J Neuropathol Exp Neurol
Copyright Ó 2011 by the American Association of Neuropathologists, Inc.
Vol. 70, No. 9
September 2011
pp. 770Y778
ORIGINAL ARTICLE
Transplanted Myoblasts Can Migrate Several Millimeters
to Fuse With Damaged Myofibers
in Nonhuman Primate Skeletal Muscle
Daniel Skuk, MD, Marlyne Goulet, CLT, and Jacques P. Tremblay, PhD
Abstract
A major restriction of the intramuscular transplantation of myoblasts is that the grafted cells fuse mostly with myofibers along the
injection trajectories. This has been attributed to a ‘‘lack of migration
ability’’ of the grafted myoblasts. It has been assumed that grafted
myoblasts remain motionless in the sites of delivery and fuse only
with myofibers with which they come into contact. In the present
study, we analyzed this phenomenon in 17 cynomolgus monkeys.
We found that intramuscularly injected myoblasts within 1 hour after
their injection are mainly located in the perimysium and not distributed along the injection trajectories. This suggested that the
grafted myoblasts later migrate from the perimysium to fuse with
myofibers that are damaged by the injections. Therefore, we analyzed
whether A-galactosidaseYlabeled myoblasts injected subcutaneously
over skeletal muscles migrate in needle-damaged and nondamaged
muscle regions. We observed that grafted myoblasts migrated up to
1 cm in depth from the muscle surface into the muscles, although
they seemingly fused mainly with damaged myofibers. Our findings
suggest that myoblast transplantation is not necessarily restricted
by a ‘‘lack of migration ability’’ of the grafted cells but by the fact
that myoblasts fuse with regenerating myofibers and not with
undamaged myofibers.
Key Words: Cell migration, Myogenic cell transplantation, Myoblasts,
Nonhuman primates, Skeletal muscle.
INTRODUCTION
Intramuscular transplantation of cells with the ability
to differentiate into myofibers is a potential therapeutic
approach for the treatment of some myopathies, mainly those
of genetic recessive origin (1, 2). To be useful in this context,
the injected cells need to fuse with the myofibers of the
recipient, thereby adding exogenous nuclei that can express
therapeutic genes in myofibers that have a specific genetic
From the Neurosciences Division Y Human Genetics, CHUQ Research
Center Y CHUL, Quebec, Canada.
Send correspondence and reprint requests to: Daniel Skuk, MD, Unité de
recherche en Génétique humaine, Centre Hospitalier de l’Université
Laval, 2705 Blvd Laurier, Quebec, Canada G1V 4G2; E-mail: Daniel.
[email protected]
This work was supported by grants of the Jesse’s Journey Foundation for
Gene and Cell Therapy of Canada to Dr Skuk.
770
disease-causing abnormality. Myoblasts were the first myogenic cells to be proposed for this therapeutic approach (3). In
postnatal life, myoblasts originate from satellite cells, the
committed stem cells of skeletal muscles. Satellite cells can be
isolated from muscle biopsies by standard cell culture techniques and can then be easily expanded in vitro to produce
large numbers of myoblasts that can fuse with existing myofibers or form new myofibers (4). Myoblasts have been
transplanted in mice (5), dogs (6), monkeys (7), rabbits (8),
and pigs (9) and were the first myogenic cells to be transplanted in human clinical trials (10).
We previously conducted a clinical trial of normal adult
myoblast transplantation in patients with Duchenne muscular
dystrophy (11Y13), which is caused by the deficiency of the
protein dystrophin and is the most frequent and one of the
most severe genetic myopathies. We observed donor-derived
dystrophin expression in 3.5% to 26% of myofibers of 8 of
9 patients 1 month after the intramuscular implantation of
myoblasts obtained from normal donors. The myoblasts had
proliferated in vitro and were transplanted using an accurate
method of cell delivery (11, 13). In another patient, biopsies
of a gastrocnemius region that was grafted with normal
myoblasts showed expression of donor-derived dystrophin in
27.5% of the myofibers 1 month after transplantation and in
34.5% 18 months after transplantation (12).
A main constraint of the intramuscular implantation of
myogenic cells, however, is that the fusion of the injected
cells with the host myofibers seemingly takes place only along
the injection trajectories. This is clearly observed in muscle
cross sections after myoblast transplantation experiments in
nonhuman primates. Indeed, myofibers expressing donorderived proteins are distributed in irregular stripes that are
oriented parallel to the original cell injection trajectories
(14Y16). A reasonable explanation for this observation is that
the injected myoblasts remained collected in the injection
trajectories and thus fused only with the myofibers in close
contact with them.
This restricted fusion of the implanted myoblasts in the
sites of cell injections has been attributed to a ‘‘lack of
migration ability’’ of the implanted cells (17, 18). This conventional dogma says that cultured myoblasts have an intrinsic inability to migrate once they are implanted into a muscle;
thus, they are unable to fuse with more myofibers than those
that are close to them. Having assumed that the ‘‘lack of
migration ability’’ of implanted cells was a limiting factor for
J Neuropathol Exp Neurol Volume 70, Number 9, September 2011
Copyright © 2011 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
J Neuropathol Exp Neurol Volume 70, Number 9, September 2011
myoblast transplantation, promoting the migration of the
transplanted cells was an important research topic to improve
the success of this approach (17Y20).
The findings in the biopsy of one patient in our clinical
trial of myoblast transplantation led us to suspect that the cells
did not always remain only in the injection trajectories.
Indeed, there was a large accumulation of small myofibers
that entirely expressed donor-derived dystrophin located in
the perimysium, outside the injection trajectories (11, 13).
These donor-derived small myofibers seemed to have been
formed by the fusion of the injected myoblasts among themselves, as if a large collection of myoblasts was formed in the
perimysium when they were injected.
In the present study, we first analyzed the behavior
of the cell suspension after the intramuscular injection in
a nonhuman primate species. Unexpectedly, most injected
myoblasts were observed 1 hour after the injection in the
perimysium rather than in the intrafascicular injection trajectories. This suggested that grafted myoblasts later migrate
from the perimysium to fuse with the myofibers damaged by
the injections. To investigate this possibility, we analyzed
whether myoblasts implanted subcutaneously over a skeletal
muscle migrate into the underlying muscle.
MATERIALS AND METHODS
Migration of Grafted Myoblasts in Monkey Muscles
neomycin resistance gene. The transduced cells were selected
twice with 600 Kg/mL Geneticin (Invitrogen, Burlington,
Ontario, Canada). Within 4 days, they proliferated until confluent and were then frozen to be stored in liquid N2. A sample
of the cells to be frozen was analyzed to determine the percentage of CD56-positive cells as an indicator of the percentage of myoblasts in the culture. These cells were
incubated with a phycoerythrin-coupled anti-CD56 antibody
(Beckman Coulter, Fullerton, CA) and analyzed by flow
cytometry: 90% to 99% of cells were CD56-positive.
Cell Transplantation
For transplantations, the cells were thawed, allowed to
proliferate during 1 or 2 passages in culture, detached from
the flasks using 0.1% trypsin and washed 3 times with
HBSS. The final cell pellets were resuspended in the same
saline solution and injected in 2 ways, depending on the
experiment. The muscles used for the experiments were
mostly the biceps brachii but also included the quadratus
femoris and gastrocnemius.
Experiment 1: Intramuscular Implantation to
Analyze the Distribution of the Grafted Cells
This experiment was done in 7 monkeys. The cell injection procedure was done percutaneously by making parallel
equidistant injections (1 or 2 mm of distance between each
Animals
Adult cynomolgus monkeys (Macaca fascicularis)
(n = 17), males and females, were used as recipients of myoblast allotransplantations. For transplantations and biopsies,
the animals were kept under general anesthesia using isofluorane (1.5%Y2% in oxygen), after induction with intramuscular ketamine (10 mg/kg) and glycopyrrolate (0.05 mg/kg).
Buprenorphine (0.01 mg/kg twice a day for 3 days) was
given for postoperative analgesia. In monkeys in which multiple muscle samples were needed, the animals were killed
at the end of the experiment by intravenous administration of
a pentobarbital overdose (120 mg/kg) after anesthesia using
intramuscular ketamine (15 mg/kg). The Laval University
Animal Care Committee authorized these procedures.
Cells
Cell cultures were obtained from skeletal muscle
biopsies done previously in 2 cynomolgus monkeys. The
biopsy samples were minced with fine scissors in fragments
of less than 1 mm3 and then dissociated with 0.2% collagenase (Sigma-Aldrich, St Louis, MO) in Hanks balanced salt
solution (HBSS) for 1 hour, followed by 0.125% trypsin
(Gibco, Grand Island, NY) in HBSS for 45 minutes. The
isolated cells were subcultured in vitro in MB-1 medium
(Hyclone, Logan, UT) with 15% fetal bovine serum
(Hyclone), 10 ng/mL of basic fibroblast growth factor (Feldan,
St Laurent, Quebec, Canada), 0.5 mg/mL of bovine serum
albumin (Sigma), 0.39 Kg/mL of dexamethasone (Sigma), and
5 Kg/mL of human insulin (Sigma). The cells were infected
twice in vitro with a replication-defective retroviral vector
LNPOZC7 (gift from Dr Constance Cepko, Harvard Medical
School, Boston, MA), encoding a LacZ reporter gene and a
TABLE. Experiment 2: Intramuscular Migration of
Subcutaneously Implanted Myoblasts
Muscle
No. Cells
Injected per Site
1
BB
18 106
2
BB
20 106
3
BB
30 106
4
5
6
QF
QF
BB
17 106
17.5 106
11 106
Monkey
G
7
8
9
10
BB
BB
BB
BB
23 106
20 106
20 106
16 106
Site: Needle
Size*
Distance of
Migration and
Fusion†‡
1: no
2: 27 gauge
1: no
2: 27 gauge
1: no
2: 27 gauge
1: 27 gauge
1: 22 gauge
1: 27 gauge
2: 27 gauge
3: 23 gauge
4: 23 gauge
1: 27 gauge
1: 27 gauge
1: 27 gauge
1: 27 gauge
1: 1.6 mm
2: 8.4 mm
1: 0
2: 1.5 mm
1: 0
2: 7.3 mm
1: Q7.6 mm
1: Q8.4 mm
1: Q10.6 mm
2: Q10.4 mm
3: Q6.9 mm
4: Q9.4 mm
1: Q8.2 mm
1: 0.3 mm
1: 4.0 mm
1: Q7.9 mm
*The number of sites of subcutaneous implantation per monkey varied from 1 to 4
(no more than 2 per muscle). Sizes of needles used for the muscle damage are indicated.
Sites indicated as ‘‘no’’ were not submitted to needle penetration previous to the
subcutaneous implantation.
†Distances from the muscle surface to the deepest A-GalYpositive myofibers. This
does not include the epimysium thickness.
‡The symbol ‘‘Q’’ indicates that A-GalYpositive myofibers reached the deepest
portion of the biopsy; there could have been more deeply located A-GalYpositive
myofibers.
BB, biceps brachium; G, gastrocnemius; QF, quadratus femoris.
Ó 2011 American Association of Neuropathologists, Inc.
Copyright © 2011 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
771
Skuk et al
J Neuropathol Exp Neurol Volume 70, Number 9, September 2011
other), perpendicular to the muscle surface and forming a square
grid of approximately 1 cm2. Injections were done with 50-KL
syringes (Hamilton, Reno, NV) using 27-gauge needles 0.5 in
in length. The procedure involved penetration of the needle
until its maximum depth and slow homogeneous delivery of
approximately 5 KL of the cell suspension during the needle
772
withdrawal. An OpSite sterile transparent dressing with a
5-mm grid (Smith & Nephew, Hull, UK) was adhered to
the skin to control the pattern of needle penetration. Each injection site was identified after the cell injections by placing
2 points of nonabsorbable polypropylene suture on the muscle
surface on both sides.
Ó 2011 American Association of Neuropathologists, Inc.
Copyright © 2011 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
J Neuropathol Exp Neurol Volume 70, Number 9, September 2011
Experiment 2: Subcutaneous Implantation
This experiment was done in 10 monkeys (Table). Limited regions of skeletal muscles were mechanically damaged
mostly with 27-gauge but also 23- and 22-gauge needles (Table).
This needle damage consisted in performing 100 parallel percutaneous intramuscular penetrations of approximately 1 cm
depth, forming a square grid of approximately 1 cm2 (as for
intramuscular cell transplantation). Between 30 and 90 minutes later, 0.5 mL of a myoblast suspension was injected
subcutaneously over the damaged region and, in some cases,
over nondamaged regions. We waited more than 30 minutes
between muscle damage and subcutaneous transplantation to
permit sufficient time for hemostasis at the points of damage. The amount of cells injected per site in each monkey
depended on the amounts of cells available for the day of the
experiment (Table). In other muscle regions of some of these
monkeys, a control site was intramuscularly injected with the
same myoblasts and a pattern of injections was made similar
to the pattern of needle damage, that is, 100 parallel equidistant
injections per squared centimeter, perpendicular to the muscle
surface and throughout roughly 1 cm3 of muscle. In those cases,
we used a 250-KL Hamilton syringe attached to a PB600-1 repetitive device (Hamilton) (15) with 27-gauge needles and delivering the cell suspension homogeneously during each needle
withdrawal. An OpSite sterile transparent dressing with a 5-mm
grid was also adhered to the skin to control the pattern of percutaneous needle penetrations and intramuscular grafts. Each
site was identified by placing 2 points of nonabsorbable suture (mostly polypropylene, sometimes silk) on the muscle
surface at both sides, taking care to avoid leakage of the
subcutaneously injected cell suspension through the incisions
made to place these sutures. Among the 3 muscles used, the
biceps brachium was preferable for this experiment for several
reasons: the skin is sufficiently loose to allow the formation of
a pocket of cell suspension over the site of damage, the epimysium is delicate, and the muscle shape (a cylinder of wellparallel myofibers) and size (large enough to observe several
millimeters of migration in tissue sections) are optimal. By contrast, the skin over the gastrocnemius was sometimes more
tense so the cell suspension spread horizontally over a large
surface during subcutaneous injection. Moreover, the distal
portion of the gastrocnemius is not large enough. On the
other hand, the quadratus femoris has a thick fascia that could
be an additional barrier to cell migration. However, these fac-
Migration of Grafted Myoblasts in Monkey Muscles
tors did not prevent myoblast migration when these muscles
were used.
Immunosuppression
An intramuscular formulation of tacrolimus (a generous
gift from Astellas Pharma Inc., Osaka, Japan) was administered for immunosuppression, beginning 5 to 7 days before
transplantation and maintained until the end of the experiment. Tacrolimus was injected once a day, beginning at
0.5 mg/kg per day and adjusting the dosage to target blood
levels of more than 50 Kg/L, as quantified in blood samples
with an IMx tacrolimus II kit for microparticle enzyme
immunoassay (Abbott, Wiesbaden, Germany).
Sampling
In Experiment 1, biopsies were performed in the muscle
regions injected with cells at 1 hour after transplantation. In
Experiment 2, biopsies were performed 1 month after implantation under the regions transplanted subcutaneously and in
control intramuscular transplant sites. The muscle samples
were mounted in embedding medium, snap-frozen in liquid
N2, and stored at j80-C until performing serial sections of 10
to 15 Km in a cryostat at j25-C.
Histologic Analysis
Some sections were mounted in a synthetic resin and
stained with hematoxylin and eosin to analyze the general
structure of the muscle. To detect damaged myofibers in the
early stages after transplantation, some sections were stained
for 5 minutes in a 2% solution of alizarin red (Sigma, Oakville,
Ontario, Canada), pH 5.4, followed by a brief rinsing in water
at pH 5.4 and a brief passage in acetone, a brief passage for
acetone/toluene 1:1, and then toluene, to be mounted in synthetic resin. For A-galactosidase (A-Gal) detection, muscle sections were fixed for 3 minutes in 0.25% glutaraldehyde, rinsed
with phosphate-buffered saline, incubated for 24 hours at
room temperature in a solution of 0.4 mg/mL X-gal (5-bromo4-chloro-3-indolyl-A-D-galactopyranoside) (Boehringer Mannheim, Vienna, Austria), containing 1 mmol/L MgCl2, 3 mmol/L
Ke3F(CN)6I3 mmol/L Ke4F(CN)6I3H2O in phosphate-buffered
saline, and mounted in 1:1 glycerin-jelly. The shortest distance
between the muscle surface and the deepest-situated A-GalY
positive myofibers was measured in digital images taken at
the microscope using a ruler made with digital pictures of a
hemacytometer taken at the same magnification. We also
FIGURE 1. Macaque skeletal muscle biopsies in which A-galactosidase (A-Gal)Ypositive myoblasts were implanted intramuscularly
through a matrix of parallel injections. (AYC) Serial sections of a biopsy 1 hour after transplantation stained for A-Gal histochemical
detection (A), hematoxylin and eosin (H&E) (B), and alizarin red (C). The external surface of the muscle is at the top of the fields;
epimysium is indicated with arrowheads in B. A-Gal staining (dark blue in A) corresponds to accumulations of mononuclear cells in
the perimysium around muscle bundles. With H&E, the myofibers damaged by the needle trajectories are seen as dark myofibers,
sometimes with a pale core, forming irregular tracks arranged from the surface to the lower region (B). Damaged myofibers are
evident as dark redYorange staining with alizarin red, indicating recent sarcolemmal damage and intracellular deposition of calcium
(C). (D) Magnification of the area indicated by the rectangle in B. Black arrowheads circumscribe the accumulation of mononuclear cells in the perimysium, which is dissected by the injected cell suspension. Blue arrowheads circumscribe a strip of damaged
myofibers and edema corresponding to the injury made by the injection needle. (E) At 1 month after transplantation, the pattern
of A-GalYpositive myofiber profiles (dark blue staining) in a whole cross section of a biopsy resembles the myofiber damage done by
the injection trajectories rather than the original distribution of most grafted cells. Vertical arrows in A to C and E indicate the
original direction of the cell injections. Scale bars = 1 mm (AYC, E); 100 Km (D).
Ó 2011 American Association of Neuropathologists, Inc.
Copyright © 2011 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
773
Skuk et al
J Neuropathol Exp Neurol Volume 70, Number 9, September 2011
FIGURE 2. Muscle biopsies from Monkey 1 (Table). (A) At 1 month after subcutaneous implantation of A-galactosidase (A-Gal)Y
positive myoblasts over a needle-damaged muscle region, many A-GalYpositive myofibers are seen covering several millimeters in
depth. (B) At 1 month after subcutaneous implantation of A-GalYpositive myoblasts over a muscle region not submitted to needle
penetrations, some A-GalYpositive myofibers are seen in a small region, possibly due to superficial damage produced by the tip of
the needle used for the subcutaneous injection. Red dots in A and B indicate the muscle surface. (C) A region transplanted
intramuscularly with A-GalYpositive myoblasts is shown for comparison as a positive control. Arrows indicate the path of migration
(A, B) and the path of intramuscular myoblast injections (C). All sites received 18 106 cells in 0.5 mL of cell suspension. Scale
bars = 1 mm. Magnification is the same for the 3 images.
FIGURE 3. Histograms illustrating numbers of A-galactosidase (A-Gal)Ypositive myofibers (x axes) versus the distance from
the surface of the muscle (y axes, in mm) in muscle biopsies from sites damaged with a needle and grafted subcutaneously with
A-GalYpositive myoblasts. Monkey 8 is not included owing to small numbers of A-GalYpositive myofibers found and their very
short distances from the muscle surface. The x axes are at the same scale with the exception of Monkeys 1 and 10, owing to the
larger numbers of A-GalYpositive myofibers present. The arrow in each graph indicates the deepest limit of each biopsy. There
is no arrow for Monkey 9 because the depth of the sample was approximately 14 mm. Counts were done in the same sections
used to measure the distance between the muscle surface and the deepest A-GalYpositive myofibers (Table); in some cases (e.g.
Monkey 5), they do not correspond to the section with the largest amount of A-GalYpositive myofibers. Images in the lower
portion are cross sections of whole biopsies stained for histochemical detection of A-GalYpositive myofibers (dark blue)
that illustrate different patterns of migration and fusion. Outer surfaces of the muscle (red dots) are at the top. In Monkey 6,
the A-GalYpositive myofibers reached the deepest surface of the gastrocnemius (black dots). BB indicates biceps brachium; G,
gastrocnemius. Scale bars = 1 mm for all images.
774
Ó 2011 American Association of Neuropathologists, Inc.
Copyright © 2011 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
J Neuropathol Exp Neurol Volume 70, Number 9, September 2011
Migration of Grafted Myoblasts in Monkey Muscles
Ó 2011 American Association of Neuropathologists, Inc.
Copyright © 2011 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
775
J Neuropathol Exp Neurol Volume 70, Number 9, September 2011
Skuk et al
counted in digital images the number of A-GalYpositive myofibers according with the distance from the muscle surface,
quantified per band of 1 mm from this surface.
concomitantly in sites in which intramuscular cell transplantation had been done at the same time as a positive control
with the same myoblasts and following the same pattern of
needle penetration (Fig. 2).
RESULTS
Distribution of the Grafted Cells After
Intramuscular Implantation
At 1 hour after transplantation, implanted cells were
mostly observed forming compact accumulations of mononuclear cells in the grafted muscle (Figs. 1A, B). Most of
these accumulations had an elongated cross-sectional shape,
were variably sized, and generally located in the perimysium
(Figs. 1B, D). They were present in regions in which the
damage produced by the injection trajectories was evident,
but their distribution differed from the pattern of needleinduced damage (Figs. 1A, C). Damaged myofibers exhibited
typical morphologic features of early necrosis, that is, they
were darker than the normal myofibers in hematoxylin and
eosinYstained sections and were calcium-positive in alizarin
redYstained sections (Figs. 1BYD). Together with the presence
of focal edema due to the mechanical damage of the needle,
the injection trajectories are easily identified. Often, the largest accumulations of implanted cells were located deeply in
the grafted region; few A-GalYpositive cells were observed
close to myofibers damaged by the cell injection site. In some
sections, accumulations of A-GalYpositive cells were also
observed outside the external surface of the muscle, beneath
or in the deepest regions of the epimysium. The distance and
position of these accumulations of A-GalYpositive cells in the
epimysium suggest that they represented leakage of the cell
suspension in the injection points. Biopsies at 1 month after
transplantation showed most A-Gal staining inside muscle
bundles (A-GalYpositive myofibers) rather than in the perimysium (Fig. 1E).
Subcutaneous Myoblast Implantation
Biopsies of nondamaged muscle under subcutaneously
myoblast-implanted sites were performed in 3 monkeys: A-GalY
positive myofibers were not observed in Monkey 2, scarce
A-GalYpositive myofibers were observed in Monkey 3, and few
A-GalYpositive myofibers were found in a small region of the
muscle surface in Monkey 1 (Table; Fig. 2).
A-GalYpositive myofibers were observed at variable
distances from the muscle surface in all biopsies of needledamaged muscle under subcutaneously myoblast-implanted
sites (Table). The distances ranged from only 2 or 3 myofibers
deep (Monkey 8) to the deepest border of the biopsy, that
is, at least 7 to 10 mm from the surface (Figs. 2 and 3). In
several animals, the depth of the A-Gal expression seemed to
encompass the entire length of the needle trajectory (approximately 1 cm of penetration). Although the topographical
distribution of the A-GalYpositive myofibers, their density,
and the intensity of labeling varied among different samples
(Figs. 2 and 3), their patterns seemed to follow the needle
trajectories. The distribution of A-GalYpositive myofibers in
these biopsies was never as homogeneous as in biopsies done
776
DISCUSSION
We show that transplanted cultured myoblasts have the
intrinsic capacity to migrate in skeletal muscle of macaques.
These results contradict the dogma that these cells do not
migrate from their site of transplantation. For several years,
our laboratory has used nonhuman primates for myogenic
cell transplantation studies. The close phylogenetic relationship between humans and macaques (both are primates of
the parvorder Catarrhini) provides optimal anatomic, physiologic, antigenic, and immunologic similarities for preclinical
transplantation research (21Y25). Moreover, because muscle
regeneration is more efficient in mice than in primates (26),
clinical extrapolation of myogenic cell transplantation results
in mice may be problematic. In our experience, myoblast
isolation, culture, scale-up, and final cell populations for
transplantation are similar in humans and macaques, which is
not the case for other less phylogenetically related mammals
such as mice and dogs. Therefore, the present results likely
can be extrapolated to humans.
Our earlier myoblast transplantation studies in macaques confirmed that the injected cells fuse with myofibers
in circumscribed areas around the injection sites (27). Therefore, to optimize myoblast grafting throughout large skeletal
muscles, we performed cell injections close to each other,
parallel between them, perpendicular to the myofibers, penetrating the whole muscle, and delivering the cells homogeneously in each injection trajectory (28). In transverse
sections of muscle biopsies 1 month later, myofiber profiles
expressing donor-derived proteins were arranged in parallel
stripes oriented from the muscle surface to deep regions
(14Y16, 29). We believe that because the cell suspension was
distributed homogeneously along each injection trajectory,
the implanted myoblasts remained in optimal positions to
participate in segmental regeneration of the neighboring
myofibers that were damaged by the needle (10).
Here, we found that even if they had been delivered
carefully along the injection trajectories, most grafted myoblasts did not remain along these trajectories but formed
capricious distribution patterns mainly in the perimysium. A
minor proportion accumulated in the epimysium but we rarely
observed A-GalYpositive cells in muscle bundles in contact
with myofibers that had been damaged by the injection.
Because the cell suspension in saline is a liquid with
low viscosity, the injected liquid is submitted to shear compressive stress by the host tissue and pushed toward weak
points. For this reason, some leakage is frequently produced
at injection points. However, our present observations indicate that the leakage is produced mostly inside the muscle,
toward cleavage planes dissected by the injected liquid in the
perimysium. We speculate that the perimysium is more susceptible to dissection than the endomysium and/or shear
compressive stress is greater inside muscle bundles. When
Ó 2011 American Association of Neuropathologists, Inc.
Copyright © 2011 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
J Neuropathol Exp Neurol Volume 70, Number 9, September 2011
the saline is absorbed, the cell accumulations become more
compact and less susceptible to intramuscular displacement.
Because we observed myoblast fusion with intrafascicular myofibers in the injection needle trajectories at
1 month after implantation, our present observations imply
either (i) that fusion with the myofibers is done by a few
myoblasts remaining in the trajectories, which may not always
be visible in the histologic sections, and/or (ii) grafted myoblasts migrate from the perimysium toward the myofibers
damaged by the injection. For the second possibility to be
valid, the grafted myoblasts must be more mobile than previously thought.
To assess whether transplanted myoblasts in macaques
migrate into skeletal muscles to fuse with damaged myofibers,
we needed a boundary to measure the starting point of the
intramuscular migration. By implanting the myoblasts subcutaneously over the epimysium of the muscle, the starting
point (i.e. the external surface of the muscle) was clear.
The A-GalYpositive myofibers detected under the subcutaneous implantation sites showed that the A-GalYpositive myoblasts were generally competent to penetrate several millimeters
in the underlying muscle to fuse with recipient myofibers in the
case of muscle damage. In 2 biopsies, the distance from the
muscle surface to the deepest-situated A-GalYpositive myofibers
was negligible (Monkey 8) or short (Monkey 2), whereas many
grafted cells fused with myofibers several millimeters deep in
the muscle. The distribution of A-GalYpositive myofibers indicated that this fusion took place in the regions damaged by the
needle and was probably with the damaged myofibers. Some
superficial A-GalYpositive myofibers were observed in 2 of the
3 sites in which no needle penetrations were done, but there
may have been superficial muscle damage in those foci caused
by the needle tip during the subcutaneous injection.
Our findings suggest that damaged myofibers attract the
grafted myoblasts to repair themselves. During myofiber regeneration, myoblasts move guided by gradients of chemoattractant factors released by the damaged myofibers and
macrophages that are recruited by the damage (30). Macrophages induce strong chemoattraction on myoblasts by
secreting platelet-derived growth factor, transforming growth
factor-A, basic fibroblast growth factor, and leukemia inhibitory factor (31). Other myoblast chemoattractants released by
myofiber damage include hepatocyte growth factor (32) and
thymosin b4 (33). Moreover, intact myofibers may have
repulsive interactions for myoblasts (34), which might prevent
the fusion of grafted myoblasts with intact myofibers.
The distribution and amount of A-GalYpositive myofibers
after subcutaneous transplantation were very different among
biopsies and less homogeneous than the intramuscular transplantation of myoblasts. Different factors could be responsible
for these differences. The procedure creates a large subcutaneous collection of cells, many of which may die by ischemia (35).
Thus, differences in cell survival could explain differences in
engraftment. The presence of clots blocking migration or the
random subcutaneous infiltration of the cell suspension could
also explain these differences.
This is the first demonstration in an animal phylogenetically close to humans that myoblasts proliferated in vitro
from primary cultures and then transplanted can migrate
Migration of Grafted Myoblasts in Monkey Muscles
several millimeters to fuse with myofibers in regeneration.
Migration of transplanted myoblasts to areas of injury was
previously reported in mouse muscles using the continuous
mouse cell line C2C12 (36), but these results are difficult
to extrapolate to humans for at least 2 reasons. First, the
immortal C2C12 cells have a greater migration capacity
than mouse myoblasts obtained from primary cultures (17).
Second, this migration was observed in minute muscles (thus,
for a very short distance) of small rodents that also may have a
greater capacity for muscle regeneration than primates (26).
The ‘‘lack of migration ability’’ is still referred as the
only factor explaining the local fusion of grafted myoblasts
in skeletal muscles (37). However, we mentioned a second
possibility several years ago: muscle regeneration is mandatory to draw the grafted cells to fuse with the recipient’s
myofibers (10, 38). Indeed, in normal muscles, this occurs
generally only in the regions damaged by the injections. The
belief that the ‘‘lack of migration ability’’ was the key factor
to explain the local fusion lead to studies attempting to promote migration of the grafted cells (18Y20, 39Y42). Some
studies reported an improved migration of myoblasts under
particular experimental conditions in vitro and in vivo in mice.
Nonetheless, exposing macaque myoblasts to motogenic factors enhanced their migration in vitro and in some in vivo
conditions (i.e. in immunodeficient mice) but did not change
the fact that they fused only in the injection trajectories after
intramuscular transplantation in macaques (42). These apparently contradictory results could be explained by observations
of the present study. Improving the intrinsic migrating capacity of grafted myoblasts would facilitate a phenomenon that
is already present, that is, they are recruited by the influx of
chemoattractants to sites of myofiber damage.
In conclusion, the limiting factor of myoblast transplantation is not an intrinsic inability of the grafted cells to
migrate. The problem is whether it would be possible to obtain
what might be called, ‘‘intelligent migration’’ of myoblasts, that
is, a coordinated infiltration of the muscle bundles allowing fusion with most myofibers in the absence of myofiber
regeneration. Because myoblasts are not supposed to have the
physiological function of migrating to fuse indiscriminately
with nondamaged myofibers (and this is probably the case for
other potential myogenic cells), it is pertinent to consider
whether this can be achieved in cell transplantation therapies.
We may have no other choice than trying to tailor transplantation strategies to the physiological properties. Otherwise, to
obtain ‘‘intelligent migration,’’ we should be able to create a
complex transplantation context that may include (a) appropriate chemoattractant gradients to make myogenic cells spread
throughout the muscle, (b) induction in cells with the ability to
penetrate the basal lamina without the help of inflammatory
cells, (c) inhibition of potential repulsive factors in intact myofibers, and (d) induction in quiescent myofibers of the molecular signals required for cell fusion interactions.
REFERENCES
1. Skuk D, Tremblay JP. Intramuscular cell transplantation as a potential
treatment of myopathies: Clinical and preclinical relevant data. Expert
Opin Biol Ther 2011;11:359Y74
Ó 2011 American Association of Neuropathologists, Inc.
Copyright © 2011 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
777
Skuk et al
J Neuropathol Exp Neurol Volume 70, Number 9, September 2011
2. Negroni E, Vallese D, Vilquin JT, et al. Current advances in cell therapy
strategies for muscular dystrophies. Expert Opin Biol Ther 2011;11:
157Y76
3. Partridge TA, Grounds M, Sloper JC. Evidence of fusion between host
and donor myoblasts in skeletal muscle grafts. Nature 1978;273:306Y8
4. Konigsberg IR. The differentiation of cross-striated myofibrils in short
term cell culture. Exp Cell Res 1960;21:414Y20
5. Watt DJ, Morgan JE, Partridge TA. Use of mononuclear precursor cells
to insert allogeneic genes into growing mouse muscles. Muscle Nerve
1984;7:741Y50
6. Bartlett RJ, Sharp NJ, Hung WY, et al. Molecular markers for myoblast
transplantation in GRMD. Adv Exp Med Biol 1990;280:273Y78
7. Kinoshita I, Vilquin JT, Gravel C, et al. Myoblast allotransplantation in
primates. Muscle Nerve 1995;18:1217Y18
8. Boubaker el Andalousi R, Daussin PA, Micallef JP, et al. Changes in
mass and performance in rabbit muscles after muscle damage with or
without transplantation of primary satellite cells. Cell Transplant 2002;
11:169Y80
9. Holzer N, Hogendoorn S, Zurcher L, et al. Autologous transplantation of
porcine myogenic precursor cells in skeletal muscle. Neuromuscul Disord
2005;15:237Y44
10. Skuk D. Myoblast transplantation for inherited myopathies: A clinical
approach. Expert Opin Biol Ther 2004;4:1871Y85
11. Skuk D, Goulet M, Roy B, et al. Dystrophin expression in muscles of
Duchenne muscular dystrophy patients after high-density injections of
normal myogenic cells. J Neuropathol Exp Neurol 2006;65:371Y86
12. Skuk D, Goulet M, Roy B, et al. First test of a ‘‘high-density injection’’
protocol for myogenic cell transplantation throughout large volumes of
muscles in a Duchenne muscular dystrophy patient: Eighteen months
follow-up. Neuromuscul Disord 2007;17:38Y46
13. Skuk D, Roy B, Goulet M, et al. Dystrophin expression in myofibers of
Duchenne muscular dystrophy patients following intramuscular injections of normal myogenic cells. Mol Ther 2004;9:475Y82
14. Skuk D, Goulet M, Roy B, et al. Efficacy of myoblast transplantation in
nonhuman primates following simple intramuscular cell injections: Toward
defining strategies applicable to humans. Exp Neurol 2002;175:112Y26
15. Skuk D, Goulet M, Tremblay JP. Use of repeating dispensers to increase
the efficiency of the intramuscular myogenic cell injection procedure.
Cell Transplant 2006;15:659Y63
16. Quenneville SP, Chapdelaine P, Skuk D, et al. Autologous transplantation of muscle precursor cells modified with a lentivirus for muscular dystrophy: Human cells and primate models. Mol Ther 2007;15:
431Y38
17. El Fahime E, Torrente Y, Caron NJ, et al. In vivo migration of transplanted myoblasts requires matrix metalloproteinase activity. Exp Cell
Res 2000;258:279Y87
18. El Fahime E, Mills P, Lafreniere JF, et al. The urokinase plasminogen
activator: An interesting way to improve myoblast migration following
their transplantation. Exp Cell Res 2002;280:169Y78
19. Caron NJ, Asselin I, Morel G, et al. Increased myogenic potential and
fusion of matrilysin-expressing myoblasts transplanted in mice. Cell
Transplant 1999;8:465Y76
20. Lafreniere JF, Mills P, Tremblay JP, et al. Growth factors improve the in
vivo migration of human skeletal myoblasts by modulating their endogenous proteolytic activity. Transplant 2004;77:1741Y7
21. Geluk A, Elferink DG, Slierendregt BL, et al. Evolutionary conservation
of major histocompatibility complexYDR/peptide/T cell interactions in
primates. J Exp Med 1993;177:979Y87
778
22. Jaeger EE, Bontrop RE, Lanchbury JS. Structure, diversity, and evolution
of the T-cell receptor VB gene repertoire in primates. Immunogenetics
1994;40:184Y91
23. Rose SM, Blustein N, Rotrosen D. Recommendations of the expert panel
on ethical issues in clinical trials of transplant tolerance. National Institute
of Allergy and Infectious Diseases of the National Institutes of Health.
Transplantation 1998;66:1123Y25
24. Kirk AD. Transplantation tolerance: A look at the nonhuman primate
literature in the light of modern tolerance theories. Crit Rev Immunol
1999;19:349Y88
25. Kirk AD. Crossing the bridge: Large animal models in translational
transplantation research. Immunol Rev 2003;196:176Y96
26. Borisov AB. Regeneration of skeletal and cardiac muscle in mammals:
Do nonprimate models resemble human pathology? Wound Repair
Regen 1999;7:26Y35
27. Skuk D, Roy B, Goulet M, et al. Successful myoblast transplantation in
primates depends on appropriate cell delivery and induction of regeneration in the host muscle. Exp Neurol 1999;155:22Y30
28. Skuk D, Goulet M, Roy B, et al. Myoblast transplantation in whole muscle
of nonhuman primates. J Neuropathol Exp Neurol 2000;59:197Y206
29. Richard PL, Gosselin C, Laliberte T, et al. A first semimanual device for
clinical intramuscular repetitive cell injections. Cell Transplant 2010;19:
67Y78
30. Moens PD, Van-Schoor MC, Marechal G. Lack of myoblasts migration
between transplanted and host muscles of mdx and normal mice. J
Muscle Res Cell Motil 1996;17:37Y43
31. Robertson TA, Maley MA, Grounds MD, et al. The role of macrophages
in skeletal muscle regeneration with particular reference to chemotaxis.
Exp Cell Res 1993;207:321Y31
32. Bischoff R. Chemotaxis of skeletal muscle satellite cells. Dev Dyn 1997;
208:505Y15
33. Tokura Y, Nakayama Y, Fukada S, et al. Muscle injuryYinduced thymosin beta4 acts as a chemoattractant for myoblasts. J Biochem 2011;
149:43Y48
34. Siegel AL, Atchison K, Fisher KE, et al. 3D timelapse analysis of muscle
satellite cell motility. Stem Cells 2009;27:2527Y38
35. Skuk D, Paradis M, Goulet M, et al. Ischemic central necrosis in pockets
of transplanted myoblasts in nonhuman primates: Implications for celltransplantation strategies. Transplantation 2007;84:1307Y15
36. Watt DJ, Karasinski J, Moss J, et al. Migration of muscle cells. Nature
1994;368:406Y7
37. Meng J, Muntoni F, Morgan JE. Stem cells to treat muscular dystrophiesVWhere are we? Neuromuscul Disord 2011;21:4Y12
38. Skuk D, Tremblay JP. Myoblast transplantation: The current status of a
potential therapeutic tool for myopathies. J Muscle Res Cell Motil 2003;
24:285Y300
39. Lafreniere JF, Mills P, Bouchentouf M, et al. Interleukin-4 improves the
migration of human myogenic precursor cells in vitro and in vivo. Exp
Cell Res 2006;312:1127Y41
40. Mills P, Dominique JC, Lafreniere JF, et al. A synthetic mechano growth
factor E peptide enhances myogenic precursor cell transplantation success. Am J Transplant 2007;7:2247Y59
41. Mills P, Lafreniere JF, Benabdallah BF, et al. A new pro-migratory activity
on human myogenic precursor cells for a synthetic peptide within the E
domain of the mechano growth factor. Exp Cell Res 2007;313:527Y37
42. Lafreniere JF, Caron MC, Skuk D, et al. Growth factor co-injection
improves the migration potential of monkey myogenic precursors without
affecting cell transplantation success. Cell Transplant 2009;18:719Y30
Ó 2011 American Association of Neuropathologists, Inc.
Copyright © 2011 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.