Research Article
1285
Muscle regeneration by reconstitution with bone
marrow or fetal liver cells from green fluorescent
protein-gene transgenic mice
So-ichiro Fukada1, Yuko Miyagoe-Suzuki2, Hiroshi Tsukihara1, Katsutoshi Yuasa2, Saito Higuchi1,
Shiro Ono3, Kazutake Tsujikawa1, Shin’ichi Takeda2 and Hiroshi Yamamoto1,*
1Department
2Department
of Immunology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan
of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo 187-8502,
Japan
3Division
of Oncogenesis, Biomedical Research Center, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
*Author for correspondence (e-mail: [email protected])
Accepted 19 December 2001
Journal of Cell Science, 115, 1285-1293 (2002)  The Company of Biologists Ltd
Summary
The myogenic potential of bone marrow and fetal liver cells
was examined using donor cells from green fluorescent
protein (GFP)-gene transgenic mice transferred into
chimeric mice. Lethally irradiated X-chromosome-linked
muscular dystrophy (mdx) mice receiving bone marrow
cells from the transgenic mice exhibited significant
numbers of fluorescence+ and dystrophin+ muscle fibres. In
order to compare the generating capacity of fetal liver cells
with bone marrow cells in neonatal chimeras, these two cell
types from the transgenic mice were injected into busulfantreated normal or mdx neonatal mice, and muscular
generation in the chimeras was examined. Cardiotoxininduced (or -uninduced, for mdx recipients) muscle
regeneration in chimeras also produced fluorescence+
muscle fibres. The muscle reconstitution efficiency of the
bone marrow cells was almost equal to that of fetal liver
cells. However, the myogenic cell frequency was higher in
fetal livers than in bone marrow. Among the neonatal
Introduction
During the past decade, mesenchymal stem cells in adult bone
marrow, although forming a heterogeneous population, have
been characterized by their ability to differentiate into various
types of tissue-specific cells (Pereira et al., 1995; Prockop,
1997; Pittenger et al., 1999; Liechty et al., 2000) (reviewed by
Clark and Keating, 1995; Bianco and Cossu, 1999; Bianco and
Gehron-Robey, 2000). These cells produce colonies of
fibroblasts, osteogenic cells or adipocytes in vitro. In vivo
experiments have shown that they differentiate to form fibrous
tissue, bone or cartilage when transplanted into the appropriate
animal.
Wakitani et al. (Wakitani et al., 1995) were the first to show
that mesenchymal stem cells from rat bone marrow have the
ability to differentiate into myotubes when they are cultured in
the presence of 5-azacytidine. In vivo transplantation of such
cells into the muscles of X-chromosome-linked muscular
dystrophy (mdx) mice induces dystrophin-positive muscle
fibres (Saito et al., 1995). These results prompted investigators
to speculate that the transplanted bone marrow cells can move
into damaged muscle and grow into new muscle fibres in mice
chimeras of normal recipients, several fibres expressed
the fluorescence in the cardiotoxin-untreated muscle.
Moreover, fluorescence+ mononuclear cells were observed
beneath the basal lamina of the cardiotoxin-untreated
muscle of chimeras, a position where satellite cells are
localizing. It was also found that mononuclear
fluorescence+ and desmin+ cells were observed in the
explantation cultures of untreated muscles of neonatal
chimeras. The fluorescence+ muscle fibres were generated
in the second recipient mice receiving muscle single cells
from the cardiotoxin-untreated neonatal chimeras. The
results suggest that both bone marrow and fetal liver cells
may have the potential to differentiate into muscle satellite
cells and participate in muscle regeneration after muscle
damage as well as in physiological muscle generation.
Key words: Muscle regeneration, Transplantation, Satellite cell
(for a review, see Cossu and Mavilio, 2000). This also has
clinical implications, not only for muscular dystrophy but also
for various tissue-specific hereditary disorders. Recently,
several investigators have reported that transplanted bone
marrow cells participate in the muscle regeneration process in
irradiated recipient mice (Ferrari et al., 1998; Gussoni et al.,
1999; Bittner et al., 1999). In these studies, bone marrow cells
were distinguished from recipient cells by donor-specific gene
expression signals such as nuclear β-galactosidase or the
presence of a Y chromosome. However, muscle fibres
containing donor-cell-derived chromosomes never exceeded
1% of the total fibres in an average muscle.
It is widely accepted that postnatal growth and repair of
skeletal muscle are normally mediated by the satellite cells that
surround muscle fibres. Satellite cells are mononucleate
precursor cells that are located beneath the basal lamina and
sarcolemma of myofibres (Mauro, 1961) (for reviews, see
Bischoff, 1994; Cullen, 1997). Satellite cells first appear in the
limbs of mouse embryos between embryonic day 16 and 18,
and the cell number reaches a peak in neonatal mice. In adult
mice, less than 5% of the total nuclei are satellite cells, and this
1286
Journal of Cell Science 115 (6)
gradually declines with age (Cossu et al., 1983) (for reviews,
see Campion, 1984; Mazanet and Franzini-Armstrong, 1986;
Bischoff, 1994). In chicken, satellite cells appear during late
embryogenesis (Hartley et al., 1992). Adult bone marrow
contains mesenchymal stem cells from which muscle precursor
cells can be supplied; however, there is no definitive evidence
as to whether or not satellite cells are recruited from bone
marrow to muscle under physiological or pathological
conditions. Moreover, it has not yet been clarified whether the
bone-marrow-derived cells exist as satellite cells in the
radiation chimera experiments.
In this report we demonstrate that the injection of bone
marrow or fetal liver cells from EGFP-transgenic (GFP-Tg)
mice into neonatal mice establishes tolerance to allogeneic
donor cells, and the muscle-reconstituting efficiencies of these
two cell populations were compared. Moreover, we provide the
first report that neonatally injected cells are recruited to the
muscle, where they probably reside as satellite cells, and
participate in normal muscle development. The combined
procedure – neonatal tolerance induction with cells from GFPTg mice – is a useful strategy for further studies of satellite cell
recruitment and muscle regeneration.
Materials and Methods
Animals and cells
Specific pathogen-free C57BL/6 mice aged between 6 to 8 weeks were
purchased from Charles River Japan (Yokohama, Japan). Specific
pathogen-free mdx mice (of C57BL/10 background) were provided by
Central Laboratories of Experimental Animals (Kanagawa, Japan) and
maintained in our animal facility by brother-sister matings.
Heterozygous EGFP transgenic (GFP-Tg) mice with a C57BL/6
background (Okabe et al., 1997) were maintained in our animal facility
by mating with normal C57BL/6 mice. The C57BL/6 or GFP-Tg mice
were mated at night, and females were examined the next morning.
The day on which a vaginal plug was found was considered day 0 of
embryonic development. Gestational day 12 or 13 fetuses were
obtained from pregnant normal C57BL/6 mice time-mated with GFPTg heterozygous male mice. GFP-Tg fetuses were identified by brief
exposure to 365 nm UV light as previously described (Okabe et al.,
1997). Single cell suspensions of fetal liver cells from GFP-Tg
embryos were prepared by gentle teasing of the tissue. Bone marrow
cell suspensions were prepared by flushing the marrow from the
femora and tibiae of adult GFP-Tg mice, followed by gentle pipetting.
mdx/scid mice were produced by mating mdx mice with scid mice
(C57BL/6 background, purchased from Charles River Japan). The scid
phenotype was determined by measuring serum immunoglobulin level.
Bone marrow or fetal liver cell reconstitution
Bone marrow chimeras were established by the usual method for
radiation chimeras. Adult (8 week old) mdx mice were lethally
gamma-ray irradiated (10 Gy) using Gamma Cell (137Cs source,
Nuclear Canada, Ontario, Canada). 40×106 bone marrow cells from
GFP-Tg mice were injected intravenously into the recipient mice
within 3 hours after radiation. The recipient mice had received
ampicillin (product of Sigma Chemicals Co., St. Louis, MO) in their
drinking water (1 g/L) from 1 week before radiation to 2 weeks after
radiation and reconstitution. 4 weeks after reconstitution, peripheral
blood was obtained and tested for chimerisms by measuring GFP+
cells with a flowcytometer. 12 weeks after reconstitution, the muscles
and lymphoid organs of the mice were examined for muscle
regeneration and chimerism, respectively.
Neonatal chimeras were established as follows (Fig. 1). Time-
Experimental protocol for neonatal chimera
Busulfan injection
(20-40mg/kg)
test for
chimerism
pregnant (E17-18)
C57BL/6 mice
neonatal
mice
bone marrow or fetal liver
cells (from GFP-Tg mice)
injection (intra-hepatically)
4 weeks
test for
chimerism
4-13 weeks
Cardiotoxin
injection (i.m.)
(at 4wk old)
histological
analyses
Fig. 1. Experimental protocols for chimeras. For more details see the
Materials and Methods section.
mated pregnant C57BL/6 or mdx mice (at gestational day 17/18)
received 20-40 mg/kg busulfan (Sigma Chemicals Co.)
subcutaneously. Busulfan-treated neonatal mice were injected
intrahepatically with either 0.1-0.2×106 fetal liver cells or 1.0-10×106
bone marrow cells from GFP-Tg mice according to the method of
Yoder et al. (Yoder et al., 1997). 4 weeks after reconstitution, blood
samples were collected from the tail vein and used for chimerism tests.
Chimeric mice with more than 5% donor-derived GFP+ cells among
their blood mononuclear cells were selected and used for further
studies of muscle regeneration. 4 to 13 weeks later (age 8-17 weeks),
the muscles and lymphoid organs were examined.
Histological analysis
Chimeric mice were sacrificed by cervical dislocation. Tibialis
anterior (TA) and vastus lateralis (VL) muscles, or diaphragms, were
isolated and frozen in liquid nitrogen-cooled isopentane. Cryosections
(6 µm) were examined for GFP+ muscle fibres under a confocal laser
scanning microscope (model MRC1024ES, Bio-Rad Laboratories,
Hercules, CA). Excitation/emission wavelengths for GFP were 488
nm/522 nm. Sections were then stained with hematoxylin/eosin (HE).
Immunohistochemistry
For immunohistochemical examinations, serial transverse
cryosections (6 µm) or cultured tissues were stained with various
antibodies. Alexa-568-conjugated monoclonal anti-dystrophin
(MANDRA-1, Sigma) was a gift from M. Imamura (National Institute
of Neuroscience, Tokyo, Japan). Polyclonal rabbit anti-desmin (ICN
Pharmaceuticals, Inc.-Cappel Products, Aurora, OH) and anti-laminin
(MEDAC GmbH, Wedel, Germany), together with rhodamineconjugated goat anti-rabbit IgG (ICN Pharmaceuticals), were used to
stain muscles. The signals were recorded photographically using
an Axiophot microscope (Carl Zeiss, Oberkochen, Germany) and
laser scanning confocal imaging system MRC1000 (Bio-Rad
Laboratories).
Test for blood chimerism
Blood samples of 4-week-old busulfan-treated neonatal chimeras
were examined for chimerism. For flowcytometric analyses of
chimerism, mononuclear cells from the blood, spleen and thymus of
chimeras were stained with monoclonal antibodies, phycoerythrinor biotin-conjugated anti-CD4 (RM4-5) and anti-CD8 (53-6.7)
(PharMingen, San Diego, CA) for donor-derived T lymphocytes, and
analyzed together with GFP and phycoerythrin-Cy5-streptavidin
(Cederlane, Westbury, NY) (Kawakami et al., 1999a; Kawakami
et al., 1999b). GFP fluorescence was measured at the same
Muscular regeneration in neonatal chimeras
1287
Fig. 2. Flow cytometric analyses of leukocyte chimerisms. (A) Splenocyte chimerisms of radiation-bone marrow chimera. (B) GFP+CD4+ and
GFP+CD8+ T cells of the gated fraction of (A). (C) GFP+ cells in the peripheral blood of neonatal-bone-marrow chimera 4 weeks after
reconstitution (4 weeks old). Splenic GFP+ cells (D) and splenic GFP+CD4+ and GFP+CD8+ T cells (E) of mouse C 9 weeks after
reconstitution. (F) GFP+ bone marrow cells of mouse (C). (G) GFP+ cells in the peripheral blood of a neonatal-fetal liver cell chimera 4 weeks
after reconstitution (4 weeks old). Thymic GFP+ cells (H), and CD4 and CD8 stainings of thymocytes (I) of mouse G at 10 weeks after
reconstitution. Splenic GFP+ cells (J) and its GFP+CD4+ and GFP+CD8+ T cells (K) of mouse G. (L) GFP+ bone marrow cells of mouse G.
excitation/emission wavelength as FITC. Flow cytometric profiles
were determined with a FACSCalibur analyzer and CELLQuest
software (Becton Dickinson Immunocytometry Systems, Mountain
View, CA).
Muscle regeneration
Selected neonatal chimeras at 4 weeks of age, with >5% donorderived GFP+ cells, were anesthetized with sodium pentobarbital (75
mg/kg) (Abbott Laboratories, North Chicago, IL), and in some
experiments (Table 1), muscle regeneration was induced by injecting
75 µl cardiotoxin (CTX; 10 µM, Latoxan, Rosans, France) into the
TA muscle to necrotize the muscle according to the method of Davis
et al. (Davis et al., 1993). Some mice were left CTX-untreated. Four
to 13 weeks later, muscle regeneration and chimerism were examined.
Muscle fibre explantation
In order to detect muscular satellite cells in the reconstituted chimeras,
muscle fibres from extensor digitorium longus (EDL) muscles from
CTX-untreated neonatal chimeras were prepared and cultured
essentially according to the methods of Bischoff (Bischoff, 1986) and
Rosenblatt et al. (Rosenblatt et al., 1995). Briefly, dissected muscle
was incubated with 0.5% type I collagenase (Worthington
Biochemical, Lakewood, NJ) in Dulbecco’s modified Eagle’s medium
(Gibco BRL, Grand Island, NY) at 37°C for 90 minutes. The muscle
was transferred to fresh growth medium, high-glucose DMEM
(Gibco BRL) containing 10% FCS (Boehringer-Mannheim GmbH,
Mannheim, Germany) and penicillin (200 U/ml)/streptomycin (200
µg/ml) (Gibco BRL), and incubated in a humidified incubator at 37°C,
5% CO2 for 16 hours. The muscle mass was triturated with a firepolished wide-mouth Pasteur pipette. Fibres were transferred to a
Matrigel (Collaborative Biomedical, Bedford, MA)-coated Lab-Tek
chamber (Nalge Nunc International, Naperville, IL) and cultured for
4 days. The fibres and attached cells were fixed with 4%
paraformaldehyde in phosphate-buffered saline at room temperature
for 10 minutes. They were then permeabilized with 0.25% Triton X100 (Nacalai Tesque, Kyoto, Japan) at room temperature for 20
minutes, then non-specific binding was blocked by incubation with
5% skim milk (in PBS) for 10 minutes. The fixed fibres and cells were
stained with anti-desmin antibody at 4°C overnight and then with
rhodamine-conjugated goat anti-rabbit IgG. Samples were examined
for GFP+ and/or rhodamine+ cells under a confocal laser scanning
microscope. Excitation/emission wavelengths for rhodamine are 554
nm/573 nm.
Preparation of muscle-derived cells
Freshly isolated muscle-derived cells from neonatal chimeras were
prepared according to the method of Rando and Blau (Rando and
Blau, 1994). CTX-untreated VL muscles from neonatal chimeras were
isolated, minced and digested with dispase II (2.4U/ml) (BoehringerMannheim) and 1% collagenase P (Boehringer-Mannheim)
supplemented with CaCl2 to a final concentration of 2.5 mM. The
slurry, maintained at 37°C for 45 minutes, was triturated every 15
minutes and passed through a 37 µm nylon mesh. Single cell
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Journal of Cell Science 115 (6)
Fig. 3. Detection of GFP+
muscle fibres in the radiation
bone marrow chimeras.
H-E staining (A,C,E) and
GFP fluorescence (B,D,F) of
TA (A,B) and VL (C,D)
muscles and diaphragm (E,F)
of radiation-bone-marrow
chimera. (G) GFP+ TA
muscle fibre; (H) dystrophin
staining (Texas-Redconjugated anti-dystrophin
antibody); (I) mixed color of
(G) and (H); (J) GFP
fluorescence; (K) dystrophinstaining; (L) the mixed color
of (J) and (K) from the TA
muscle of a normal C57BL/6
mouse as a control. Bars,
100 µm (A-F) and 50 µm
(G-I).
suspension was washed and injected into CTX-treated (24 hours
before) TA muscles of mdx/scid mice. 4 weeks later, muscle sections
were histologically examined.
Results
GFP+ muscle fibre generation in radiation-bone-marrow
chimeras
Twelve weeks after reconstitution, splenic lymphocytes from
radiation-bone-marrow chimeras were examined for GFP
expression. As shown in Fig. 2A, about 85-87% of splenocytes
from chimeras expressed GFP (see also Table 1). A small
unidentified fraction (~13-15%), which expressed no GFP, may
represent either host-derived radio-resistant cells or rapidly
dividing donor cells (Kawakami et al., 1999b). CD4+ and CD8+
T cells showed normal lymphocyte development in the mice
(Fig. 2B). Thymic lymphocytes and splenic B cells were also
replaced by GFP+ cells at similar levels to total splenocytes
(data not shown).
Several GFP+ fibres were readily identified in TA (Fig.
3A,B), VL (Fig. 3C,D) muscles and diaphragm (Fig. 3E,F)
sections and are clearly distinct from surrounding GFPnegative fibres. Because one of the GFP+ fibres expressed
dystrophin, as shown in Fig. 3G-I, the fibre is thought to be
newly generated from donor-derived precursor cells (Fig. 3JL; this figure shows positive control sections of normal
C57BL/6 muscle). Although the reason for the difference of
dystrophin expression in each fibre is not yet known, GFP+ but
dystrophin-negative fibre was also seen.
GFP+ muscle fibre regeneration in neonatal chimeras
Lymphocyte chimerisms of neonatal chimeras were then
examined. In order to enhance both tolerance and blood
chimerism, mice received busulfan, an anticancer drug for
lympho-myeloid neoplasms, at embryonic day 17/18, then
received either adult bone marrow or fetal liver cells from
GFP-Tg mice within 16 hours of birth (Fig. 1). Typical
flowcytometry studies of the chimeras are shown in Fig. 2C-L.
4 weeks after neonatal injection of donor bone marrow (Fig.
2C) or fetal liver cells (Fig. 2G), the proportions of GFP+ blood
leukocyte were 26% or 18%, respectively. The frequency of
chimeras ranged from 1 to 64%, and mice having more than
5% donor-derived GFP+ cells in their blood leukocytes were
selected. At the time of muscle examination, GFP+ splenocytes
(Fig. 2D,E,J,K), thymocytes (Fig. 2H,I) and bone marrow cells
(Fig. 2F,L) were also examined for chimerisms. Normal
patterns of splenic mature T cells or immature thymocytes
were observed. The donor-derived GFP+ cells were found to
be recruited to the recipient bone marrow. The chimerisms
ranged from 5 to 78% at the time when mice were killed (see
Table 1).
CTX-induced muscle regeneration of neonatal chimeras is
shown in Fig. 4. The GFP+ muscle fibres of a neonatal bone
marrow chimera (Fig. 4B) or a fetal liver cell chimera (Fig. 4D,
F) could be detected. The frequencies of GFP+ fibres are
summarized in Table 1. It is evident that the frequencies of
GFP+ fibres in radiation-chimeras do not exceed 2%. They
show better reconstitution efficiency in their lymphocyte
chimerisms than neonatal chimeras. It is interesting to note that
the donor bone marrow or fetal liver cell number of the
neonatal chimeras was only 1:4-1:40 (for bone marrow cells)
or 1:200-1:400 (for fetal liver cells) of adult radiation-chimera
experiments; however, the frequencies of GFP+ fibres in mice
with lower lymphoid chimerisms are similar to those of
radiation chimeras. GFP+ fibres were also observed in the
CTX-untreated neonatal chimeras (mdx as recipients) (Table 1;
Experiment 6 and 7). This result suggested that the donor-
Muscular regeneration in neonatal chimeras
1289
Fig. 4. Detection of GFP+ muscle fibres and possible satellite cells in neonatal chimeras. H-E staining (A,C,E) and GFP fluorescence (B,D,F) of
CTX-treated TA muscles of neonatal chimeras. (A,B) Neonatal bone marrow chimera; (C-F) neonatal fetal liver cell chimera; (G,H) a single
GFP+ fibre with peripheral nucleus in a intact (CTX-untreated) neonatal bone marrow chimera; (I) detection of a GFP+ mononuclear cell
beneath the laminin layer (rabbit anti-laminin plus rhodamine-conjugated goat anti-rabbit IgG) (neonatal bone marrow chimera); (J-O) desminstained explanted fibres (J,L,M,O) were examined for GFP fluorescence (J,K,M,N). A GFP+ cell attaching to a single fibre was stained with
desmin (arrowheads of (L) and (O)). (P) Positive control of explanted muscle cells from GFP-Tg mouse (mixed color of desmin and GFP);
(Q-T) are muscle sections of the secondary mdx/scid recipient. Single cell suspension from intact (CTX-untreated) muscles of neonatal bone
marrow chimera was obtained and injected into CTX-treated TA muscle of the mdx/scid recipient. GFP+ fibres were observed (arrowheads of
(R) and (T)). Bar, 50 µm (A-H and Q-T).
derived bone marrow (Experiment 6) or fetal liver (Experiment
7) cells participate in the normal muscular regeneration in mdx
mice. Muscle reconstituting efficiencies of adult bone marrow
and fetal liver cells were compared. As summarized in Fig. 5,
when the efficiencies were calculated as GFP+ fibres/injected
cell numbers, fetal liver gave better results than bone marrow
1290
Journal of Cell Science 115 (6)
Table 1. Chimerisms of lymphocytes and muscle fibres
Experiment
number
(strain/condition)
Total
fibre(2)
GFP+
fibre
%
reconstitution(3)
%(spleen)
chimerism(4)
Weeks after
reconstitution(5)
CTX(6)
1,670
1,947
1,405
25
25
15
1.50
1.28
1.07
85.8
87.1
86.9
12
12
12
1×106
BM
CTX
2,498
1,767
1,645
12
4
7
0.48
0.23
0.43
7.5
5.9
12.8
8
8
8
1
2
3
4
5
6
0.2×106
FL
CTX
2,636
2,144
1,313
820
1,606
2,163
3
15
16
4
1
16
0.11
0.70
1.22
0.49
0.06
0.74
16.0
31.7
37.9
27.4
5.0
21.2
8
9
10
14
10
12
4
(B6/neonate)
3
5
6
4×106
BM
CTX
1,144
2,261
2,126
12
28
19
1.05
1.24
0.89
13.0
44.9
18.9
12
9
9
5
1
4
5
10×106
BM
CTX
2,619
1,934
1,854
69
26
20
2.63
1.34
1.08
41.5
62.7
65.4
17
17
17
6
(mdx/neonate)
1
2
3
4
6×106
BM
None
1,771
1,850
1,282
1,485
19
20
21
24
1.07
1.08
1.64
1.62
39.8
34.2
36.8
28.2
13
8
8
13
7
(mdx/neonate)
1R
1L
3L
4L
6L
0.1×106
FL
None
2,307
1,452
1,411
1,683
2,119
8
15
13
21
17
0.35
1.03
0.92
1.24
0.80
18.6
–
63.6
68.4
77.9
11
–
10
8
12
1
(mdx/radiation)
2
(B6/neonate)
3
(B6/neonate)
(B6/neonate)
Mouse
number(1)
Donor
cells
1
4
5
40×106
BM
3
9
10
Treatment
(1) Left VL (Experiment 1), right TA (Experiments 2-6) or left (L) or right (R) TA (Experiment 7) muscles were examined.
(2) Total fibre numbers were counted.
(3) GFP+ fibre×100/total fibre was calculated.
(4) % chimerisms in the spleen (GFP+ lymphocytes×100/total splenic lymphocytes) were calculated.
(5) Mice were killed and examined at the indicated weeks after the reconstitution.
(6) Left VL (Experiment 1) or right TA (Experiments 2-5) muscles were treated with cardiotoxin (CTX) and muscles were examined.
GFP+ mononuclear cells in the intact muscles of
neonatal chimeras
During the course of the study, we observed several GFP+
fibres in the CTX-untreated muscle of neonatal bone marrow
(Fig. 4H) (C57BL/6 as recipient) as well as fetal liver cell
chimeras (data not shown), but the frequency was low. It is
evident from the H-E stained picture that the GFP+ fibre has a
peripheral nucleus, suggesting that the fibre was generated
without damage by CTX. We speculate that these fibres were
derived from GFP+ satellite cells in the normal muscle. In
accordance with this observation, we detected GFP+
mononuclear cells residing beneath the laminin-positive basal
lamina of another CTX-untreated TA muscle (Fig. 4I). This is
the typical position in which muscle satellite cells reside. To
Fig. 5. Muscle-reconstituting efficiencies of bone marrow and fetal
liver cells. Muscle-reconstituting efficiencies of neonatal bone
marrow and fetal liver cells were compared as a factor of GFP+
muscle fibres/injected cell numbers. From the data shown in Table 1,
both CTX-treated normal C57BL/6 and CTX-untreated mdx recipient
mice were calculated.
investigate which of the cells are myogenic progenitor cells,
possibly satellite cells, and whether they contributed to muscle
generation, we performed a muscle explantation study. After 4
days of explantation culture, desmin+ and GFP+ mononuclear
cells were found attached to a single muscle fibre (Fig. 4J-L,
M-O).
In order to confirm whether the GFP+ mononuclear cells
of neonatal chimeras are myogenic progenitors, single cell
(GFP positive fibers / injected cells number) × 10-6
cells. Fetal livers may contain a higher frequency of muscle
progenitor cells than bone marrow.
250
0.002
0.0006
200
150
100
50
0
BM
FL
C57BL/6
(CTX-treated)
BM
FL
mdx
(untreated)
Muscular regeneration in neonatal chimeras
suspension from CTX-untreated VL muscles was obtained and
transplanted into CTX-treated TA muscles of mdx/scid mice.
As shown in Fig. 4R and T, muscles of the second recipients
showed GFP+ fibres that were derived from intact muscles of
neonatal chimeras. Collectively, these results suggest that the
GFP+ cells are located in the muscle as possible satellite cells
and may be participating in muscular generation upon muscle
damage, as well as under physiological condition.
Discussion
The chicken beta-actin promoter and cytomegarovirus
enhancer-driven EGFP gene transgenic mouse strain is a
powerful tool for studies tracing cell migration and
differentiation in animals. The muscles of these mice express
ubiquitous and strong fluorescence (Okabe et al., 1997), and
this prompted us to apply these mice to bone marrow
transplantation and muscle regeneration experiments. In
contrast to earlier bone marrow transplantation studies that
used nuclear β-galactosidase, the Y chromosome (Ferrari et al.,
1998; Gussoni et al., 1999; Bittner et al., 1999) or GFP with
the aid of anti-GFP antibody (Orlic et al., 2001) as markers for
the detection of donor-derived skeletal or cardiac muscle fibres,
GFP fluorescence can easily be detected by flow cytometry or
fluorescence microscopy (Chalfie et al., 1994; Kawakami et al.,
1999a) without any cofactor for light emission or any specific
staining procedures. As shown in this report, although the
frequency was not high enough, GFP+ fibres could be detected
in the muscles of chimeric mice.
It has long been known that the neonatal injection of
allogeneic cells induces tolerance to alloantigens of the donor
cells and frequently establishes a chimeric state. Very recently,
Liechty et al. (Liechty et al., 2000) described the
transplantation of human mesenchymal stem cells in utero into
sheep at an early gestational period and the site-specific
differentiation of these cells into to a variety of tissues, such
as chondrocytes, adipocytes, myocytes, cardiomyocytes, and
thymic and bone marrow stromas, without any detectable
immune response against the human cells. Butler et al. (Butler
et al., 2000) showed that neonatally induced tolerance to
alloantigens in rats persisted, and skeletal tissue allografts
survived, even though the rate of blood cell chimerism was
around 3%. Thus transplantation to neonatal or embryonic
animals is a promising approach for stem cell therapy for
hereditary diseases in animal models. The origin of satellite
cells and the time at which they first migrate into muscle tissues
are not yet clear. Cossu et al. (Cossu et al., 1983) reported that
satellite cells have a different sensitivity to phorbol ester than
myoblasts. Taking advantage of this characteristic, the authors
showed that satellite cells appear between day 16 and 18 of
embryonic development in mice. Muscle satellite cells account
for about 30% of sublaminar muscle nuclei in neonatal mice,
and this level decreases to less than 5% in adult mice (for
reviews, see Campion, 1984; Mazanet and FranziniArmstrong, 1986; Cossu and Molinaro, 1987; Bischoff, 1994).
These observations strongly suggested to us the idea of
introducing muscle precursor cells at the earliest possible time,
for example at birth. In the present study, neonatal injection of
allogeneic bone marrow or fetal liver cells combined with
busulfan treatment successfully induced a chimeric state in
both muscular precursor cells and hematopoietic cells.
1291
Neonatal mice make it necessary to limit the cell dose and
volume of intravenous or intrahepatic injections; however,
neonatal chimeras produced similar muscular reconstitution to
adult radiation chimeras, although neonates have less efficient
blood chimerisms. The immune response against proteins to
viral vectors or to newly introduced gene products presents a
new obstacle for both gene therapy and cell therapy. Once the
mice achieve radiation-induced or neonatally induced
immunological tolerance, they can be challenged by another
injection of cell transplantation without any immune response.
In order to examine whether donor cells migrate into
muscles, we searched for GFP+ cells in the CTX-untreated
muscles of neonatal chimeras. As shown in Fig. 4H,I, GFP+
fibre (H) as well as the GFP signal beneath the laminin-positive
layer (I) was present. We then explanted muscle fibres and
cultured them for 4 days in vitro under non-differentiating
conditions. It was reported that naïve desmin+ single human
muscle satellite cells in culture develop into two types of cells,
one fuses into myotubes and the other persists for weeks
among the myotubes (Baroffio et al., 1996). The former
expresses alpha sarcomeric actin, whereas the latter expresses
desmin. It is well documented that quiescent satellite cells in
the muscle express neither muscle-specific markers, such as
desmin and myosin, nor the MyoD family of muscle-specific
regulatory molecules. However, after muscle injury (Helliwell,
1988; Saito and Nonaka, 1994; Rantanen et al., 1995; Molnar
et al., 1996) or short term culture in vitro (Allen et al., 1991;
Kaufman et al., 1991; Creuzet et al., 1998), satellite cells
became desmin+ and MyoD+. This suggests that the desmin+
cell that attaches to a single fibre is probably a muscle satellite
cell. Such cells were observed in the muscle fibre cultures in
our present study (Fig. 4L,O), and they are GFP+. GFP+ donor
bone marrow or fetal liver cells may be recruited as muscle
satellite cells and may participate in muscle regeneration in
mdx or CTX-treated muscle.
We then examined whether the fibre-attached cells express
M-cadherin (Donalies et al., 1991; Irintchev et al., 1994;
Bornemann and Schmalbruch, 1994; Beauchamp et al., 2000),
but they were negative in our experiments (data not shown).
Beauchamp et al. (Beauchamp et al., 2000) reported that some
(~20%) fibre-attached satellite cells in vitro do not express Mcadherin. We observed a few fibre-attached cells, so it is
possible that M-cadherin-negative fibre-attached satellite cells
were detected in these earlier experiments. To examine whether
the GFP+ mononuclear cells become GFP+ fibres, we
transplanted the single cell suspension from muscles of
neonatal chimeras into immunodeficient mdx/scid secondary
recipients. As shown in Fig. 4R,T, GFP+ fibres were generated.
Muscle is a highly vascularised tissue and therefore
contamination from circulating blood cells can not be
excluded; however, the results suggest that the GFP+ cells
residing in the CTX-untreated muscles are muscle precursor
cells, probably satellite cells.
Recently, De Angelis et al. (De Angelis et al., 1999) reported
that the large majority of mouse clones showing typical
satellite cell morphology were derived from the dorsal aorta
not from somites. Since the cells express several vascular cell
markers, the postnatal satellite cells may be derived from a
vascular lineage. In the fetal liver, aorta-gonad-mesonephros
(AGM)-area-derived hemangioblasts differentiate to both
endothelial cells for liver vessels and hematopoietic stem cells
1292
Journal of Cell Science 115 (6)
(Miyajima et al., 2000). Endothelial precursors also arise from
the AGM (Ohneda et al., 1998). Because the AGM area
appears to be attached in clusters to the dorsal aorta, the
precursors of muscle satellite cells, vascular endothelial cells
and hematopoietic cells may derive from the same origin at an
early embryonic stage. They may move to fetal liver and then
to bone marrow in adults. Thus, the bone marrow and fetal liver
may have similar cell populations from which various types of
cells arise. As shown in Fig. 5, fetal liver cells exceeded bone
marrow cells in their myogenic potentials, and it suggests
that fetal livers contained higher proportion of myogenic
progenitors than adult bone marrows.
The frequency of donor-derived muscle fibres is still too low
for the treatment of muscular dystrophies. There are several
approaches to overcome these problems. First, it is necessary
to enrich the muscular precursor cells from bone marrow or
fetal liver cells by using various monoclonal antibodies against
cell-type-specific surface markers, such as c-Met, VEGFreceptor, Sca-1 and so on. Further trials to search for novel
markers are also necessary. Second, it is not yet known what
kind of adhesion molecules are required for the migration of
precursor cells into muscle. Neonatal or radiation-induced
tolerance, combined with injections of various monoclonal
antibodies against adhesion molecules, will answer this
question. Lastly, developing or regenerating muscle may
produce certain kinds of chemokines or cytokines that control
the trafficking of precursor cells. These mediators and their
receptors are likely to be critical for their migration. HGF and
TGFβ are possible chemotactic factors for adult muscle
satellite cells of the rat (Bischoff, 1997), but it is not yet known
what kind of factors are responsible for bone marrow- or
fetal liver-derived precursors. Although the establishment of
tolerance may not be immediately applicable to clinical trials,
the experimental system, together with the above-mentioned
strategies for the proper reconstitution of muscle, will provide
valuable information for future clinical application.
We thank M. Imamura and M. D. Ohto for the antibody used in this
study and for helpful advice, respectively. This work is supported by
grants-in-aid from the Ministry of Education, Culture, Sports, Science
and Technology, and the Ministry of Health, Labour and Welfare of
Japan.
References
Allen, R. E., Rankin, L. L., Greene, E. A., Boxhorn, L. K., Johnson, S. E.,
Taylor, R. G. and Pierce, P. R. (1991). Desmin is present in proliferating
rat muscle satellite cells but not in bovine muscle satellite cells. J. Cell
Physiol. 149, 525-535.
Baroffio, A., Hamann, M., Bernheim, L., Bochaton-Piallat, M. L.,
Gabbiani, G. and Bader, C. R. (1996). Identification of self-renewing
myoblasts in the progeny of single human muscle satellite cells.
Differentiation. 60, 47-57.
Beauchamp, J. R., Heslop, L., Yu, D. S., Tajbakhsh, S., Kelly, R. G.,
Wernig, A., Buckingham, M. E., Partridge, T. A. and Zammit, P. S.
(2000). Expression of CD34 and Myf5 defines the majority of quiescent
adult skeletal muscle satellite cells. J. Cell Biol. 151, 1221-1234.
Bianco, P. and Cossu, G. (1999). Uno, nessuno e centomila: searching for the
identity of mesodermal progenitors. Exp. Cell Res. 251, 257-263.
Bianco, P. and Gehron-Robey, P. (2000). Marrow stromal stem cells. J. Clin.
Invest. 105, 1663-1668.
Bischoff, R. (1986). Proliferation of muscle satellite cells on intact myofibers
in culture. Dev. Biol. 115, 129-139.
Bischoff, R. (1994). The satellite cell and muscle regeneration. In Myogenesis
(ed. A. G. Engel and C. Franszini-Armstrong), pp. 97-118. New York:
McGraw Hill.
Bischoff, R. (1997). Chemotaxis of skeletal muscle satellite cells. Dev. Dyn.
208, 505-515.
Bittner, R. E., Schofer, C., Weipoltshammer, K., Ivanova, S., Streubel, B.,
Hauser, E., Freilinger, M., Hoger, H., Elbe-Burger, A. and Wachtler, F.
(1999). Recruitment of bone-marrow-derived cells by skeletal and cardiac
muscle in adult dystrophic mdx mice. Anat. Embryol. Berl. 199, 391-396.
Bornemann, A. and Schmalbruch, H. (1994). Immunocytochemistry of
M-cadherin in mature and regenerating rat muscle. Anat. Rec. 239, 119125.
Butler, P. E., Lee, W. P., van de Water, A. P. and Randolph, M. A. (2000).
Neonatal induction of tolerance to skeletal tissue allografts without
immunosuppression. Plast. Reconstr. Surg. 105, 2424-2430.
Campion, D. R. (1984). The muscle satellite cell: a review. Internat. Rev.
Cytology 87, 225-251.
Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W. and Prasher, D. C. (1994).
Green fluorescent protein as a marker for gene expression. Science 263, 802805.
Clark, B. R. and Keating, A. (1995). Biology of bone marrow stroma. Ann.
NY Acad. Sci. 770, 70-78.
Cossu, G. and Mavilio, F. (2000). Myogenic stem cells for the therapy of
primary myopathies: wishful thinking or therapeutic perspective? J. Clin.
Invest. 105, 1669-1674.
Cossu, G. and Molinaro, M. (1987). Cell heterogeneity in the myogenic
lineage. Curr. Top. Dev. Biol. 23, 185-208.
Cossu, G., Molinaro, M. and Pacifici, M. (1983). Differential response of
satellite cells and embryonic myoblasts to a tumor promoter. Dev. Biol. 98,
520-524.
Creuzet, S., Lescaudron, L., Li, Z. and Fontaine-Perus, J. (1998). MyoD,
myogenin, and desmin-nls-lacZ transgene emphasize the distinct patterns of
satellite cell activation in growth and regeneration. Exp. Cell Res. 243, 241253.
Cullen, M. J. (1997). Muscle regeneration. In Dystrophin: Gene, Protein and
Cell Biology. (ed. S. Brown, and J. Lucy), pp.233-273. Cambridge:
Cambridge University Press.
Davis, H. L., Miche, M. L. and Whalen, R. G. (1993). DNA-based
immunization induces continuous secretion of hepatitis B surface antigen
and high levels of circulating antibody. Human Mol. Genet. 2, 1847-1851.
De Angelis, L., Berghella, L., Coletta, M., Lattanzi, L., Zanchi, M.,
Cusella-De-Angelis, M. G., Ponzetto, C. and Cossu, G. (1999). Skeletal
myogenic progenitors originating from embryonic dorsal aorta coexpress
endothelial and myogenic markers and contribute to postnatal muscle
growth and regeneration. J. Cell Biol. 147, 869-878.
Donalies, M. Cramer, M., Ringwald, M. and Starzinski-Powitz, A. (1991).
Expression of M-cadherin, a member of the cadherin multigene family,
correlates with differentiation of skeletal muscle cells. Proc. Natl. Acad. Sci.
USA 88, 8024-8028.
Ferrari, G., Cusella-De-Angelis, G., Coletta, M., Paolucci, E., Stornaiuolo,
A., Cossu, G. and Mavilio, F. (1998). Muscle regeneration by bone
marrow-derived myogenic progenitors. Science 279, 1528-1530.
Gussoni, E., Soneoka, Y., Strickland, C. D., Buzney, E. A., Khan, M. K.,
Flint, A. F., Kunkel, L. M. and Mulligan, R. C. (1999). Dystrophin
expression in the mdx mouse restored by stem cell transplantation. Nature
401, 390-394.
Hartley, R. S., Bandman, E. and Yablonka-Reuveni, Z. (1992). Skeletal
muscle satellite cells appear during late chicken embryogenesis. Dev. Biol.
153, 206-216.
Helliwell, T. R. (1988). Lectin binding and desmin staining during
bupivicaine-induced necrosis and regeneration in rat skeletal muscle. J.
Pathol. 155, 317-326.
Irintchev, A., Zeschnigk, M., Starzinski-Powitz, A. and Wernig, A. (1994).
Expression pattern of M-cadherin in normal, denervated, and regenerating
mouse muscles. Dev. Dyn. 199, 326-337.
Kaufman, S. J., George-Weinstein, M. and Foster, R. F. (1991). In vitro
development of precursor cells in the myogenic lineage. Dev. Biol. 146, 228238.
Kawakami, N., Nishizawa, F., Sakane, N., Iwao, M., Tsujikawa, K., Ikawa,
M., Okabe, M. and Yamamoto, H. (1999a). Roles of integrins and CD44
on the adhesion and migration of fetal liver cells to the thymus. J. Immunol.
163, 3211-3216.
Kawakami, N., Sakane, N., Nishizawa, F., Iwao, M., Fukada, S.-I.,
Tsujikawa, K., Kohama, Y., Ikawa, M., Okabe, M. and Yamamoto, H.
(1999b). Green fluorescent protein-transgenic mouse:Immune functions and
its application to the studies of lymphocyte development. Immunol. Lett. 70,
165-171.
Muscular regeneration in neonatal chimeras
Liechty, K. W., MacKenzie, T. C., Shaaban, A. F., Radu, A., Moseley,
A.-M. B., Deans, R., Marshak, D. R. and Flake, A. W. (2000). Human
mesenchymal stem cells engraft and demonstrate site-specific
differentiation after in utero transplantation in sheep. Nat. Medicine 6,
1282-1286.
Mauro, A. (1961). Satellite cells of skeletal muscle fibers. J. Biophys.
Biochem. Cytol. 9, 493-495.
Mazanet, R. and Franzini-Armstrong, C. (1986). The satellite cell. In
Myology, vol. 2 (ed. A. G. Engel and B. Q. Banker), pp. 285-307. New York:
McGraw Hill.
Miyajima, A., Kinoshita, T., Tanaka, M., Kamiya, A., Mukouyama, Y. and
Hara, T. (2000). Role of Oncostatin M in hematopoiesis and liver
development. Cytokine Growth Factor Rev. 11, 177-183.
Molnar, G., Ho, M. L. and Schroedl, N. A. (1996). Evidence for multiple
satellite cell populations and a non-myogenic cell type that is regulated
differently in regenerating and growing skeletal muscle. Tissue Cell. 28,
547-556.
Ohneda, O., Fennie, C., Zheng, Z., Donahue, C., La, H., Villacorta, R.,
Cairns, B. and Lasky, L. A. (1998). Hematopoietic stem cell maintenance
and differentiation are supported by embryonic aorta-gonad-mesonephros
region-derived endothelium. Blood. 92, 908-919.
Okabe, M., Ikawa, M., Kominami, K., Nakanishi, T. and Nishimune, Y.
(1997). ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett. 407,
313-319.
Orlic, D., Kajstura, J., Chimenti, S., Jakoniuk, I., Anderson, S. M., Li, B.,
Pickel, J., McKay, R., Nadal-Ginard, B., Bodine, D. M., Leri, A. and
Anversa, P. (2001). Bone marrow cells regenerate infarcted myocardium.
Nature 410, 701-705.
Pereira, R. F., Halford, K. W., O’Hara, M. D., Leeper, D. B., Sokolov, B.
P., Pollard, M. D., Bagasra, O. and Prockop, D. J. (1995). Cultured
1293
adherent cells from marrow can serve as long-lasting precursor cells for
bone, cartilage, and lung in irradiated mice. Proc. Natl. Acad. Sci. USA 92,
4857-4861.
Pittenger, M. F., Mackay, A. M., Beck, S. C., Jaiswal, R. K., Douglas, R.,
Mosca, J. D., Moorman, M. A., Simonetti, D. W., Craig, S. and Marshak,
D. R. (1999). Multilineage potential of adult human mesenchymal stem
cells. Science 284, 143-147.
Prockop, D. J. (1997). Marrow stromal cells as stem cells for
nonhematopoietic tissues. Science 276, 71-74.
Rando, T. A. and Blau, H. M. (1994). Primary mouse myoblast purification,
characterization, and transplantation for cell-mediated gene therapy. J. Cell
Biol. 125, 1275-1287.
Rantanen, J., Hurme, T., Lukka, R., Heino, J. and Kalimo, H. (1995).
Satellite cell proliferation and the expression of myogenin and desmin in
regenerating skeletal muscle: evidence for two different populations of
satellite cells. Lab. Invest. 72, 341-347.
Rosenblatt, J. D., Lunt, A. I., Parry, D. J. and Partridge, T. A. (1995).
Culturing satellite cells from living single muscle fiber explants. In Vitro
Cell. Dev. Biol Animal 31, 773-779.
Saito, T., Dennis, J. E., Lennon, D. P., Young, R. G. and Caplan, A. I.
(1995). Myogenic expression of mesenchimal stem cells within myotubes
of mdx mice in vitro and in vivo. Tissue Engineering 1, 327-343.
Saito, Y. and Nonaka, I. (1994). Initiation of satellite cell replication in
bupivacaine-induced myonecrosis. Acta Neuropathol. 88, 252-257.
Yoder, M. C., Hiatt, K. and Mukherjee, P. (1997). In vivo repopulating
hematopoietic stem cells are present in the murine yolk sac at day 9.0
postcoitus. Proc. Natl. Acad. Sci. USA 94, 6776-6780.
Wakitani, S., Saito, T. and Caplan, A. I. (1995). Myogenic cells derived from
rat bone marrow mesenchymal stem cells exposed to 5-azacytidine. Muscle
Nerve 18, 1417-1426.