1 Tissue Stem Cell Research Team, Mitsubishi Kagaku Institute of Life Sciences,
11 Minamiooya, Machida, Tokyo 194-8511, Japan
2 Fine Structure Analysis Section, Mitsubishi Kagaku Institute of Life Sciences,
11 Minamiooya, Machida, Tokyo 194-8511, Japan
3 Kitasato University, School of Science, Department of Biosciences, 1-15-1
Kitasato, Sagamihara, Kanagawa 228-8555, Japan
* Author for correspondence (e-mail: nao{at}libra.ls.m-kagaku.co.jp)
Accepted 11 August 2004
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SUMMARY |
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Key words: Myoblast transfer therapy, Muscle regeneration, Muscle satellite cell
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Introduction |
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Skeletal muscle exhibits a great capacity for regeneration after injury.
Muscle stem cells, also known as muscle satellite cells, are responsible for
the repair and regeneration of adult skeletal muscle tissue. Histopathological
analysis has shown that satellite cells differentiate into myotubes and
myofibers exclusively (Saito and Nonaka,
1994). However, the multipotentiality of satellite cells was
suggested by studies of both primary cultured mouse myoblasts and an
immortalized mouse myoblastic cell line
(Asakura et al., 2001
;
Katagiri et al., 1994
;
Teboul et al., 1995
;
Yamamoto et al., 1997
).
Furthermore, our previous clonal analyses indicated that mouse and human
muscle satellite cell-derived clones actually preserve the ability to undergo
myogenic, osteogenic and adipogenic terminal differentiation in vitro
(Wada et al., 2002
).
It has been suggested that satellite cells are highly heterogeneous in
nature. Satellite cells have been shown to exhibit diversity in relation to
the type of fibers in which they reside. Satellite cells originating from slow
or fast muscles form myotubes in vitro that express different isoforms of
myosin heavy chains (Qu et al.,
2000; Rosenblatt et al.,
1996
). After transplantation, myoblasts fuse with host myofibers
expressing the same type of myosin heavy chain
(Qu et al., 2000
). In
addition, clonal analysis suggests that satellite cells within the same muscle
proliferate at different rates in vitro
(Molnar et al., 1996
;
Schultz et al., 1985
;
Yablonka-Reuveni et al.,
1987
). However, whether multipotentiality is a common property of
all satellite cells or is preserved in only a limited fraction of satellite
cells remains unknown.
Two populations of satellite cell-derived myogenic cells have been
identified in rat muscle (Rantanen et al.,
1995; Schultz,
1996
): a minority of cells divide slowly and represent a
population with stem cell-like properties, whereas the majority divide rapidly
and are committed to terminal differentiation. The former remain
undifferentiated when cultured under differentiation-inducing conditions, and
are involved in the generation of rapidly dividing cells. Thus, these
stem-like cells were designated reserve cells
(Schultz, 1996
). The two
populations were also identified in myogenic cultures derived from a single
cell (Baroffio et al., 1996
).
Myogenic cells with stem cell-like properties might be necessary for muscle to
regenerate on transplantation (Beauchamp et
al., 1999
). However, their ability to reconstitute skeletal muscle
has not been determined because they have not been identified and purified
from the primary myogenic culture prior to transplantation.
We have established a unique primary culture system of mouse and human
myogenic cells, in which muscle satellite cells located on isolated single
myofibers are able to grow clonally and form colonies containing only their
descendants (Wada et al.,
2002). This culture system enables us to follow the sequence of
events occurring in each colony derived from a single satellite cell. Using
this culture system, the present study indicates that the majority of mouse
muscle satellite cell clones retain both myogenic and osteogenic
differentiation potential in vitro. We have identified two distinct cell types
in single-satellite cell-derived colonies: `round cells', which divide slowly,
and `thick cells', which divide rapidly. Round cells, representing activated
satellite cells that are the immediate descendants of quiescent satellite
cells, are capable of self-renewal and the generation of rapidly dividing
progeny, thick cells. We showed that round cells can efficiently contribute to
muscle regeneration when transplanted into injured adult mouse host muscle.
The identification and isolation of activated satellite cells with stem
cell-like properties from adult muscles shown here has significant
implications for successful myogenic cell transfer in human muscle
diseases.
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Materials and methods |
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To enhance clonal growth of round cells, single fibers were cultured in pmGM supplemented with human basic fibroblast growth factor (bFGF; 10 ng ml-1; PeproTech EC, London, UK) and mouse leukemia inhibitory factor (LIF; 1000 U ml-1; AMRAD Biotech, Victoria, Australia).
For induction of osteogenic terminal differentiation, cells were cultured in pmGM supplemented with recombinant human bone morphogenetic protein 2 (BMP2; 250 ng ml-1; PeproTech EC), for the indicated period.
Time-lapse recording
For time-lapse observation, single myofibers isolated from gastrocnemius
muscles of an 8-week-old C57Bl/6 mouse were plated on a 35-mm collagen-coated
dish and cultured in pmGM for 8 days. Cells were then placed in a humid
chamber (Olympus, MI-MIBC), maintained at 37°C, and observed under a
microscope (Olympus, IX-81) with a 10x Plan Apo Fluor objective lens.
Time-lapse images were taken by a CCD camera (CoolSnap HQ; Roper Scientific,
Atlanta, GA), with MethaMorph image analysis software (Roper Scientific).
Immunofluorescence and immunocytochemical analyses
For immunofluorescence or immunocytochemical analysis, sections and
paraformaldehyde-fixed cultured cells were incubated for 12 to 36 hours at
4°C with mouse monoclonal antibodies to Pax7
(Ericson et al., 1996)
(Developmental Studies Hybridoma Bank, Iowa City, IA), sarcomeric myosin heavy
chain (MHC) (Bader et al.,
1982
) or dystrophin (Sigma, St Louis, MO), or rabbit polyclonal
antibodies to green fluorescence protein (GFP; Medical Biological Laboratory,
Nagoya, Japan) or myogenin (Hashimoto et
al., 1994
). Cy3-labeled antibodies to mouse immunoglobulin G or
biotinylated-labeled antibodies to rabbit immunoglobulin G (Jackson
ImmunoResearch Laboratory, Bar Harbor, ME) were used as secondary antibodies.
The biotinylated antibodies were detected with streptavidin-conjugated
Alexa488. Nuclei of cells were visualized by staining with
2,4-diamidino-2-phenylindole dihydrochloride n-hydrate (DAPI) (0.5
µg ml-1; Sigma, St Louis, MO).
Alkaline phosphatase (ALP) activity was detected as described previously
(Wada et al., 2002).
Intramuscular cell transplantation
Round cells were detached from the culture vessel without enzymatic
treatment because they are loosely attached to the substratum. Round cells
were washed with Dulbecco's modified phosphate buffered saline [PBS: 0.8 g/l
NaCl, 0.2 g/l KCl, 1.15 g/l Na2HPO4, 0.28 g/l
KH2PO4 (pH 7.4)] and detached by gentle pipetting in PBS
supplemented with 0.02% EDTA. Cells were collected by centrifugation,
suspended in hDMEM supplemented with 10% FBS, and centrifuged again. Then the
cells were resuspended in 100 µl Leibovitz-15 (L-15; Sigma) and an aliquot
used for counting cell numbers. Finally, cells were centrifuged again,
resuspended in L-15 at 1000 cells µl-1, and kept on ice until
transfer.
Thick cells were detached by incubation in 0.05% trypsin and 0.53 mM EDTA (Invitrogen, San Diego, CA), collected by centrifugation, and then resuspended in L-15 at 1000 cells µl-1 for the transfer of 5000 cells, or at 5x104 cells µl-1 for the transfer of 106 cells. Each gastrocnemius muscle of 8- to 10-week-old host C57Bl/6 or mdx-nude (kindly provided by Drs Takahashi and Partridge) mice was injected with 50 µl of 10 µM cardiotoxin (CTX; Wako Pure Chemical Industries, Osaka, Japan), on the day before transplantation to induce muscle regeneration. Five microliters of L-15 containing 5000 cells were injected longitudinally into each gastrocnemius muscle of the host mice using a gel-loading tip (Sorensen BioScience, Salt Lake City, UT). To transfer a large number of cells into a muscle, 20 µl of L-15 containing 106 thick cells was injected using a Hamilton syringe with a 27 G needle. Host mice were sacrificed at 14 or 28 days after cell transfer, and gastrocnemius muscles were isolated. Isolated muscles were fixed with 4% paraformaldehyde overnight, embedded in OCT compound (Sakura Finetechnical, Tokyo, Japan), frozen, and sectioned at a thickness of 12 µm with a cryostat.
Scanning electron microscopy
Cells were fixed with 2.5% glutarardehyde in 0.1 M cacodylate/HCl buffer
overnight at 4°C, and were then treated as described previously
(Takeuchi et al., 1995).
Ion-coated samples were viewed with a scanning electron microscope (S4500,
Hitachi).
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Results |
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Histochemical analysis indicated that ALP expression was induced exclusively in thick cells but not in round cells (Fig. 4A,B). The temporal change of BMP2 responsiveness of satellite cell descendants in the myofiber culture paralleled the time course of the round cell to thick cell conversion (Fig. 4C,D). The conversion occurred earlier in the single-fiber culture of soleus muscle (Fig. 1B). Actually, even when exposed to BMP2 on days 2-4, ALP was induced in certain soleus colonies containing thick cells (13.3%; Fig. 4D).
The present results suggest that round cells represent activated satellite
cells, whereas thick cells represent multipotent progenitor cells
(multiblasts) (Wada et al.,
2002). Taken together, the results suggest that round cells are
converted to thick cells prior to both myogenic and osteogenic terminal
differentiation.
Basic FGF and LIF synergistically suppress both round cell to thick cell conversion and myogenic terminal differentiation
Differences in responsiveness to differentiation signals between round
cells and thick cells support the notion that the efficiency of their
contributions to muscle regeneration in vivo on transplantation differs in the
two cell types of satellite cell descendants. However, we were unable to
obtain a sufficient number of round cells to test this because round cells are
spontaneously converted into thick cells during culture. To achieve continuous
clonal expansion of round cells without conversion to thick cells, we cultured
isolated myofibers from gastrocnemius muscles of adult mice in pmGM
supplemented with various growth factors. Basic FGF (bFGF) or LIF markedly
inhibited the expression of MHC (Fig.
5A-C). When combined, bFGF and LIF synergistically inhibited
myogenic differentiation (Fig.
5D,E). In addition, bFGF, but not LIF, markedly enhanced the
proliferation of round cells (Fig.
5E).
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Round cells have the ability to restore dystrophin in myofibers of mdx mice
To determine whether round cells have the capacity to restore dystrophin in
the muscle of mdx nude mice, which lack functional dystrophin due to a genetic
mutation (Sicinski et al.,
1989), 5000 round cells derived from GFP-transgenic mice were
injected into gastrocnemius muscles of mdx nude mice that had received an
intramuscular injection of CTX on the day before transplantation. We did not
find any revertant fibers in the gastrocnemius muscles in this series of
experiments (Fig. 8A). After 28
days, GFP-positive myofibers (approximately 10-30/gastrocnemius muscle) were
identified in cryosections of host muscles
(Fig. 8B). Immunofluorescence
analysis shows that dystrophin was restored in approximately 10% of
GFP-positive myofibers (Fig.
8C). Thus, round cells representing immediate descendants of
quiescent satellite cells have the capacity to restore dystrophin in the
muscle of mdx nude mice.
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Discussion |
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In many previous studies, more than 105 myoblasts have been transferred intramuscularly because only a minor fraction with stem cell-like characteristics contributed to muscle reconstruction. Thus, myoblast cultures must be expanded to collect sufficient numbers of the stem-like cells, but the stem-like cells can be lost during extensive culture because they divide slowly, whereas the other cells divide rapidly. Round cells, however, can efficiently regenerate skeletal muscle after intramuscular injection even when the transferred cell number is as small as 5000. Therefore, the isolation of cells with stem cell-like properties is required to obtain sufficient numbers of these cells in culture.
The presence of two subpopulations of myogenic cells has been demonstrated
in postnatal muscle: a majority of rapidly dividing cells and a minority of
slowly dividing reserve cells (Rantanen et
al., 1995; Schultz,
1996
). The latter stem-like subpopulation has been identified
within primary myogenic cultures derived from single cells
(Baroffio et al., 1995
;
Baroffio et al., 1996
) and the
myogenic cell line C2C12 (Hashimoto and
Ogashiwa, 1997
; Yoshida et
al., 1998
). These results suggest that the term `myoblast' has
been used simply to describe a mononucleated, undifferentiated cell with the
potential to undergo myogenic terminal differentiation
(Beauchamp et al., 1999
). We
identified round cells in our single-fiber culture system that can grow
satellite cells clonally, excluding contamination of non-myogenic cells
through careful isolation of single myofibers
(Wada et al., 2002
). Round
cells divide slowly, whereas thick cells divide rapidly. In addition, we found
that round cells generate thick cells after self-renewal. Thus, round cells
retain the stem cell-like properties suggested in previous studies:
self-renewing, slowly cycling, and generating a rapidly dividing progeny that
undergo terminal differentiation. Stem-like cells in human myogenic cells
decreased in number with successive passages
(Baroffio et al., 1996
). We
also found that round cells are gradually lost during prolonged culture and
successive passages. Round cells have the capacity of regenerating skeletal
muscle in vivo, a capability that is attributed to a subpopulation with stem
cell-like characteristics in myoblast culture
(Beauchamp et al., 1999
). Taken
together, round cells correspond to the stem-like subpopulation described
previously both in vitro (Baroffio et al.,
1996
) and in vivo (Beauchamp et
al., 1999
).
The stem cell-like subpopulation has been identified only retrospectively
by its ability to survive in vivo and its apparent lack of distinct phenotypic
traits under differentiation-inducing conditions in vitro. In contrast to the
previous studies, we can identify round cells by their rounded appearance, and
can follow their fate progressively both in vitro and in vivo. Using this
culture system, we found a recapitulation of the ability to respond to
terminal differentiation-inducing signals in a muscle satellite cell lineage.
Round cells do not differentiate even when cultured under myogenic or
osteogenic differentiation-inducing conditions, whereas their progeny, thick
cells, respond to these conditions and undergo myogenic or osteogenic terminal
differentiation. Therefore, round cells and thick cells are distinct cell
types at different stages of maturation/differentiation in a muscle satellite
cell lineage: round cells are activated satellite cells that preserve
multipotentiality but do not differentiate; their progeny, thick cells, are
multipotent progenitors, previously designated multiblasts
(Wada et al., 2002), that
retain the ability to respond to differentiation-inducing signals.
Our clonal culture system reveals that round cells with stem cell-like properties appear in fiber culture independently of type of fiber origin. The present study also indicates that multipotentiality is preserved in the majority of satellite cells despite their type of fiber origin. In culture, muscle satellite cell descendants derived from slow muscle spontaneously undergo myogenic terminal differentiation earlier than those from fast muscle, although the processes of myogenesis, colonization of round cells, conversion of round cells to thick cells, and myotube formation by thick cells, are conserved among them. The finding that the number of round cells in each colony declines earlier in cultures originating from slow muscle suggests that round cells derived from slow muscle retain relatively low potential for self-renewal. It is well known that severe atrophy is induced in slow muscle rather than fast muscle under unloaded conditions such as low gravity and bed rest. Differences in the self-renewal activity of round cells between slow and fast muscles might be involved in the symptoms of atrophy.
Various factors in donor cells may affect the myofiber reconstruction upon transplantation: cell type of muscle-derived cells, stage of cell differentiation, cell modification during culture, and number of transplanted cells. Pre-treatment of host muscle (injection of CTX, irradiation, or none) also affects muscle reconstruction. In the present study, we compared the efficiency of muscle regeneration produced by cells at different stages of maturation/differentiation in a satellite cell lineage under basically identical conditions. Although the survival rate of the transplanted cells was not monitored, the number of GFP-positive myofibers and the expression level of GFP should indicate the extent of survival, proliferation and differentiation of the transplanted cells in vivo. Five thousand round cells had the capacity to reconstitute myofibers expressing GFP at a high level in host muscles efficiently at 28 days after transplantation, whereas these cells had formed only a few myofibers expressing GFP at a reduced level at 14 days. The results imply that transplanted round cells continue to proliferate and provide myonuclei after transplantation.
Quiescent satellite cells actually preserve a prominent capacity for muscle
regeneration, as was also suggested by whole or sliced muscle transplantation
(Schultz et al., 1988;
Smythe et al., 2001
). The
required number of transplanted cells for the formation of a single myofiber
in vivo is estimated to be 6.6 quiescent satellite cells. Round cells also
preserve an ability to reconstruct host muscle that, although much lower than
that of quiescent satellite cells, is approximately 20-50 times that of thick
cells. The number of quiescent satellite cells is approximately 3-5% of nuclei
in skeletal muscle (Gibson and Schultz,
1983
) (R. Umeda and N.H., unpublished). Thus, it does not seem
possible to collect a sufficient number of quiescent satellite cells from
skeletal muscle directly. Transplantation of a large number of thick cells is
another strategy to improve muscle repair by cell transplantation. However,
numbers of transplanted cells are limited by host muscle capacity and by the
culture scale for cell preparation. The present study suggests that
transplanted round cells contribute to new myofibers for a prolonged period in
vivo. Thus, round cells may be a possible cell source of myogenic cell
transfer therapy in the future.
The restoration of dystrophin was observed in approximately 10% of
GFP-positive fibers after transplantation of round cells to mdx nude mice.
Incorporated donor nuclei may supply only a limited amount of dystrophin that
covers the limited area designated the nuclear domain of a host myofiber
(Kong and Anderson, 2001).
Therefore, incorporation of a large number of donor nuclei into host myofibers
is required to produce amounts of dystrophin adequate to be distributed over
the whole area of the plasma membrane of a host fiber. By contrast,
incorporation of a relatively small number of donor nuclei from GFP-round
cells may be sufficient to produce fluorescence along the whole length of a
host myofiber because GFP is a very diffusible protein and is highly expressed
in donor nuclei.
The present results indicate that control of the conversion from round cell to thick cell is crucial to expand the number of round cells with stem cell-like properties in culture. We found that bFGF and LIF synergistically enhance the proliferation of round cells and suppress their conversion to thick cells. However, the present culture conditions cannot prevent the conversion during a prolonged culture period and successive passages. Further improvement of culture conditions is required to obtain sufficient numbers of round cells for cell transfer therapy. Time-lapse recording of round cell behavior reveals that round cells are converted to thick cells without cell division and that the conversion is triggered by cell-to-cell contact (Fig. 2). These observations suggest that culture conditions capable of avoiding cell-to-cell contact may inhibit the conversion.
In a clinical trial of human myoblast transfer on a patient with DMD, many
nuclei of transplanted myoblasts were incorporated into host myofibers but did
not express dystrophin (Gussoni et al.,
1997). The clinical trial, however, suggested that donor factors,
including the ability of cells to proliferate, differentiate, and function in
vivo, may be crucial for successful myoblast transfer therapy. Rapid death of
transferred cells is a serious problem in DMD muscle. Therefore, isolation and
maintenance of a subpopulation of human muscle satellite cell descendants
equivalent to the mouse round cells presented here could greatly enhance the
potential of myogenic cell transfer therapy. Pax7 is an essential
transcription factor for muscle satellite cell specification
(Seale et al., 2000
) and is
exclusively expressed in adult satellite cells
(Wada et al., 2002
). The high
level of Pax7 expression in mouse stem-like cells has significant implications
for the identification and isolation of human satellite cell descendants with
stem cell-like properties.
Our findings raise a simple question. Why did many previous studies not
describe the stem-like cells that are similar to round cells? Only a few
studies have reported results showing the presence of myogenic cells with a
morphology similar to that of round cells
(Praud et al., 2003;
Torrente et al., 2001
). Based
on the present results, both high cell density and a relatively low
concentration of growth factors may enhance the conversion of round cells to
thick cells in early passages. It is conceivable that most `myoblast' cultures
reported previously had lost round cells in early passages.
Taking the above results together, we conclude that round cell transfer has significant potential for the improvement of myogenic cell transfer therapy in human muscle disorders.
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ACKNOWLEDGMENTS |
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