Functional improvement of damaged adult mouse muscle by implantation of primary myoblasts.

Abstract

1. Myoblasts from expanded primary cultures were implanted into cryodamaged soleus muscles of adult BALB/c mice. One to four months later isometric tension recordings were performed in vitro, and the male donor cells implanted into female hosts were traced on histological sections using a Y-chromosome-specific probe. The muscles were either mildly or severely cryodamaged, which led to reductions in tetanic muscle force to 33% (n = 9 muscles, 9 animals) and 70% (n = 11) of normal, respectively. Reduced forces resulted from deficits in regeneration of muscle tissue as judged from the reduced desmin-positive cross-sectional areas (34 and 66% of control, respectively). 2. Implantation of 10(6) myogenic cells into severely cryodamaged muscles more than doubled muscle tetanic force (to 70% of normal, n = 14), as well as specific force (to 66% of normal). Absolute and relative amount of desmin-positive muscle cross-sectional areas were significantly increased indicating improved microarchitecture and less fibrosis. Newly formed muscle tissue was fully innervated since the tetanic forces resulting from direct and indirect (nerve-evoked) stimulation were equal. Endplates were found on numerous Y-positive muscle fibres. 3. As judged from their position under basal laminae of muscle fibres and the expression of M-cadherin, donor-derived cells contributed to the pool of satellite cells on small- and large-diameter muscle fibres. 4. Myoblast implantation after mild cryodamage and in undamaged muscles had little or no functional or structural effects; in both preparations only a few Y-positive muscle nuclei were detected. It is concluded that myoblasts from expanded primary cultures-unlike permanent cell lines-significantly contribute to muscle regeneration only when previous muscle damage is extensive and loss of host satellite cells is severe.

Full text

Page 1

Journal of Physiology (1997), 500.3, pp.775-785
Functional improvement of damaged adult mouse muscle by
implantation of primary myoblasts
A. Irintchev, M. Langer, M. Zweyer, R. Theisen and A. Wernig *
Department of Physiology, University of Bonn, Wilhelmstraf3e 31, D-53111 Bonn,
Germany
1.
Myoblasts from expanded primary cultures were implanted into cryodamaged soleus muscles
of adult BALB/c mice. One to four months later isometric tension recordings were
performed in vitro, and the male donor cells implanted into female hosts were traced on
histological sections using a Y-chromosome-specific probe. The muscles were either mildly or
severely cryodamaged, which led to reductions in tetanic muscle force to 33% (n = 9 muscles,
9 animals) and 70% (n = 11) of normal, respectively. Reduced forces resulted from deficits in
regeneration of muscle tissue as judged from the reduced desmin-positive cross-sectional
areas (34 and 66% of control, respectively).
Implantation of 106 myogenic cells into severely cryodamaged muscles more than doubled
muscle tetanic force (to 70% of normal, n = 14), as well as specific force (to 66% of normal).
Absolute and relative amounts of desmin-positive muscle cross-sectional areas were
significantly increased indicating improved microarchitecture and less fibrosis. Newly
formed muscle tissue was fully innervated since the tetanic forces resulting from direct and
indirect (nerve-evoked) stimulation were equal. Endplates were found on numerous
Y-positive muscle fibres.
As judged from their position under basal laminae of muscle fibres and the expression of
M-cadherin, donor-derived cells contributed to the pool of satellite cells on small- and large-
diameter muscle fibres.
Myoblast implantation after mild cryodamage and in undamaged muscles had little or no
functional or structural effects; in both preparations only a few Y-positive muscle nuclei were
detected. It is concluded that myoblasts from expanded primary cultures - unlike
permanent cell lines - significantly contribute to muscle regeneration only when previous
muscle damage is extensive and loss of host satellite cells is severe.
2.
3.
4.
It has long been known that whole muscles and muscle
minces can be transplanted from animal to animal with
considerable success (for review see Carlson, 1972). In
contrast, transplantation of cultured myogenic cells (MCs)
has been generally much less efficient in both humans and
animals (Hoffman, 1993; Partridge & Davies, 1995). This
difference may be due to the effects of incubation on the
behaviour and viability of cultured myoblasts in vivo as it
has been found that the vast majority of cultured myoblasts
die soon after implantation, whilst non-cultured myoblasts
from muscle slices survive for long periods after trans-
plantation (Huard, Acsadi, Jani, Massie & Karpati, 1994;
Fan, Beilharz & Grounds, 1996).
Implantation of normal MCs has been considered as possible
therapy for genetic muscular disorders, and, in addition,
genetically engineered myoblasts might deliver missing
gene products (for reviews see Partridge, 1991; Hoffman,
1993; Partridge & Davies, 1995). In all cases, the fate of
the implanted cells in terms of degrees of survival and
proliferation, fusion capacity, immunogenicity and the
possibility of malignant growth in animal models are of
interest, especially in view of the inefficacy of myoblast
implantations revealed in clinical trials with Duchenne
dystrophy patients (Gussoni et
Bouchard, Malouin, Richards & Tremblay, 1992; Karpati et
al. 1993; Tremblay et al. 1993; Mendell et al. 1995). Previous
studies with permanent mouse muscle cell lines in our
laboratory have provided evidence for formation of muscle
tissue in vivo, innervation of the newly formed muscle fibres
and contribution to the contractile strength of the implanted
muscles, but also formation of tumours at later stages
(Wernig, Irintchev, Hartling, Stephan, Zimmermann &
Starzinski-Powitz, 1991 a). MCs from primary cultures have
been shown to survive and differentiate after implantation
in the mouse (Watt, Lambert, Morgan, Partridge & Sloper,
al. 1992; Huard, Roy,
*To whom correspondence should be addressed.
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A. Irintchev, M. Langer, M. Zweyer, R. Theisen and A. Wernig
1982;
Labrecque, Dansereau, Robitaille & Tremblay, 1991; Rando
& Blau, 1994; Huard et al. 1994) but functional effects have
not been investigated. Only one study shows improvement
of contractile parameters of damaged and X-ray irradiated
muscles in the rat (Alameddine, Louboutin, Dehaupas,
Sebille & Fardeau, 1994). It should be pointed out that in
most previous studies in the mouse, mixtures of different
cell types, rather than pure myoblast cultures, have been
implanted. Methods of obtaining mouse myoblast cultures
with high purity have only recently been developed and
used for implantation (Rando & Blau, 1994; present study).
The present investigation concerns the question of whether,
and under what circumstances, implanted primary MCs
contribute to contractile force in adult muscle. In particular,
we have examined the role that existing muscle damage
plays in promoting the incorporation of new cells into
regenerating muscle.
Morgan,
Hoffman &
Partridge,
1990;
Huard,
METHODS
Animals and animal care
Inbred BALB/c mice, purchased from Charles River Wiga (Sultzfeld,
Germany) or bred in the laboratory and kept under standard
laboratory conditions, were used for experiments. Donor satellite
cells were isolated from 4- to 13-day-old male animals, grown in
culture and then implanted into female animals aged 2-7 months.
The sex of the donor animals was determined first by examination
(longer anogenital distance in males) and later verified by in situ
hybridization of cultured cells or cryostat sections from donor liver
with
procedures were performed in accordance with the German law for
protection of experimental animals.
a Y-chromosome-specific
probe(see below).
All animal
Preparation of primary myoblast cultures
Young animals were killed by cervical dislocation, dipped briefly in
cool alcohol (4 °C, 70% v/v), fixed on a disinfected pad and washed
again with alcohol, then with cool (4 °C) sterile phosphate-buffered
saline (PBS; composition (mM): 137 NaCl, 2 7 KCl, 851 Na2HPO4,
1l5 KH2PO4) containing penicillin G-streptomycin (1000 U ml-'
and
1000 jug ml-',
respectively;
(1 25 jug ml-, Fungizone; Gibco). Under a dissecting microscope,
the hindlimb skin was removed, the musculature dissected out and
placed in a 60 mm diameter plastic tissue culture dish (Falcon,
Becton Dickinson, Heidelberg, Germany) containing 5 ml Hanks'
balanced salt solution (HBSS; composition (mM): 137 NaCl, 0'5 KCl,
0'3 Na,HPO4, 04 KH P04, 4'2 NaHCO3, 5'0 D-glucose, 25 Hepes)
supplemented with Ca2' (0'1 mm CaCl2) and Mg2+ (0'5 mMMgCl,2
0'4 mm MgSO4). The tissue was minced with fine scissors under a
lamina flow hood to a diameter of 1 mm or less, then dissociated in
a mixture of 2 ml collagenase solution (0'07% w/v in HBSS,
385 U ml-', Type Ia; Sigma) and 6 ml Ham's nutrient mixture (FIO
medium;
Gibco)
supplemented
(100 U ml-' and 100 jug ml-', respectively; Sigma) for 30 min in a
cell culture incubator (37 °C, 5% CO2). The suspension
transferred to a 50 ml plastic tissue culture tube (Greiner, Solingen,
Germany), and triturated using a 10 ml plastic pipette (30 times in
and out) and then allowed to settle. The supernatant, containing
mostly desmin-negative (non-muscle) cells (see Quality control of
myoblast cultures), was discarded. The pellet was resuspended in
5 ml crude trypsin solution (1: 250; Serva, Heidelberg, Germany)
diluted to 0 25% (w/v, i.e. 5S5 U ml-' with HBSS), incubated in a
Sigma),
and amphotericin B
with
penicillin-streptomycin
was
shaking water-bath (37 °C; shake frequency, -2 s-1) for 10 min and
triturated with a plastic pipette as above. After trypsin activity was
blocked by addition of 5 ml F10 medium supplemented with 20%
fetal bovine serum (FBS; PAN Systems, Aidenbach, Germany) and
penicillin-streptomycin (100 U ml-' and 100 jug ml-', respectively),
the cell suspension was filtered through sterile 40#smpolyamide
nylon mesh (neoLab, Heidelberg, Germany), centrifuged (250 g for
15 min at room temperature, 20-25 °C) and the pellet resuspended
in 3 ml F10-FBS-antibiotic solution. Cells were seeded in 60 mm
diameter dishes coated with collagen Type 1 (3 mg ml-', collagen S;
Boehringer Mannheim,
diluted
incubated at 37 °C in CO2 (5%)-enriched air.
Myoblast culture growth
Initially, growth medium consisted of Eagle's minimum essential
medium supplemented with D-valine (80%; Gibco), FBS (20%) and
antibiotics. More recently, F10 medium (80%) supplemented with
FBS (20%), antibiotics and basic fibroblast growth factor (bFGF,
human recombinant, 2'5 ng ml-'; Serva, Heidelberg or Pharma
Biotechnology, Hannover, Germany) has been used (Rando & Blau,
1994).
Sixteen to twenty-four hours after initial plating most of the
myogenic cells (desmin positive and small in diameter; see below
and also Baroffio, Aubry, Kaelin, Krause, Hamann & Bader, 1993)
remain unadhered whilst numerous fibroblast-like cells (desmin
negative and large in diameter) spread on the bottom of the dish.
The non-adherent cells were collected by aspiration and replated.
The culture medium was changed every 2-3 days and if numbers
of fibroblast-like cells appeared to increase preplating (Richler &
Yaffe, 1970) was performed. Within 7-10 days cultures typically
consisted of >90% desmin-positive myogenic cells and the cell
numbers derived from the hindlegs of each neonatal mouse
amounted to 106. During the first 10 days culture growth was not
apparently dependent on presence ofbFGF or the type of medium.
After this period cell proliferation ceased or was much reduced, but
after 2-3 weeks active proliferation resumed and the culture was
expanded to more than 107 cells. The period of restrained culture
growth appeared to be shorter when bFGF was present in the
medium and we now routinely use FlO medium supplemented with
bFGF (Rando & Blau, 1994) for culture growth. Unless otherwise
indicated, the cells used for implantations were passaged 2-6 times.
The potential for expansion of these cell cultures was large: three
preparations were cultured up to passage 40 without loss of their
proliferative capacity.
Quality control of myoblast cultures
Culture purity. Double staining of samples for desmin and cell
nuclei (see below) was routinely performed to control purity of
myogenic cell cultures from the point of tissue dissociation to the
time of implantation. Quantification was performed as follows:
fields containing cell nuclei (bis-benzimide staining, UV filter) were
selected at random at high magnification (x 500, Axiophot; Zeiss)
and counted, after which the fluorescence filter was changed to
visualize desmin staining (rhodamine excitation) and desmin-
positive cells were counted. Whenever possible, at least 100
(typically 150-200) cell nuclei per preparation were evaluated. The
cell cultures used in this study consisted of 94-99% desmin-
positive cells.
Differentiation. To test the potential of the cultured cells to form
myotubes, prior to each implantation a fraction of the cells was
seeded into 35 mmn diameter collagen-coated Petri dishes and
cultured in growth medium until 70-80%confluency was reached.
Growth medium was then replaced by differentiation medium
consisting of 98% Dulbecco's modified Eagle's medium (4'5 g
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1 :10 with
0-1 N HCl) and
-'
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Implantation of primary myoblasts
glucose; Gibco), 2% horse serum (PAN Systems), and penicillin-
streptomycin. Between 3 and 7 days after medium switch, cultures
were examined daily with phase-contrast optics (x 400, Axiovert;
Zeiss) for the presence of multinucleated cells. In all cultures
numerous myotubes, some of which contracted spontaneously, were
observed.
Control for micro-organism contamination of cultures. Cultures
wereroutinelychecked under
contamination with micro-organisms. In addition, cells from each
culture were grown for 2 weeks without antibiotics to reveal
possible latent bacterial infection. Bis-benzimide (Hoechst 33258;
Sigma) staining of monolayer cultures was used to detect
mycoplasma contaminations.
the
microscope
for
signs
of
Cell implantations
Cell implantations were performed
(Wernig et al. 1991 a; Wernig, Irintchev & Lange, 1995). Surgery
was done under neuroleptanalgaesia with fentanyl (Fentanyl-
Janssen; Janssen, Neuss, Germany), droperidol (Dehydrobenz-
peridol;Janssen) and diazepam
Grenzach-Wyhlen, Germany) at doses of 0 4, 10%0 and 5 0 mg kg-'
i.p., respectively. The soleus muscle of the right limb was exposed
along its entire length and frozen by a single application of the flat
end (3 0 mm x 0 7 mm) of a copper rod pre-cooled in liquid
nitrogen (cryode) onto the muscle surface midway between the
tendons for 10 s (mild cryodamage). Immediately after thawing
each muscle was implanted with myoblasts (4 ,u
containing 1 x 106 cells in HBSS without Ca2+ and Mg2+; n = 13
muscles, 13 animals) or injected with HBSS (4,ul,control, n= 11).
After implantation wounds were
polyamide threads (Ethicon, Norderstedt, Germany). The animals
were kept warm for several hours on a thermostatically controlled
plate (37 °C) and allowed to recover.
as previously
described
(Valium
10 Roche;Roche,
cell suspension
closed
with
7-0 Ethilon
In two other groups freeze-thawing of the soleus muscle was
repeated 3 times to induce severe muscle damage. During each
freezing stage, the cryode was placed first on the proximal half of
the muscle for 10 s and then immediately moved to the distal half
for 10 s. After the final thawing of the muscle, the animals of one
group (n= 14) were each implanted with 106 myoblasts and those
in the other (control) group (n = 9) were injected with HBSS only.
Finally, in some animals (n= 6) undamaged soleus muscles were
injected with myoblasts.
In vitro isometric tension measurements
Contraction measurements were performed 1-4 months after cryo-
damage and cell implantation or cryodamage alone as previously
described (Irintchev, Draguhn & Wernig, 1990; Wernig, Irintchev
& Weisshaupt, 1990; Wernig et
algaesia
(see
above),
soleus nerve-muscle
dissected out. Muscles were mounted in Lucite® chambers perfused
with aerated Tyrode solution (composition (mM): 125 NaCl, 1-0
MgCl2,
1-8 CaCl2,
5-4 KCl, 24 NaHCO3,
connected to a force transducer. Muscle contractions were evoked
either directly via a pair of silver electrodes in the bath or
indirectly via nerve suction electrodes. Muscle length was adjusted
so that maximal twitch tension was produced upon single stimuli.
Voltage amplitudes were set to twice the lowest values sufficient for
maximum twitch stimulation (final values 20-25 V for direct and
4-6 V for indirect muscle stimulation). Single pulses (0 5 ms
duration for direct and 0-1 ms for indirect stimulation) and tetani
(20, 50 and 100 Hz for 2 s) were used for stimulation.
The temperature was kept at 25-0 + 0 5 °C throughout the
measurements. Muscles and nerves were stimulated alternately
Downloaded from J Physiol (
al. 1991 a). Under neuroleptan-
preparationswere
10 D-glucose) and
with single pulses and tetani in a sequence which was kept constant
in all experiments. A minimum of 3 min was allowed for muscle
recovery between stimulations. Signals were stored in a digital
oscilloscope (model HM 208; Hameg, Frankfurt, Germany) and
plotted on paper.
ACh sensitivity was tested by rapid exchange of the normal
perfusion solution with ACh-containing Tyrode solution (5 and
50 mg 1-' acetylcholine perchlorate; Sigma). The amplitude of the
evoked contracture was expressed as a fraction (%) of the amplitude
of a preceding tetanus (100 Hz).
After the contraction measurements muscles were gently blotted
and weighed with parts of the distal and proximal tendons present.
Histological procedures
Both soleus muscles of each animal were fixed at approximately
resting length on a piece of formalin-fixed turkey liver and frozen
in isopentane pre-cooled in liquid nitrogen (Irintchev, Zweyer &
Wernig, 1995; Wernig et
thickness) were collected on chrome-gelatin or silane-coated slides.
Cell smears or cells grown on coverslips were used for staining of
cultured cells.
al. 1995). Serial cross-sections (6 1um
Histology and histochemistry. For general histology sections
were stained with an aqueous solution of Toluidine Blue (1% w/v)
and borax
(1% w/v). Tovisualize endplates,
Acetylcholinesterase (AChE) activity and ACh receptors were
performed (Wernig
benzimide was used to reveal cell nuclei, and, in cultured cell
preparations, mycoplasma contamination.
stainings
for
et
al. 1991 a). Staining of DNA with bis-
Immunofluorescence. The following primary monoclonal anti-
bodies were used: anti-desmin (clone D33; Dako, Hamburg,
Germany; purified mouse IgG diluted to 1 jug ml-'), anti-NCAM
(neural
Deagostini-Bazin, Hirsch & Goridis, 1981; rat IgG, hybridome
supernatant diluted 1 :100), anti-dystrophin (clone DYS2; Novo
Castra Labs, Newcastle-upon-Tyne, UK; mouse IgG, 1:20) and
anti-laminin (clone LAM-1; ICN Biomedicals, Eschwege, Germany;
purified rat IgG, 5 jug ml-'). Affinity-purified polyclonal antibody
against M-cadherin was raised in rabbits as described by Rose et al.
(1994) and its specificity was proved using Western blots, enzyme-
linked immunoabsorbent assays and immunofluorescent stainings
(data not shown). No cross-reactivity with other adhesion molecules
(NCAM, N-cadherin and E-cadherin tested) was detected.
Affinity-purified biotin-conjugated goat anti-rat, anti-mouse and
anti-rabbit IgG; rhodamine-conjugated anti-mouse and anti-rat
IgG; normal sera and IgGs; and 5-((dichlorotriazin-2-yl)amino)-
fluorescein (DTAF)-streptavidin were purchased from Jackson
Immunoresearch Laboratories (Dianova, Hamburg, Germany).
Immunofluorescent staining procedures have been described
previously in detail (Wernig
Faissner & Wernig, 1993; Irintchev, Zeschnigk, Starzinski-Powitz
& Wernig, 1994; Irintchev et al. 1995). Briefly, after acetone or
methanol fixation and blocking of non-specific binding with normal
serum, sections were incubated with primary antibodies (overnight,
4 °C), and after washing with PBS reacted with either rhodamine-
conjugated second antibody or with biotin-conjugated second anti-
body followed by DTAF-streptavidin.
In
situ
hybridization.
In
Y-chromosome-specific DNA probe Y1 (or 145SC5; Nashioka, 1988)
was used to identify male donor cells implanted into female animals
(Grounds, Lai, Fan, Codling & Beilharz, 1991). Y1/pGEM7 plasmid
DNA expressed in Escherichia
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cell adhesion molecule;
clone H-28; Hirn,
Pierres,
et
al. 1991 a; Irintchev, Salvini,
situ
hybridization
with
the
coli was digested with EcoRI
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777

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A. Irintchev, M. Langer, M. Zweyer, R. Theisen and A. Wernig
Figure 1. For legend see facing page.
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Implantation of primary myoblasts
restrictase (Boehringer Mannheim), the 1P5 kb Y1 fragment was
isolated using agarose gel electrophoresis and digoxigenin labelled
with a labelling kit as suggested by the manufacturer (DIG DNA
labelling and detection kit, non-radioactive; Boehringer Mannheim).
Tissue sections were mounted on silane-coated slides, air dried,
fixed with absolute ethanol (10 min, room temperature), rehydrated
through a 70, 50 and 30% (v/v) ethanol series, washed with PBS
containing 5 mM MgC12, digested with proteinase K (0 1 8sg ml-' in
PBS for 15 min at 37 °C; Sigma), washed with 0'2% glycine-PBS
(10 min, room temperature), and fixed
formaldehyde in PBS (10 min, room temperature). Culture cells,
grown on uncoated glass slides, were washed with PBS, air dried,
fixed in paraformaldehyde, washed with PBS and air dried again.
To reduce non-specific hybridization, sections were overlaid with
prehybridization solution composed of 4x saline-sodium citrate
buffer (SSC), 50% (v/v) amberlite-deionized formamide,
Denhardt's solution, 5% (w/v) dextran sulphate and 0 5 mg ml-'
salmon testes DNA (all from Sigma) and incubated for 15 min at
37 'C. Cell preparations were only rehydrated with 2 x SSC.
Hybridization
was performed
containing labelled Y1-probe DNA (0 4-0-7 ng 1uls) overnight
(16 h) at 37 'C. After thorough washing in buffers with increasing
stringency (twice for 15 min in 4 x SSC-45% formamide, at 42 'C;
twice for 5 min in 2x SSC, at 42 'C; 15 min in 0 2x SSC, at 50 'C;
15 min in O0lx SSC, at 60 °C), labelled DNA was visualized with
the digoxigenin detection kit as suggested by the manufacturer
(Boehringer Mannheim). Finally, bis-benzimide staining of nuclei
was performed.
In the course of establishment of optimal staining conditions,
specificity controls included omission of labelled probe (negative),
omission of the antibody to digoxigenin (negative), staining of
sections from intact solei of female (negative) and male (positive)
animals. Routinely, sections from normal muscles of male and female
animals were added onto each slide with sections from experimental
muscles. The reaction product in male control muscles was confined
to nuclei as revealed by nuclear bis-benzimide staining and, in most
cases, covered only a fraction of the nucleus. Labelling efficiency of
nuclei in control muscles varied from 16 to 50% (see below).
Quantification of the labelling efficiency in sections from male
muscles was performed after each staining. Several fields were
selected at random using first the bis-benzimide filter and then the
fraction of Y-positive nuclei was counted using bright-field optics
(x 500, Axiophot; Zeiss; 100-250 nuclei quantified per muscle).
in 4% (w/v) para-
1 x
with
prehybridization
solution
Counting of Y-positive nuclei in implanted muscles was performed
on two or three complete good quality sections made through the
endplate region of each muscle (at x 500). The whole cross-sectional
area of each muscle was scanned with the photographic frame of
the microscope seen in the visual field so that no overlapping of
frames, and thus double counting of nuclei, occurred. All positive
nuclei in the section were counted. Correction of absolute numbers
was made in accordance with the staining efficiency determined in
the concomitantly stained sections ofmale muscles.
Cross-sectional areas and numbers of muscle fibre profiles.
Cross-sectional areas occupied by muscle (desmin positive) and non-
muscle tissue (desmin negative) were measured on a single complete
section from the endplate region of individual muscles as described
previously (Wernig et al. 1995). Briefly, low-power videoimages
(x 6 25 objective lens) were taken with a high-sensitivity video-
camera and enhanced with an image processor system. Areas were
measured directly on the video monitor using the software of the
image processor.
The total number of muscle fibre profiles was evaluated from
complete muscle cross-sections reconstructed from videoprints
(Toluidine Blue staining, final magnification x 382) as described
previously (Wernig et al. 1995). Due to split and branched fibres in
regenerated muscles (see Wernig et al. 1990, 1995) the number of
profiles is bound to be higher than the number of muscle fibres; no
attempt was made to correct for this (Wernig et al. 1990).
Statistical analysis
One-way analysis of variance (ANOVA) and a subsequent Tukey's
test were performed to compare mean values of more than two
groups (see Wernig et al. 1995). The accepted level of significance
was 0 05 or less. Throughout the text mean group values are given
with standard deviations.
RESULTS
Incomplete muscle regeneration after severe
cryodamage
Soleus muscles were mildly or severely cryodamaged (see
Methods). At 4 weeks after spontaneous regeneration the
amount of contractile force was particularly small after
severe cryodamage (Table 1) and there was no further
improvement after the first month (not shown): directly and
indirectly evoked maximum tetanic and twitch tension
Figure 1. Soleus muscles 1 0-2 5 months after severe cryodamage with and without MC
implantation
A-C, low power magnification of desmin-stained cross-sections through the endplate region of non-
implanted muscle (A), MC-implanted muscle (B), and intact muscle contralateral to that shown in A (C). The
regenerated non-implanted muscle has a much smaller cross-sectional area than the other two muscles and,
in addition, a fibrotic area devoid of desmin-positive profiles (arrows). The implanted muscle (B) has a cross-
sectional area roughly comparable to that of the intact muscle (C) andonlyperimyseal septa (asterisks) are
desmin negative, as also seen in the other two muscles. The scale bar in C represents 100#smand applies
also to A and B. D, dystrophin staining 2-5 months after severe cryodamage and MC implantation
delineates large-diameter (>20 ,um, left) and numerous small-diameter (<20,um) muscle fibre profiles.
Scale bar represents 20#sm.E and F, in situ hybridization with the Y-chromosome-specific probe reveals
numerous donor-derived male nuclei (1 month post implantation, E). F, Y-positive nuclei in large-diameter
fibres are more often localized centrally (6 of 8 seen in the figure), whilst the typical localization in small-
diameter fibres (arrow) is peripheral (2 months post implantation). Scale bars represent 40,um(E) and
20,um (F).
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