan,
Indianapolis, IN) (Bischoff and Heintz, 1994). After
removal of epimysium and internal connective tissue,
the muscles were minced with scissors and incubated
with constant agitation for 1 hr at 37°C in 1% Pronase
(Calbiochem, San Diego, CA) in Earle’s balanced salt
solution (EBSS). Cells were released from the tissue
fragments by vigorous trituration in Eagle’s minimal
essential medium (MEM) containing 10% horse serum.
Undissociated fragments were removed and triturated
in fresh volumes of medium for a total of 3 cycles until
no identifiable myofibers remained during microscopic
examination. Pooled cells were filtered through a 10 µm
Nitex filter (TETCO, New York, NY), centrifuged at
1,000g for 2 min and resuspended in 1.5 ml MEM with
10% horse serum before fractionation by density gradient centrifugation (Yablonka-Reuveni et al., 1987; Morgan, 1988). The cells were layered over a step gradient
of Percoll (Pharamacia, Piscataway, NJ) consisting of
3.5 ml each of 70, 50, and 35% Percoll in MEM in a 16 3
125-mm screw cap culture tube and centrifuged with
the brake off at 1,250g in a horizontal rotor for 20 min
at room temperature. Cells were collected from the
50–70% interface, corresponding to a density of 1.087
g/ml as measured with density marker beads (Sigma,
St. Louis, MO), washed in EBSS by centrifugation, and
512
BISCHOFF
Fig. 10. Phase-contrast micrographs of cultured satellite cells from
the 50–70% Percoll fraction after density gradient centrifugation. A: Cells
after 3-day culture in medium containing 10% serum and 5% embryo
extract. Most cells exhibit the plump, spindle-shaped morphology of
myogenic cells, and some have begun to align themselves. B: Cells after
an additional 2 days in medium containing 1% serum to promote
differentiation. The cells have fused to form multinucleated, branching
myotubes. Bar 5 100 µm.
suspended in growth medium consisting of MEM with
10% horse serum, 5% chick embryo extract, and 1%
antibiotic-antimycotic mixture. The cells were grown
for 3 days in gelatin-coated tissue culture dishes with
twice daily medium changes and removed from dishes
for experiments by 5 min incubation in trypsin (100
µg/ml, 1:250, Sigma). Muscles from one rat yielded
2.3 6 0.4 3 106 cells after 3 days growth (average of four
experiments). The Percoll-purified fraction consisted of
greater than 98% myogenic cells based upon morphol-
ogy (Fig. 10A) and differentiation to form multinucleated myotubes (Fig. 10B).
Chemotaxis Assay
Chemotaxis experiments were carried out in blindwell Boyden-type chambers with ten 7-mm-diameter
wells per plate (Neuro Probe, Cabin John, MD). Standard polycarbonate filters with 8 µm holes (Neuro
Probe; holes 5 6% surface area) were washed thoroughly, treated with bovine fibronectin, 25 µg/ml DW
513
CHEMOTAXIS OF SATELLITE CELLS
for 30 min, and dried. Of several extracellular matrix
proteins tested, fibronectin gave the best results in
terms of attachment, migration, and absence of drop-off
from the lower surface of the filter. In use, the bottom
wells were filled with test substance, a filter was added,
and the top wells were filled with a suspension of
satellite cells. The chambers were incubated at 37°C in
95% air and 5% CO2. After allowing for migration, the
filters were removed, fixed in alcohol:formalin:acetic
acid (20:2:1), stained in hematoxylin, and mounted on
slides.
Both a negative control, consisting of medium alone,
and a positive control, consisting of either crushed
muscle extract or chick embryo extract, was carried out
for each experiment. Drop-off of cells from the lower
surface of the filter, monitored by examining medium
from the bottom chamber in a hemocytometer, was
negligible. In early experiments, cells were scraped
from the top of the filter before counting, but for most of
the experiments reported here cells were counted on
both surfaces of the filter. Cells were counted using an
ocular grid at a magnification of 5003. The culture
medium for chemotaxis consisted of MEM with 0.5%
bovine albumin and 1% rat serum derived from plateletfree plasma prepared from fresh rat blood.
Tissue Extracts
Muscle extract was prepared by crushing whole leg
muscles of adult rats with forceps and incubating the
muscles in EBSS at 4°C for 1 hr (Bischoff, 1986). The
supernatant was concentrated about tenfold by ultrafiltration (YM10, Amicon, Beverly, MA) and used in
chemotaxis experiments as crude extract. Affinity chromatography on a column of heparin-agarose (Sigma)
was used to purify the crude extract as described
(Bischoff, 1989; Bischoff, 1990b). Embryo extract was
prepared from 11-day-old chick embryos; other tissue
culture reagents were from Sigma. Protein concentration was measured with the microbiuret method
(Munkres and Richards, 1965) using bovine albumin as
a standard.
Statistical Analysis
The data presented are from single experiments with
each factor tested in duplicate or triplicate wells and
are representative of at least two experiments. Statistical analysis of individual experiments was carried out
by comparing sample means with a two-tailed t-test
using StatS (Spreadware, Palm Desert, CA) operating
within Excel (Microsoft, Redmond, WA).
ACKNOWLEDGMENTS
Plasma and Platelet Extract
Rats were anesthetized with pentobarbital, and platelets and serum were obtained as described (Catalfamo
and Dodds, 1989). The abdominal aorta was exposed,
and blood was withdrawn using a 21-gauge needle and
collected in a syringe containing enough sodium citrate
to give a final concentration of 0.38%. The blood was
centrifuged at 550g for 2 min at room temperature, and
the platelet-rich supernatant plasma was collected.
Centrifugation was repeated 3 times, the platelet-rich
plasma was pooled, and an aliquot was diluted 1:100 for
counting in a hemocytometer. The platelets were pelleted by centrifugation at 1,800g for 15 min, and the
supernatant was used to prepare serum from the
platelet-free plasma. The platelets from the pellet were
washed twice with EBSS by centrifugation and suspended in EBSS to give 1.5 3 106 platelets/µl. Thrombin (0.5 U/ml) was added to stimulate the platelets and
cause the secretion of the alpha granules. After 10 min,
the preparation was centrifuged at 1,800g, and the
supernatant was retained as platelet extract for testing
as chemoattractant.
Antibodies and Peptides
For inhibition experiments, pan-specific TGF-b neutralizing rabbit IgG (R&D Systems, Minneapolis, MN)
was mixed with either platelet extract or TGF-b and
incubated for 1 hr at room temperature before testing
in chemotactic assays. Controls consisted of nonimmune rabbit IgG (Sigma). Purified growth factors were
obtained from both R&D Systems or Sigma and gave
comparable results regardless of source.
I thank C. Heintz and R. Frederickson for expert
assistance. This work was supported by grants from the
Muscular Dystrophy Association.
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