Vol. 8, 927-934,
August
1997
Cell Growth
Stable Integration Dystrophic (mdx) Cell Statu&
Janet
and
Smith2
Cambridge
CB2
3DY,
University
United
of Cambridge,
Downing Street,
Kingdom
Abstract We have previously described a method for the derivation of long term cuftures of undifferentiated
myoblasts
from the skeletal
muscle of dystrophic
(mdx)
mice (J. Smith and P. N. Schofield, Exp. Cell Res., 210 86-93, 1994). We now show that a clonal mdx-derived skeletal muscle cell line labeled with a retrovirus conferring fi-galactosidase actMty and G418
resistance (PD5OA) is capable of incorporation into skeletal muscle myofibers for up to 14 months with no incidence of tumor formation. After a lag period of 5 days, injected PD5OA cells disperse throughout the injected tibialis anterior muscle and take up satellite cell positions on the perimeter of myofiber bundles. PD5OA cells begin to incorporate Into fused muscle syncitium as early as 8 weeks after injection and persist for at least 14 months. We have rederived myoblasts expressing gi-galactosidase from PD5OA-injected muscles 12 months after injection, mdx
demonstrating that proliferation-competent
a reserve
of mononuclear
PD5OA cells are present in host muscle up to a year after their original introduction. These data support the contention that myoblasts derived by this cufture method are functionally representative of a class of skeletal muscle “stem cells” and thus have potential both as agents for cellular
therapy
of intransigent
diseases
such as
Introduction Cell therapy, by means of transplanting precursor or stem cells, has been suggested as a treatment for a variety of genetic diseases including Duchenne muscular dystrophy (1, 2). A prerequisite of such an approach must be the availability of stem or precursor cells appropriate to the disease
Received
Duchenne
5/15/97;
muscular
revised
The costs of publication
6/10/97;
To whom requests
333750/333783;
accepted
(3), which
is primarily
6/i 1/97.
of this article were defrayed in part by the This article must therefore be hereby marked
payment of page charges. advertisement in accordance cate this fact. 1 Supported by the Wellcorne 2
dystrophy
with Trust
for reprints
1 8 U.S.C.
Section
1 734 solely
a pathology of skeletal muscle, has been identified as an important candidate for such an approach (4) because of its relative frequency (1 of 3500 male live births) and high rate of new incidence (5). Progress in this field has, however, been restricted because of the lack of an appropriate muscle stem cell culture system. To date the majority of myogenic cells used in muscle repopulation experiments (both mouse and human) have been derived either from bulk primary cultures (1 , 2, 6), where reproducibility and cell numbers are a problem, or from established clonal muscle cell lines such as C2 or F8B (7, 8). Without
to mdi-
Foundation.
should be addressed. Phone: 01223Fax: 01223-333786; E-mail:
[email protected].
exception,
the established
lines
previ-
ously reported to incorporate into fused genic and generate rhabdomyosarcomas
muscle are tumoriin the injected
muscle
implantation.
within
the
first
6 months
after
More
recently, Morgan et al. (9) have reported the use of primary muscle cultures derived from transgenic mice carrying a thermolabile SV4O T-antigen gene (1 0). Expression of this gene at permissive temperatures immortalizes cells in culture, but as they differentiate in vivo, this approach can produce
large
numbers
of myogenic
cells
but
is probably
inappropriate for therapeutic use in humans. An additional problem that needs to be addressed is the extent of incorporation of grafted muscle cells into the target muscle fibers. Without irradiation (1 1), toxins (8, 12), or mechanical induction of muscle damage, incorporation rates of injected poor,
cells even
into differentiated in mice
and
skeletal
with
muscle
proportionally
are generally
(to muscle
size)
huge numbers of cells being injected (10 cells/muscle, Ref. 2; 5 x 1O cells per muscle, Ref. 9). The few experiments using human cells injected into human muscle (6, 13) resuIted in disappointingly low incorporation levels of injected (dystrophin-positive) largely confined
Duchenne muscular dystrophy as well as being a useful tool for the further investigation of normal muscle development.
target.
927
of an mdx Skeletal Muscle Cell Line into Skeletal Muscle: Evidence for Stem
N. Schofield
Paul
of Anatomy,
Department
& Differentiation
myoblasts, and these seemed to the area of injection. In addition,
to be either
immunosuppression, needle-assisted muscle damage, or both have been used in these studies. Injection of cells with stem cell characteristics would obviate the need for the injection
of large
because
they
appropriate
dergo
positions
repeated
therapeutic
numbers
would
of cells
within
divisions.
approach
into pre-damaged
be expected the
This
because
muscles
to migrate
injected
muscle
would the
to take
enable stem
and
up
to un-
a long-term
cell
population
would be expected to retain a small pool of undifferentiated cells, which would differentiate as required during fiber turnover. An important additional benefit of such an approach would be that true stem cells do not form tumors in an appropriate environment. We have developed a method of deriving long-term primary cultures (1 4) from the skeletal muscles of both normal and dystrophic mice, which retain the features of the original “satellite”
cell culture
cell.
Using
previously
(1 5, 16), we have
validated
been
able
principals
to readily
of stem
derive
928
Integration
of Skeletal
Muscle
Stem
Cells
Fig. 1. A, anti-skeletal myosin (red) staining of PD5OA cells in the initial stages of differentiation using tetramethylrhodamine /3-isothiocyanate-labeled second antibody. Nuclei were counterstained with DAPI, and the photograph was obtained by double exposure through rhodamine and UV filters. Labeling of these cells with anti-dystrophin and FITC-second antibody (B) demonstrates, as expected, the complete absence of these proteins. Cells were counterstained with DAPI (double exposure under FITC and UV). C: conversely, the dystrophin-related protein utrophin is present at detectable levels in all PD5OA cells. D, DAPI labeling of utrophin-positive cells in C.
clonal cell lines from such cultures without subjecting cells to the extreme selective pressure used previously the
isolation
of skeletal
lactosidase
tagging,
apparent
stem
muscle we
cell
myoblasts
have
properties
been
(1 7). Using
able
of one
to
the for j3-ga-
demonstrate
of these
cell
the
in vivo.
lines
myosin
(Fig.
clonally
derived
1A).
staining positive
with anti-dystrophin (18) antibodies for an antibody (1 9) directed against
related
protein,
To select infected
Results Isolation Myoblast rived
of 1J-Galactosidase Cell Lines. Clonal
from
established
undifferentiated,
passaged
but
blasts as described capacity of these
for calcium
and to reduce clones. both
prior
stage,
that
skeletal
SMS to
fusion,
the
cells
Skeletal were de-
stocks mdx
passaged FCS. The
primary culture
CaCI2)
to inhibit
LM
the likelihood
the morphological
densities,
primary
fusion-competent,
(70
Myoblasts
entiated
mdx lines
cell
(dfdl3)
skeletal
of myo-
elsewhere (14). To retain the proliferative cell lines, we cloned them in conditioned
medium prepared from supplemented with 20% pleted
Expressing myoblast
of selecting
have
to
cells.
become form
begin
a
cultured
retain
skeletal
containing
(pec/plRV)
known
were
as utrophin of
medium
taken
presence
of
filament
desmin.
selection cells
began
dishes
to
die.
enough
(20-50
off and expanded to
At this
a further
panded the
Surviving
clones
from
pIRV
lines
cells)
stage,
further.
round
and thus
of
derived
until
Six of these
cbonal
characterized.
placed
were
selection
One expressed
myo-
cells retained intermediate
were
colonies
cells expres-
in pIRV-infected
for 2 weeks
for picking
50%
producer
virus) cultures. until resistant
several
we with
Transient
colonies
control (0% in G41 8 selection
C-D).
1,
-gaIactosidase,
Polybrene.
cultures
cultured
except
maintained
large
Remaining
at 36 h and
non-
(Fig. 1B) and the dystrophin-
putative
was observed
and
cultures
uniformly
(Fig.
expressing
cultures
of J3-galactosidase
mdx
lines
blasts fixed at 36 h after infection, and these their immunogenicity for the skeletal muscle
at
reduced
primary
(SMS)
stably
in the
pipette.
At this muscle-specific
syncitium.
also clones
of undiffer-
and line up end-to-end,
multinucleate
to express
fusion
in this way
properties
When
bipolar
myoblast
for fusion-impaired
cultured
and antigenic
muscle
cells
been
myoblasts medium was demdx
passaged
subconfluent
virus sion
Both myoblast
G418
into
noninfected
observed
in all
Cultures colonies
off with
were were
a drawn-glass
per dish
were
picked
cultures
were
subject
before
of these cytoplasmic
was
they
were
PD5OA. -galactosid-
ex-
All of
Cell Growth
Survival
A
..
of PD5OA
Myoblasts
in mdx
& Differentiation
TA3: 0-8
929
weeks.
To determine the effect of graft size on survival of PD5OA cells in vivo and to determine the minimum number of cells
.c$’#,*’
ic#{149}
required
to obtain
muscle,
we
4000,
and 40000
avoid
potential
used mdx mice
(one
incorporation
three
different
for all of these
male
and
one
were
f3-galactosidase
the
each
after
we Two
of these
injection,
to complete
histochemistry.
TA. To
system,
experiments.
from
5 days
subject
into (400,
into mouse
immune
injection
female)
killed
and left TAs were
cells
with
mice
of PD5OA
concentrations
per 1 0 Ml) of PD5OA complications
groups
injection
right
reasonable
injected
serial
In no cases
three
and
both
section
and
did we observe
staining in the sections from the contralateral (right TA) muscles. Examination of the injected (left TA) muscles (Fig. 3) blue
revealed
that
site after
5 days
were
grafts
apparently
The majority stage (Fig. 3, E-G).
cells
mean
number
to the number
division
material characteristic
of this cell line.
ase protein as assessed by histochemistry; lected for injection experiments because intense
cytoplasmic
Morphologically, mdx passaged rived.
staining these
2B) for -gaIactosidase.
(Fig.
cells
primary
were
cultures
PD5OA was seof its uniform and
indistinguishable
from
which
they
from
the
were
de-
of 398
line,
after
injection,
which
blue-staining
the injected
of cells
were
not associated
lines
NIH-3T3TK
Polybrene.
In no experiment under
did we find
surviving
colonies
presence integration
of virus. We thus conclude in the PD5OA cell line was
either
G41 8 selection
blue
indicative
cells
or
of the
that j3-galactosidase stable and that helper
was not present.
Examination these
of metaphase cells
were
chromosomal revealed mosomal
essentially
number that
there
material
of 40.
was
a small
on two
somes.
This
may
smaller
chromosomal
satellite
material.
spreads
with
Closer
of the smaller
material
The karyotype karyotypes from those
acrocentric represent
presented
which
modal (Fig.
of additional
a reduplication
or may
in this line for the time during
revealed
a stable
examination
amount
be due to either
experimentally, i.e., PD5OA do not differ
of PD5OA
diploid
2B) chro-
chromo-
a small
different
bolus were with
observed of cells.
dispersed to be incor-
of nucleated,
tions
around
the
periphery
with
j3-galactosidase-positive
These point
and
changes.
with increased muscle fibers.
levels of No other
animals
cells
of mature
the
injected
with
of PD5OA within some sections
position
enabled single
time
of inflammatory
the distribution we counterstained the
(Fig. 3!), which injection.
at this
between
to illustrate sections
cells
observed
any signs
were
of these
presence
were
By 8 weeks
were
of the original
only
numbers
hematoxylin
nation
of this experi-
cell lines. each group)
appeared
of undispersed
To further characterize mdx TA at this time point, with
suggests
injection
cells
some
These foci were not associated PD5OA incorporation into adjoining differences
concenor that
cells
cells.
a
virtu-
muscle fibers (Fig. 3H). Blue-staining cells in satellite cell positions and adjacent to
foci
cell
in cell
(PD5OA)
small
fibroblast
that
against
of PD5OA
TA, and
cells
mouse
of the
3C) yielded
(Fig. 3H). In muscles that had been injected concentrations of cells (4,000 and 40,000), we
muscle fibers the higher
with
noted
injected of blue
This finding
or uncloned (one from
injection
to be the remnants
on the
occur
(Fig.
at this of cells
muscle
suggested
cell death.
may
after
porated into fused were also observed
of the
per TA (Fig.
in vivo selection
ments using primary cultures An additional three animals
throughout
cells
graft
of cells originally the number
sections cells
with
appeared
and NR6, as well as the parent myoblast cell line Dfdl3, at a range of concentrations up to 1 00% and in the presence of
stable
pace
cloned
at 2 months
injection
although
cells injected (at least at this almost 1 week after injection
of extensive
killed
at the
in the larger
1 7 (SD), which
±
the absence cell
400
also
these
that
had kept
counting
in serial
To determine the stability of (3-galactosidase retrovirus into the PD5OA line, we tested supematant from
integration
virus
and
with
ally all of the PD5OA tration) had survived Fig. 2. Isolation and characterization of PD5OA cells showing PDSOA cells demonstrating strong and uniform cytoplasmic staining for -gaIactosidase (A). B, karyotype of PD5OA demonstrating the normal diploid chromosomal content of these cells. Arrows, additional chromosomal
intact observed,
to disperse
A-C),
present
injected
largely
dispersal
of cells appeared to be unfused The size of the blue staining area
was roughly related (Fig. 3, compare staining
little
beginning
3D).
two mice
remained
with
of nuclei. confirmation
situated skeletal
cytoplasmic
Examiof the
in satellite
posi-
muscle
fibers
staining
and
of the additional
in Fig. 2 has been
it has been used
of cells of early passage of later passage cultures.
The
abbreviations
growth
medium;
3
2-phenylindole.
used
are:
TA,
tibialis
DAB, 3,3’-diaminobenzidine;
anterior;
mGM,
myoblast
DAPI, 4,6-diaminidino-
of
930
Integration
of Skeletal
Muscle
Stem
Cells
.,
I ‘,
6,
-
,,
‘
‘4
I I
Fig. 3. Distribution of PD50A cells after injection into mdx TA muscle. Shown are frozen muscle sections, fixed in glutaraldehyde and stained histochemically for (3-galactosidase, and counterstained with Metanil Yellow. A-G, 5 days after injection of 40,000 PD5OA cells (X 64; A), 4,000 cells (x252; B), 400 cells (x252; C), demonstrating graft integrity and cell survival at this time point; D, 40,000 cells (x252) showing the beginnings of dispersal at the edges of this graft (arrows). E-C3, higher power view (x1600) ofthe same grafts. E, 400 cells; F, 40,000 cells; G, 4,000 cells at 5 days after injection, demonstrating that the majority of cells remain unfused at this time point. Arrows in E-F, single cells. Integration of PD5OA cells into muscle fibers (H) 2 months after injection of PD5OA cells into mdx TA (xi600) is shown. Single blue cells in satellite cell positions (arrows) can also be observed. I, a residual bolus of undispersed PD5OA cells in the TA of an animal injected with 40,000 cells 2 months previously (x 1000).
newly
fused
within
mature
(3-galactosidase-positive myofiber
Long-Term mice
were
bundles
Incorporation.
14 months (n tumor formation
cells
freely
or other
appeared mixing
technique
(Fig.
4B)
these
single-nucleate,
myofibers
sections confirmed
in combination
clearly demonstrated pressing cells were
peared
with
into
already
with this
of of
of these
tibody
(Fig.
observed
animals
demonstrate myoblasts
in muscle
stained
with
to -gaIac-
of these
Using
from
from
the presence in injected mdx
mice
cells.
Both
2-5% (PD5OA)-injected
ap-
whereas
ase,
derived
no such
from
cells
were
line
(PDR).
injected injected
the
the
then
right
Muscle.
placed
staining into
contralateral
was
an-
To unequiv-
previously
noninjected
(saline-injected)
the
of single proliferating TA, we have cultured
1 2 months and
around
the perimysium.
myoblasts, (Fig. 4E).
TA.
were
which Approxi-
from the left 13-galactosid-
observed
G41 8 selection
with
muscles
of myoblasts from cell cultures TA stained positively for
mately
this
sections
cells
PD5OA
position
anti-f3-galactosidase
cultured and readily yielded primary were then stained for -gaIactosidase
muscle Labeling
TA
cell
but within
4D). of PD5OA from Injected
Recovery ocally PD5OA
bundles
fused
that uninucleate 3-gaIactosidase-exalso present in injected muscles (Fig. 4C), -galactosidase-positive
was
legs
in the satellite fiber
mdx
present.
antibodies
of muscle
cells in these the entire TA.
observation.
H&E staining
to be located
No staining
injected
= 1), and
of the left TA of injected animals at after injection of PD5OA revealed the
to be integrating
with
paraformaldehyde-fixed tosidase
(n
We found no evidence graft-related pathology in any
presence of f3-galactosidase-positive these were well dispersed throughout
PD5OA and
four
= 2), 12 months
= 1) and autopsied.
these mice. Examination both 1 0 and 1 4 months extensive muscles;
(n
present
periphery
The remaining
at 10 months
killed
myofibers 4A).
(Fig.
in cultures Some
of these
to isolate
a cell
Cell Growth
Table
1
Aepopulation
of mdx TA with
PD5OA
931
& Differentiation
muscle
stem
cells
Number of fJ-galactosidase-positive fiber bundles expressed as mean percentage of positive fiber ±SD of the injected TA of mdx mice injected with PD5OA cells. Animals were sacrificed between 1 and 14 months after intramuscular injection with PD5OA. The six animals killed at 5 days were not assessed in this manner (see “Results”). Time after injection (mo)
No. of cells injected
Gender
1 1 1
400 4,000 40,000
Male Female Female
2
400
B
10 10 14#{176}
:4 a
,.t‘‘
Extent
,
‘
I
C
-
number .
S
#{149}
also
t
#{149}1
,
obtained
from
muscle
stained
10 17 12 31 18 18 6.3
± ± ±
± ±
antibody.
The
histochemically
for
To
into and
mdx
assess TA,
nonpositive
we
the
efficiency
have
counted
fibers
present
of the
(Table
in nonad-
1). Nonadjacent
for two reasons: (a) to maintain conreports of such estimates (20), which
strategy, more
muscle
and
than
throughout
(b) to minimize
once
the
and
to
the
ensure
muscle.
PD5OA
..
positive
than
5 days
(7 in total).
from
The
20 to 71 % with
However,
percentage a mean
the level
over time, was
quency nificantly
fibers.
of PD5OA influenced
in animals
Neither
cells
and
in
dispersal
fibers
of 39.6
±
evenness
of
at 1 0 months colonization of
sacrificed
unevenness
of
(13 anifor more
of positive
incorporation
of incorporation
whereas
considerable
PD5OA-positive
.
number
a representa-
found in all of the injected muscles examined in total) and in all of the animals maintained
muscle I
this
spread
there
, .
52 34 30
distribution were greater in animals sacrificed onward (see Table 1 ), suggesting a gradual
4
%f
254 230 1071
were mals
1 5.7%.
#{149}
Female Male Male
of injected
counted
varied
‘
71
anti-j3-galactosidase
were counted with previous
used
tive
, S
D
113i
with
of positive
fibers S
Female
Incorporation.
sections
sections sistency
S.,
#{149}
39 31 20
-
, b
320 414 989
stained
incorporation
jacent
‘
of
PD5OA
.
#{149}
‘
I
were
% positive fibers (±SD)
S
I
.
in muscle
remaining data j3-galactosidase.
‘*
-
Assessed
4,000 40,000 4,000
No. of fibers counted
at 2 months
the
distribution
of
nor colonization
fre-
cells in TA muscles appeared by the gender of the animal
to be siginjected. To
our surprise, we also found that the extent of colonization those muscles injected with 400 cells was comparable that
seen
found
in muscles
injected
no -gaIactosidase
with
4,000
or 40,000
incorporation
cells.
in adjacent
3T3TK taken and
Fig. 4. Long-term incorporation of PD5OA into mdx TA muscle newly fused fJ-galactosidase-positive (arvows) myofiber (A) showing a long extension of striated -galactosidase-positive cytoplasm of three fused
PD5OA cells. The counterstain
with hemotoxylin
demonstrates
or Dfdl3 injected from
did
cells
animals rederived
not stain
incubated taken
(PDR) positively
with
either
at sacrifice
blood
or with
cell cultures were for p-galactosidase.
We
muscles
or in the muscles of the contralateral leg control, which examined carefully in every animal. Furthermore, from
in to
was NIH
samples
supernatant
killed by G41 8 These data
also demonstrate the absence of systemic or local free a-galactosidase retrovirus and, therefore, helper virus, in PD5OAinjected
mice.
the posi-
tion of nuclei in a TA injected 2 months previously. B, extensive colonization of mdx TA muscle fiber bundles (labeling is DAB-visualized anti-agalactosidase) 1 4 months after injection. C, -galactosidase-positive uninucleate cell (arrow, DAB staining) in a TA muscle injected 14 months previously with PD5OA cells. D, contralateral leg control for B and C. E, f3-galactosidase-positive cells in culture after reisolation from a muscle injection 1 2 months previously.
Discussion We injected myoblasts. fibers dIes.
1 3 animals (6 males All of these animals
and single Retention
cells
located
of unfused
and 7 females) with PD5OA had blue-staining muscle
on the periphery marked
cells
has
of fiber been
bun-
reported
of Skeletal
Integration
Muscle
Stem
Cells
previously as a rare event following injection of immortalized cells (9) and also after injection of uncloned bulk primary cell cultures (2), where clonal selection might be expected. None of our injected
animals
developed
tumors,
despite
maintain-
ing four of these animals for more than I 0 months. This is in direct contrast to injections of C2 cells (the best studied of injectable muscle cell lines) which, in common with other myogenic cell lines (8), will form sarcomas as early as 2 weeks after their injection into mouse skeletal muscle (7) with virtually
100%
frequency.
We
suggest
that
the
nontumori-
genic nature of PD5OA cells is likely to be a result of their close relationship to muscle satellite or “stem” cells, as demonstrated by their normal karyotype, positive staining for utrophin,
and
the
presence
of single,
uninucleate
PD5OA
cells located around the periphery of muscle fiber bundles in muscles injected many months previously. We have demonstrated that in vivo PD5OA is able to contribute both to differentiated myofibers and the mononuclear stem cell population over 14 months. Proliferation competent cells can be recovered from the muscle after this period of time,
indicating
the functional
stem
cell status
of this
clone.
was the lag period observed between PD5OA injection and dispersal and fusion of the injected cells. The gender of the animals did not appear to affect the percentage of incorporation of PD5OA cells, and the injection of 400 cells was found to be comparable to the injection of 4 x i03 or 4 x 10 cells in inducing widespread incorporation of PD5OA into muscle fibers. This
An important
feature
observation
of these
is potentially
experiments
of great
importance
apeutic strategies and suggests autoregulated in vivo. It is important
determined miffed
to
differentiate
to form specific
muscle
that
stem
cells
between
muscle with
to future
PD5OA
cells
ther-
may be
progenitor
fiber
a wider
cells
and com-
isotypes
potential
range
of
fates. The experiments of Hughes and Blau (21) demonstrate that there may be several uninucleate cell types in neonatal and adolescent rodent skeletal muscle of which only some will have characteristics of pluripotential stem cells; the remainder are determined to form specific fiber isotypes. Within
the context
of adult
or neonatal
muscle,
these
popu-
lations are termed respectively “muscle stem cells” or “myoblasts.” This distinction may go some way to explaining the wide
variation
in repopulation
efficiencies
observed
in many
experiments using primary cultured muscle cells or partially differentiated myoblast or myogenic cell lines (1 , 2, 22, 23). We have also recently identified a potential further complication of cell replacement studies, that of increased programmed cell death or apoptosis (24). Programmed cell death is up-regulated in dystrophic (mdx) skeletal muscle and is under the control of specific peptide growth factors. This
phenomenon
cell lines derived
is reflected from
these
in the muscles,
stem
cell
suggesting
cultures that
and it is the
survival and proliferation of this group of cells that is perturbed in dystrophic muscle. Consequently, further investigation of the control of this process in vivo would be of importance in establishing the significance of these data for future therapeutic strategies.
Although
ically repaired
repopulation
myogenic
of muscle
cells seems
with
wild-type
or genet-
to be the most powerful
approach, injection of cells into either human or mdx dystrophic muscle have to date resulted in very poor colonization (25). The induction of muscle damage by X-irradiation facilitates repopulation, which has lead to the suggestion that poor results from the injection of unlesioned muscle are related to the state of the host muscle rather than the intrinsic capacity of the injected cells to act as stem cells. Our data suggests that it is the intrinsic stem cell status of the grafted cells that determines the efficiency of colonization. Similar considerations have been important in the isolation of stem cells and committed progenitors in the immune system (15). The dispersal of PD5OA cells from the graft site is much slower than that reported for the injection of primary muscle cells or established cell lines (2, 9), and many previous experiments have been carried out over a much shorter time course, the end point often determined by the time taken to form
tumors
(8). It is apparent
from
our
data
that
complete
dispersal and incorporation of injected stem cells into muscle can take weeks to months. Earlier experiments may have, therefore, underestimated the potential extent of repopulation of injected cell grafts. However, to date, we have not observed
migration
of cells
from
the injected
muscle
(TA) to
underlying or adjacent muscles such as the extensor digitorum longus, as reported by Watt et a!. (26). We have shown, however, that after an 8-12-week lag period, PD5OA cells can disperse very efficiently throughout the target muscle and are able to contribute stably to host muscle fibers for more than one-half of the life span of the mdx mouse. Furthermore, it is clear that once injected, these cells will persist without pathological consequences for some considerable time. It is likely that this persistence is due to the ability of these cells to remain in an undifferentiated, uninucleate state within
the
injected
The retention transformation
muscle.
of proliferative of cells
assisted
enormously
PD5OA
and enabled
carried
clones
capacity
derived
under
in estimating
conditions
the repopulation
experiments out than previously. Although PD5OA and are, therefore, a powerful tool for transferring
into dystrophic
much
in the absence these
muscle,
these
longer
data
term
also
have
useful
of has
ability
of
to be similar genes implica-
tions
for the derivation of cell lines for human therapeutic program and the time course over which such program should be extended. These studies also provide insight into the behavior of normal adult muscle stem cells in vivo, which may have important implications for tumors of skeletal muscle origin. Materials
and
of
Methods
Isolation PD5OA. mdx skeletal explant cultures were established and maintained as described previously(14). Along term (uncboned) passaged primary culture (designated dfdl3) was derived from the skeletal muscle of 5-week-old mdx mice by this method and was frozen at passage 11. Myoblast cultures were maintained in mGM (a 50:50 mix of DMEM and
Ham’s F-12 medium inactivated
FCS,
supplemented
with 20% (v/v) batch tested,
heat-
2 mM glutamine, 100 units/mI penicillin, and 100 pg/mI streptomycinj. To maintain survival and an undifferentiated phenotype when cultured at low densfties(10-1O celIs/cm), myobbasts were grown in a 50:50 mix of mGM and myoblast conditioned medium (see Ref. 14 for further details). To introduce the (3-galactosidase tag to PD5OA cells, we infected cells with plRV (27, 28). pIRV is a replication-defective retrovirus that carries the genes for both (3-galactosidase and 0418 resistance,
Cell Growth
driven
from an internal
promoter
independent
of the retroviral
transcription
machinery. The viral genome is integrated into host DNA but does not carry a nuclear localization construct; therefore, all ofthe -galactosidase activity detected in a cell line will be cytoplasmic rather than nuclear. To infect mdx myoblasts with the plRV retrovirus, dfdl3 cells were plated out in 60-mm tissue culture dishes at a density of 5 x I O cells/cm2
and allowed to attach ovemight. This plating regime yielded subconfluent cultures of dfdl3 myoblasts. Preincubation with Polybrone to enhance viral infectivity was not corned out on these cultures because proionged treatment of myoblast cultures with Polybrene was found to be detrimentab to their
survival.
virus producer tional Institute
At the same
time,
subconfluent
cultures
of the pIRV
cell line (pec/pIRV, a gift from Dr. Rosa Beddington, Naof Medical Research, Mill Hill, London, United Kingdom)
were incubated overnight in mGM containing (3 pg/m Polybrene to encourage shedding of virus. The following morning medium was colbected from Polybrene-treated pec/pbRV cultures, and this was then fibtered to remove cell debris. Filtered viral supornatant was then added to subconfluent cultures of dfdi 3 myoblasts at the following concentrations: 0, 25, 50, 75, and 100% viral supematant made up to 100% with mGM supplemented with (3 pg/mI) Pobybrene. Plates were then incubated at 37#{176}C/5%CO2 for 2 h, when they were rated with mGM without Polybrene.
After an additional
36-h incubation,
medium
was replaced
with G418
selection medium for2 weeks untilsizabbe coionies ofG4i8-resistant cells had formed in the dishes and the remaining cells had been shed. 6418 selection medium was 50% mGM and 50% myobbast conditioned medium with 800 pg/mI G418. 6418 was replenished every 2 days during selection. Colonies were then picked off the dish using a drawn Pasteur pipette and expanded in myoblast conditioned medium. These cultures were then subject to a further round of cbonab selection in 641 8-containing medium. One colony per original infection culture was expanded until sufficient cells were available for freezing. A series of (3-galactosidase-
expressing
cell lines were derived
by this method.
In all of thorn, the
(3-galactosidase was found to be cytoplasmic. One of these cell lines (PD5OA) was used in in vivo experiments and was maintained in culture from frozen stocks and used between passages 4 and 16. To avoid cell culture adaptation and transformation or senescence, no attempts were made to use PD5OA at passage numbers beyond 20. All cell lines used were found to be free from Mycoplasma contamination. The assay for Mycoplasma was conducted regularly in house as described previously (29). 13-GaIactosIdase Hbstochemistry. Gbutaraldehyde-fixed sections or cells (prepared as descnbed below) were washed briefly in buffered detergent [2 mM MgCl2, 0.001 % (w/v) sodium deoxychobate, and 0.002% (v/v) NP4O in PBS] before overnight incubation in reaction mixture [0.2 mg/mb 5-bromo-4-chboro-3-indolyl-(3--gabactopyranoside (Sigma Chemical Co.), 1 .6 mg/mb potassium ferricyanide, and 2.1 mg/mb potassium ferrocyanide made up in buffered detergent as above]. After air drying, slides were mounted in DePeX for microscopy. Slides were either counterstained with hemotoxybin for visualization of nuclei or with Metanib Yellow (1 % in aqueous solution; Sigma). Host Mice. C57BVIO (rndx) mouse stocks maintained in house and generated from a nucleus of animals generously provided by the IAPGR (Rosbin Research Institute, Roslin, Edinburgh) were used as hosts for injections of PD5OA Seven female and six male mdx mice, 8 weeks of age, were anesthetized with Avertin (0.1 mlstock solution per 10 g of body weight) and received injections i.rn. into the TA of the loft beg with a sterile b-iLl solution of PD5OA cells In PBS using a microinjection pump debivenng 10 pb in 8 mm. The contrabaterab (right) beg was used as a control. Animals were divided into three injection groups on the basis of the number of cells injected; male and female animals were distributed evenly among the three groups, with the extra female being placed in group 2: group 1 fri = 4) injected with 400 celbs/leftTA group 2 (n = 5) injected with 4,000 cells/left TA and group 3 (n = 4) injected with 40,000 cells/left TA. Five days after injection, one male and one female animal from each group (n = 6) were killed, and both right and left TM were frozen. Frozen sections (8 pM) were prepared from snap-frozen tissue dissected from injected and contralateral mdx TM. Frozen serial transverse sections were made of the entire muscle and placed onto gelatin-subbed slides within 24 h of removal from the animal. Sections were then fixed in 0.5% (v/v) gbutaraldehyde in PBS and subjected to (3-gabactosidase histochemistry as described above. Remaining animals were sacrificed at 2 months (n = 3) and 10 months fri = 2) after injection and were subjected to the same
& Differentiation
procedure. At 1 2 months, the TA muscles of one of the remaining two animals was placed into tissue culture as described below, whereas the other was fixed at 14 months by paraformaldehyde (4%, w/v) cardiac perfusion as described previously (24) for a detailed histological exarnination of its muscles. Blood samples were taken from 10-, 12-, and 14-month animals to exclude the generation of systemic virernia caused by recombination of the pbRV retrovirus with endogenous retroviral gonornes or helper virus effects. lmmunohlstochemlstry. For immunostaining, paraffin wax sections of paraformaldehyde (4% in PBS) fixed muscle were stained using rabbit polycbonal antibodies to (3-gabactosidase (1 :500 dilution; Capell). As doscribed previously (14), visualization was with DAB (Dakopatt), 1 mg/mb
hydrogen peroxide (0.2%) after streptavidin enhancement using the Vector Elite ABC kit (Vector Laboratories). Slides prepared in this way were generally counterstained with H&E using a standard protocol. Some injected and stained muscle sections were also stained by Van Gieson’s method to allow identification of the basement membrane. In frozen sections stained previously for (3-galactosidase using immunohistochemistry (see above), basement membrane was identified using anti-collagen (IV) antibody (Sigma) and visualized with Vector Elite and DAB as above. Myoblasts cultured on glass chamber slides were washed in Dulbecco’s PBS and fixed in formal saline (3.7% formaldehyde in PBS) for 45 mm. Cells were permeabibized at room temperature in PHEM buffer (30) for 2 mm and then incubated for 1 h at room temperature with one of the following primary antibodies: myosin (1 :80 dilution; Sigma); anti-dystrophin (peptide IV) rabbit polycbonal antibody (18); and anti-Utrophin (LDP) rabbit polycbonab antibody (19). Both anti-dystrophin and anti-Utrophin antibodies were a gift from S. Ishlura (Fokyo, Japan) and were used at 1:400 dilution. Following three PBS washes (15 mm each), slides were incubated with second antibody (FITC conjugated goat anti-rabbit, 1:150 dilution, or tetrarnethylrhodarnine (3-isothiocyanate conjugated goat antirabbit, 1 :150 dilution; both from Sigma)for 1 h at room temperature. After final washes in PBS, slides were incubated in DAPI (1 pg/mI) to counterstain nuclei. Karyotyplng. Cell lines were karyotyped and banded by incubation with trypsin using conventional methods as described previously (29). Mean chromosome number of PD5OA was determined by counting 100 metaphase spreads, and representative spreads were photographed and enlarged to identify each chromosome. Recovery PD5OA from Injected Muscle. Explant cultures were established from injected and noninjected TA muscles of mdx mice as described by Smith and Schofield (14). At the first passage, cultures (designated PDR) were divided and were either stained for (3-gaactosidase or were placed into 641 8 selection using the contrabaterai leg cultures as controls. 641 8 selections were maintained in 50% conditioned mediurn as described above, and colonies expanded until large enough to be cloned. For (3-gabactosidase staining, cells were fixed in the dish in 0.5% (v/v) glutaraldehydo in PBS for 20 mm. Assays for Helper Virus. Filtered PD5OA myobbast conditioned mediurn was assayed on mouse fibroblasts (NIH-3T3TK) in the presence of Polybrene as follows. Subconfluent cultures of PD5OA cells were fed with fresh mGM containing (3 pg/mI) Polybrene and incubated for 24 h; medium was then removed and filtered through a 0.2-pm-pore filter. This medium was added to monolayer NIH-3T3TKceII cultures (5 X 1O per crn) at concentrations of 0-100% (set up in triplicate) and incubated for 2-3 days. One-half of the plates were then fixed in 0.5% (v/v) glutarabdehyde and stained for (3-gabactosidase as described above. PD5OA cells were included as a positive control for the (3-galactosidase assay. This
of
test was done three times in total. As an additional control, this assay was repeated using two other cell lines, mouse fibroblast NR6 cells and the parent myobbast cell line dfdl3. The remaining NIH-3T3TK and dfdl3 cells treated as above were subject to 6418 selection to confirm the absence of functional virus. Filtered supernatant from pIRV, PD5OA cells, PDR cultures, contrabaterab controls, and mdx mouse serum taken from PD5OA-injected mice were overlaid onto mouse fibrobbasts and stained for f3-gabactosidase (as described above) after 48 h.
References 1 . Partridge, T. A., Morgan, J. E., Coulton, Kunkel, L M. Conversion of mdx myofibers
6. A., Hoffman, from dystrophin
E. P., and negative to
933
934
Integration
positive 1989.
of Skeletal
by injection
Muscle
Stem
of normal
Cells
myoblasts.
Nature
(Lond.),
337: 176-179,
17. Yaffe, genic
2. Yao, S-N., and Kurachi, myofibers but also survive 957-963, 1993.
K. Implanted myoblasts not only fuse as muscle precursor cells. J. Cell Sci.,
3. Moser, H. Duchenne muscular genetic prevention. Hum. Genet.,
dystrophy: 1 7-40,
66:
pathogenetic 1984.
aspects
with 105: and
4. Anderson, M. S., and Kunkel, L M. The molecular and biochemical basis of Duchenne muscular dystrophy. Trends Biochem. Sci., 1 7: 289292, 1992. 5. Partridge, T. A. Molecular and Cellular Biology pp. 12-36. London: Chapman and Hall, 1993.
of Muscular
Dystrophy,
D., and Saxel,
cells
725-727,
isolated
from
0. Serial
passaging
dystrophic
mouse
and differentiation muscle.
Nature
of myo(Lond.),
270:
1977.
18. lshiura, S., Arahata, K, Tsukahara, T., Koga, T., Anraku, T., Yamaguchi, M., Kikuchi, T., Nonaka, I., and Sugita, H. Antibody against the COOH-terminal portion of dystrophin cross-reacts with the 400 kDa protein in the pia mater of dystrophin deficient mdx mouse brain. J. Biochem., 107: 510-513, 1990. 19. Takemitsu, M., Ishiura, S., Koga, A., Kamakura, K, Arahata, K, Nonaka, I., and Sugita, H. Dystrophin-related protein in the fetal and denervated skeletal muscles of normal and mdx mice. Biochem. Biophys. Roe. Cornmun., 180: 1 170-1 186, 1991.
6. Gussoni, E., Pavlath, 6., Lanctot, A. M., Sharma, K. R., Miller, R. G., Steinman, L, and Bbau., H. M. Normal dystrophin transcripts detected in Duchenne muscular dystrophy patients after myoblast transplantation. Nature (Lond.), 356: 435-438, 1992.
20. Acsadi, G., DiCkSOn, 6., Love, D. A., Jani, A, Walsh, Gurusinghe, A, Wolff, J. A., and Davies, K E. Human dystrophin sion in mdx mice after intramuscular injection of DNA constructs. (Lond.), 352: 815-818, 1991.
7. Morgan, J. E., and Partridge, T. A. Cell transplantation and therapy in muscular dystrophy. Bioassays, 14: 641-645, 1992.
gene
21 . Hughes, S., and Bbau, H. M. Muscbefiber pattern is independent of cell lineage in postnatal rodent development. Cell, 68: 659-671 , 1992.
8. Wernig, A., brintchev, A, Hartling, A., Stephan, 6., Zimmermann, K, and StarkinskiPowitz, A. Formation of new muscle fibers and tumors after injection of cultured myogenic cells. J. Neurocytob., 20: 982-997, 1991.
22. Karparti, 6., Ajdukovic, D., Arnold, D., Gledhill, A. B., Gluttman, A., Holland, P., Koch, P. A., Shoubridge, E., Spence, D., Vanasse, M., Watters, G. V., Abrahamowicz, M., Duff, C., and Worton., A. 6. Myoblast transfer in Duchenne muscular dystrophy. Ann. Neurol., 34: 8-17, 1993.
9. Morgan, J. E., Beauchamp, J. A., Pagel, C. N., Peckharn, M., Atabiotis, P., Jat, P. 5., Noble, M. D., Farmer, K., and Partridge, T. A. Myogenic cell lines derived from transgenic mice carrying a thermolabile T antigen: a model system for the derivation of tissue specific and mutation specific cell lines. Dev. Biol., 162: 486-498, 1994. 10. Jat, P. 5., Noble, M. D., Ataliotis, P., Tanaka, Y., Yannoutsos, N., Larsen, L, and Kioussis, D. Direct derivation of conditionally immortal cell lines from an H-2Kb-tsA58 transgenic mouse. Proc. Natb. Acad. Sci. USA, 88: 5096-5100, 1991. 1 1 . Morgan, J. E., Pagel, C. N., Sherratt, T., and Partridge, T. A. Long term persistence and migration of myogenic cells injected into pre-irradiated muscle of mdx mice. J. Neurob. Sci., 1 15: 191-200, 1993. 12. Itagaki, V., Saida, K., and Iwamura, K. Regenerative capacity of mdx mouse muscles after repeated applications of myo-necrotic bupivicalne. Acta Neuropathol., 89: 380-384, 1995. 13. Tremblay, J. P., Mabouin, F., Roy, A., Huard, J., Bouchard, J. P., Satoh, A., and Richards, C. L Results of a triple blind clinical study of myobbast transplantations without immunosuppressive treatment in young boys with Duchenne muscular dystrophy. Cell Transplant., 2: 99112, 1993. 14. Smith, J., and Schofield, P. N. The effects of fibroblast growth factors in long term primary culture of dystrophic (mdx) mouse muscle myoblasts. Exp. Cell Res., 210: 86-93, 1994. 15. Dexter,
plications
T. M. Synergistic
and clinical
interactions
in hemopoiesis:
biological imuse. Eur. J. Cancer, 29 (Suppb. 3): 56-59, 1993.
16. Robertson, E. J. Embryo-derived stem cell lines. In: E. J. Robertson (ed), Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, pp. 71-112. Oxford: IRL Press, 1987.
23. Weller, observations 1993. 24. Smith, dystrophic 251, 1995.
B., Lehnert, S., Carpenter, S., and Karparti, 6. on gamma-irradiated mdx muscle. Neurology,
Long 43:
term 314,
J., Fowke, 6., and Schofield, P. N. Programmed cell death in (mdx) muscle is inhibited by IGF-ll. Cell Death Differ., 2: 243-
25. Dubowitz, V. Transferring Med. J., 305: 844-845, 1992. 26. Watt,
F. S., expresNature
myobbasts
In Duchenne
dystrophy.
Br.
J., and England, M. A. Migration of LACZ anterior to the extensor digitorum bongus muscle of the X-Iinked muscular dystrophic (mdx) mouse. J. Muscle Aes. Cell Motib., 14: 121-132, 1993.
positive
D. J., Karasinski,
cells from the tibialis
27. Beddington, A. P., Morgenstern, J. P., Land, H., and Hogan, A. An in situ transgenic enzyme marker for the midgestation mouse embryo and the visualization of inner cell mass clones during early organogenesis. Development (Camb.), 106: 37-46, 1989. 28. Morgenstem, J. P., and Land, H. A series of mammalian expression vectors and characterization of thelr expression of a reporter gene in stably and transiently transfected cells. Nucbelc Acids Res., 18: 1068, 1990. 29. Smith, J., and Hooper, M. L Dominance and independent segregation of metabolic cooperation-competence and pluripotency in an embryonab carcinoma cell hybrid. Exp. Cell Res., 181: 40-50, 1989. 30. Houbiston, E., Pickering, S. J., and Maro, B. Redistribution of microtubules and pencentriobar material during the development of polarity in mouse bbastorneres. J. Cell Biob., 104: 1299-1308, 1987.
