Journal of Neuropathology and Experimental Neurology
Copyright q 2003 by the American Association of Neuropathologists
Vol. 62, No. 9
September, 2003
pp. 951 967
Resetting the Problem of Cell Death Following Muscle-Derived Cell Transplantation: Detection,
Dynamics and Mechanisms
DANIEL SKUK, MD, NICOLAS J. CARON, MSC, MARLYNE GOULET, BRIGITTE ROY,
AND
JACQUES P. TREMBLAY, PHD
Abstract. We conducted a study in mice to reevaluate and clarify many aspects of the early survival of muscle cells following
transplantation. Male mouse muscle cells (primary-cultures and T-antigen-immortalized clones) labeled with [14C]thymidine
and ß-galactosidase were injected into female muscles. Each label was detected in the muscles after different time periods.
TUNEL, alizarin red, and immunodetection of active caspase-3 were done in muscle sections. The donor cell labels disappeared
from the muscles following donor cell death, but this was not instantaneous and even if the donor cells were killed before
transplantation, the first 6 hours were not enough to clear [14C]thymidine and Y chromosome. Using the cell pellet before
injection as the 100% baseline for cells injected to evaluate cell death can lead to misinterpretations: the Y-chromosome band
was 5-fold stronger than that of a muscle injected with cells, irrespective of whether the cells were previously killed or not.
There was no evidence of an immediate massive donor cell death. Necrosis (detected by alizarin red) and apoptosis (detected
by active caspase-3) were present among the donor myoblasts following transplantation. Necrosis seemed to be the most
important mechanism during the first hours. T-antigen immortalized cells died earlier and more massively than primarycultured cells, but the surviving cells proliferated more. Indeed, they seemed to exhibit more apoptosis and they triggered a
more rapid CD81 cell infiltration. As a result of our findings, many concepts concerning the early donor cell death following
myoblast transplantation must be reconsidered.
Key Words:
Apoptosis; Cell death; Myoblast; Necrosis; Proliferation; Transplantation.
INTRODUCTION
Cell replacement therapies are experimental approaches to the treatment of diseases based on the transplantation of normal or genetically modified cells. In the field
of neuropathology, the targets of cell-based therapies are
both pathologies of the central nervous system, such as
stroke (1) and Parkinson disease (2), and peripheral illnesses, such as myopathies (3). Neurons and glial cells
from different origins are the donor cells experimentally
used for the treatment of central diseases, while myogenic
cells (myoblasts) are those experimentally used for the
treatment of myopathies.
These cell-based therapies depend on an appropriate
knowledge of the donor cell biology after transplantation,
one of the crucial factors being the early survival of donor cells. In spite of the studies done to date, the early
survival of myogenic cells following transplantation is
not well understood. A review of the literature in this
subject can be disconcerting. As an example, the extent
of donor cell survival (as interpreted by the different
studies) varied from 0.7% of donor cells remaining at 2
days (4) to 70% at 3 days (5). The factors implicated in
the early donor cell death are controversial. First, it was
suggested that cellular effectors of an acute inflammatory
From Unité de recherche en Génétique humaine, Centre de Recherche
du Centre Hospitalier de l’Université Laval, CHUL du CHUQ, Ste-Foy,
Québec, Canada.
Correspondence to: Daniel Skuk, MD, Unité de recherche en Génétique humaine, Centre Hospitalier de l’Université Laval, 2705 boulevard
Laurier, Québec (Qué), G1V 4G2 Canada. E-mail: Daniel.Skuk@anm.
ulaval.ca
This work was supported by a grant from the Association Française
contre les Myopathies (AFM).
response killed the donor cells (6–8). Second, it was suggested that a small specific donor cell sub-population
with stem cell-like characteristics survived transplantation and proliferated, while the other cells died by an
unidentified mechanism (9). Third, it was proposed that
a combination of natural killer cells, T-lymphocytes, and
complement killed the donor cells as soon as they were
implanted (4, 10).
While some contradictions can be attributed to the differences in the methods used to detect the donor cells,
some others are especially intriguing. Two of the previous
hypotheses were based on the interpretation that a massive donor cell death occurs instantaneously after the
transplantation (4, 10), or during the minutes following
transplantation (9). However, these studies eluded an important issue: they did not explain how the labels used to
quantify the donor cells can disappear as rapidly from the
host muscles, assuming that when a cell die all their labels disappear immediately.
Another aspect that remains obscure is the mechanism
of donor cell death. There are at least 3 pathways that
can be considered: apoptosis, necrosis, and autolytic cell
death. The importance of these mechanisms was not sufficiently studied. Apoptosis was considered briefly in
only 3 studies during limited periods after myoblast transplantation (4, 7, 9). It was concluded either that apoptosis
was not present or that it was negligible. However (as
discussed later) the methods were not appropriate to
quantify apoptosis in vivo.
We conducted the present study with the aim of resetting the basis for understanding the phenomenon of early
donor cell death after muscle-derived cell transplantation
and, potentially, transplantation of other kinds of cells.
951
952
SKUK ET AL
Our idea of departure was that some previous conclusions, such as that there is a massive donor cell death
immediately after transplantation that can be immediately
detected, couldn’t be biologically supported. Our working
hypothesis was that these conclusions were based on misinterpretations or methodological insufficiencies and,
therefore, we had 4 specific goals: 1) to define the properties and limitations of the methods currently used to
quantify donor cell death and survival after transplantation; 2) to determine if there is unequivocal evidence that
most donor cells are dying immediately after transplantation; 3) to search more accurately for evidence of apoptosis, necrosis, or autolysis among the donor cells during this period; and 4) to analyze the dynamics of the
early donor cell survival according to our own data, especially in correlation with the dynamics of the immune
cell reactions.
MATERIALS AND METHODS
Host Animals
Mouse muscle-derived cells labeled with radioactive
[14C]thymidine, ß-galactosidase (ß-Gal) or Y chromosome
(male cells were transplanted in females) were injected into
mouse muscles. Cell transplantation was performed on 2- to 4month-old, female, wild type CD-1 mice. This work was conducted according to the guidelines of the Canadian Council of
Animal Care and was authorized by the Laval University Animal Care Committee.
Donor Cells
Muscle-derived cells were from primary cultured (PC) cells
from newborns and clones of T-antigen-immortalized (Tag)
myoblasts. PC cells were prepared from pups on postnatal days
2 to 3. CMVLacZ transgenic mice were used for ß-Gal expression (for details of the transgenic mice, see [8]). After enzymatic dissociation of muscles with collagenase 0.2% and trypsin 0.25%, the cells were cultured in DMEM high glucose
(Invitrogen, Burlington, ON, Canada) at 378C for 3 days. Tag
cells contained the heat sensitive T-antigen under the promoter
of a MHC gene (H-2Kb) inducible by g-interferon. These cells
were cultured at 338C and 10% CO2 in DMEM low glucose
(Invitrogen) with 15% fetal calf serum, 2% chick embryo extract (Invitrogen), and 20 U/ml of mouse recombinant g-interferon (Genzyme, Cambridge, MA). These cells express 2 ß-Gal
genes, one under the control of a retroviral long terminal repeat
and the other under the quail fast troponin I promoter (6).
labels, the muscles injected with cells were dissected and frozen
in liquid nitrogen. For time 0, the muscle of a previously killed
mouse was dissected off and the cells were injected into the
isolated muscle that was immediately frozen. For histological
studies, the muscles were embedded in OCT media (Miles Laboratories, Elkhart, IN) and immediately frozen in liquid nitrogen.
Y Chromosome QC-PCR
DNA was extracted from muscles or from cell pellets as previously described (11). Male QC-PCR analysis was done by
adding 2 ml out of each 500 ml sample to the PCR mixture
containing primers (pY2a: 59-GCATTTGCCTGTCAGAGAGAG-39, pY2c: 59-ACTGCTGCTGCTTTCCAACTA-39) engineered to amplify a 488-bp region of the mouse Y chromosome
and a 571-bp region of the pBbgpY2 competitor plasmid. Details of the PCR amplification can be found in a previous report
(11). PCR products were loaded in 1.5% agarose gel and ethidium bromide-stained DNA was scanned with an AlphaImager
digital imaging system, avoiding saturation. PCR amplicon densitometry was performed with NIH Image analysis software.
Each value was expressed as the ratio genomic/standard PCR
product densitometry.
Radiolabel Detection
Radiolabeling of myoblasts was performed by adding 0.25
mCi/ml [methyl-14C] thymidine (Amersham Biosciences, Baie
d’Urfé, QC, Canada) to the growth medium 16 to 24 hours
before cell transplantation. The amount of radiolabel in the
muscles transplanted with these cells was measured in the DNA
isolated as for the PCR. Similar aliquots of the DNA solution
were mixed with 5 ml of a liquid scintillation counting cocktail
(Sigma, Oakville, ON, Canada) and radioactivity was measured
in a counter system.
ß-Gal Assay
ß-Gal activity in the muscles was quantified as previously
described (6). Muscles were homogenized in 800 ml of Tris
buffer 0.25M pH 8.0. Samples were centrifuged 5 min at 6,500
rpm. One hundred ml of the supernatant was mixed with 3 ml
of a solution of 0.1 M MgCl2 4.5 M ß-mercaptoethanol plus 66
ml of a solution of 4 mg/ml of 0-nitrophenyl-ß-D-galactopyranoside (Sigma) diluted in 0.1 M sodium phosphate buffer pH
7.5 and 131 ml of the same buffer. Samples were incubated at
378C. The reaction was stopped by adding 150 ml of 1 M
Na2CO3. Optical density was read at 420 nm on a spectrophotometer.
Histological Analysis
Cell Transplantation
Mice were anesthetized with a solution of 10 mg/ml of ketamine (Rogar STB, Montreal, QC, Canada) and 10 mg/ml of
xylazine (Bayer, Etobicoke, ON, Canada). Cell injections (5 3
105 to 2 3 106 cells resuspended in 10–20 ml of HBSS) were
performed in the Tibialis anterior with a glass micropipette
(Drummond Scientific, Broomall, PA). In some experiments the
cells were killed by 3 cycles of freezing/thawing with liquid
nitrogen before transplantation. Mice were killed at different
periods under deep anesthesia. For quantification of the cell
J Neuropathol Exp Neurol, Vol 62, September, 2003
Serial 10-mm sections were obtained in a cryostat. Some sections were stained with hematoxylin and eosin. ß-Gal was revealed by histochemistry using X-Gal as substrate, as previously described (12). Sections were incubated for 1 hour with
1:50 monoclonal mouse anti-desmin (Dako, Mississagua, ON,
Canada), followed by a 30-min with 1:200 Alexa Fluor 488
goat anti-mouse IgG (Molecular Probes, Eugene, OR), then incubated with 0.5 mg/ml rabbit anti-human/mouse active caspase-3 (R&D Systems, Minneapolis, MN), followed by 30 min
with 1:300 biotinylated swine anti-rabbit IgG (Dako), and 30
CELL DEATH IN MYOBLAST TRANSPLANTATION
min with 1:700 Cy3-streptavidin (Sigma). Neutrophils were
identified by 1-hour incubation with 1:20 biotinylated antimouse Gr-1 (Pharmingen, Mississagua, ON, Canada), followed
by 1:700 streptavidin-Cy3 for 30 min. CD81 cells were identified using a YTS-169 hybridoma supernatant (gift of Dr.
Waldmann, Cambridge, UK) and macrophages using a rat antimouse F4/80 (Cedarlane, Hornbly, ON, Canada). Sections were
incubated 1 hour with undiluted supernatant (YTS-169) or with
anti-F4/80 (1:100) followed by 30 min with 1:300 biotinylated
anti-rat IgG antibody (Dako), and 30 min with 1:700 streptavidin-Cy3. Nonspecific binding was blocked with 10% FBS in
PBS during 20 min. The antibodies were diluted in PBS pH
7.4, with 1% FBS. Incubations were done at room temperature.
Negative controls omitting the first antibody were done. A terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay (TUNEL) was performed
with an Apoptag Plus kit (Intergen, Burlington, MA) according
to the manufacturer’s instructions, applying 1 mg/ml propidium
iodide to the mounting medium as counterstain. Some sections
were stained for 5 min in a 2% alizarin red (Sigma) solution at
pH 5.4 (13). Acid phosphatase was detected by a 378C 1-hour
incubation in a solution of Naphthol AS phosphate (Sigma),
pararosaniline hydrochloride (Sigma), and sodium nitrite in a
veronal-acetate buffer at a final pH of 4.7 to 5.0 (13).
Quantitative Histological Analysis
Computer images of the muscle sections under different histological techniques were obtained using a Pixera 1.0.2 camera
(Pixera Corporation, Los Gatos, CA) attached to a microscope.
The cross-sectional area of the donor cell pockets and the surface that was positive to a given reaction (alizarin red or immunopositive labeling) was measured using a computer image
analyzer (NIH Image 1.61, Bethesda, MD). The percentage of
propidium-iodide-positive nuclei that were TUNEL-positive in
the implanted cell clusters was analyzed this way. The number
of cells that were positive both for desmin and caspase-3 active
were directly counted in the microscope.
Statistical Analysis
Each time point in the graphics is represented as the mean
value of n 5 3 to 6 mice (generally 4–5) 6 1 SD. An ANOVA
test (using statistical software Stat View 4.01, BrainPower, Ottawa, ON, Canada) was used to discriminate significant differences.
RESULTS
Properties and Limitations of the Methods Used to
Detect Donor Cell Death
Cell Pellet vs Cell Injection of Donor Cells: The previous studies that reported a massive donor cell loss instantaneously after transplantation used the cell pellet obtained before injection to establish the baseline (100%)
value of the amount of donor cells to be injected. We
investigated whether there is a difference between this
method and our approach, which establishes the baseline
value based on the number of cells present at time 0 in
a muscle (i.e. a muscle that was dissected out of the
953
mouse, injected with donor cells, and immediately frozen). Using [14C]thymidine to detect the donor cells (Fig.
1A), only 66% of the radiolabel present in the cell pellet
was found in the muscles at time 0. In fact, 21% of radiolabel was detected in the tube after transplantation (the
radiolabel in the micropipette was not analyzed). When
we quantified the Y-chromosome by PCR, the band was
5- to 6-fold more intense in the cell pellet than in the
muscle at time 0 (an example of the PCR is shown in
Fig. 1B, top). However, when living or dead cells were
transplanted in muscles (the cells were killed by 3 cycles
of freezing/thawing before transplantation), no quantitative differences in the Y chromosome detected in the
muscles were observed at either time 0 or 1 hour (i.e.
killing the cells did not reduce the amount of Y chromosome detected). In addition, there were no significant
differences between the pellets of living cells, the pellets
of dead cells at time 0, and the pellets of dead cells left
1 hour at 378C (Fig. 1B, bottom). As we will discuss
later, these results suggest that when the DNA was extracted from muscles injected with cells rather than from
the cells alone, some factor due to the presence of the
muscle reduced the amount of Y-chromosome detected.
Consequently, in the present study we used a muscle injected with cells and immediately frozen as the reference
to quantify the 100% of donor cells.
Rapidity of the Different Labels to Detect Donor Cell
Death (Fig. 1C): As a second step, we tested the rapidity
by which the specific labels currently used to quantify
the donor cell survival ([14C]thymidine, Y chromosome
and ß-Gal) can detect the donor cell death. For this, PC
cells were killed by 3 cycles of freezing/thawing and immediately injected in the muscles of mice. A trypan-blue
exclusion test previously done in vitro confirmed that this
treatment killed all the cells. The muscles injected with
these cells were analyzed at time 0 and at 1, 6, and 24
hours post-transplantation. Using [14C]thymidine and Y
chromosome to quantify the donor cells in the host muscles, there were no significant differences between time
0 and 6 hours. In the case of ß-Gal, a loss of 60% was
observed at 1 hour and 89% at 6 hours. All labels disappeared 24 hours post-transplantation. In conclusion,
even if cells were killed before transplantation, the first
6-hour-period was not enough to clear the [14C]thymidine
and Y chromosome labels from the host muscles. ß-Gal
disappeared faster: its loss was not complete at 1 hour,
but 6 hours were enough to evidence most of the cell
death.
Equivalency of the Different Labels to Quantify Early
Donor Cell Survival (Fig. 1D): We analyzed the equivalency of the 3 methods to evaluate the donor cell survival after transplanting living PC cells into muscles and
sampling the muscles 3 days later. [14C]thymidine (which
permits to detect cell death but not cell proliferation) was
reduced to ;30% of the time 0 value. Y chromosome
J Neuropathol Exp Neurol, Vol 62, September, 2003
954
J Neuropathol Exp Neurol, Vol 62, September, 2003
SKUK ET AL
CELL DEATH IN MYOBLAST TRANSPLANTATION
955
Fig. 2. A: Detection of donor cell labels at 6 hours post-transplantation: only ß-Gal in cloned Tag cells significantly decreased.
B: During the first 3 days following transplantation, [14C]thymidine revealed a more intense cell death in Tag cells than in PC
cells. In spite of this cell death, the Y chromosome PCR showed that the whole population of cells increased (PC cells) or
remained at similar levels (Tag cells). [14C]thymidine and Y chromosome revealed that the population of PC cells was relatively
stable between day 3 and day 6. In contrast, the Tag cells disappeared in this time period. Abbreviations: D: days; H: hours.
* p , 0.05, ** p , 0.005 (when compared with the preceding value in the graphic or between values indicated by arrows).
(which quantifies the whole population of donor cells resulting of death and proliferation) increased ;30% above
the time 0 value. ß-Gal was reduced to ;35% of the time
0 value.
In theory, ß-Gal should follow the same pattern of expression as the Y chromosome, thus it should increase
instead of decrease. This discrepancy suggested that the
expression of the ß-Gal transgene was affected after
transplantation. We observed after culturing the
CMVLacZ PC cells during 3 days that although the number of cells increased ;3-fold, the expression of ß-Gal
for the whole population decreased to ;30% of the
←
Fig. 1. A: Taking the radiolabel detected in the cell pellet (before transplantation) as 100%, 66% was found in the muscles
immediately after the cell injection and 21% remained in the tube. B: Examples of the Y-chromosome PCR in the cell pellet and
in the muscles injected with cells. There were no differences between the muscles injected with living cells or cells previously
killed by freezing/thawing at time 0 and 1 hour: a homogeneous ratio between the standard and the DNA was observed (top).
For the cell pellet, in contrast, a stronger Y-chromosome signal was observed and the standard became sometimes imperceptible.
The intensity of the Y-chromosome band in the cell pellet was not significantly different, independently of whether the cells were
living or whether they passed 1 hour at 378C after being killed (bottom). C: Rapidity of different labels to detect donor cell death
after transplanting cells previously killed by freezing/thawing. [14C]thymidine and Y chromosome were unable to reveal the cell
death at 6 hours. ß-Gal decreased faster, but 6 hours were needed for a substantial reduction. D: Behavior of the labels, 3 days
following transplantation of living cells. [14C]thymidine was reduced to ;30% and Y chromosome increased by ;30%. While
ß-Gal was expected to be similar to Y chromosome, it was reduced to ;35% (see the text for the explanation). Abbreviations:
D: days; H: hours; L: living cells; K: killed cells. * p , 0.05, ** p , 0.005 (when compared with the preceding value in the
graphic).
J Neuropathol Exp Neurol, Vol 62, September, 2003
956
SKUK ET AL
original value. Therefore, proliferation in vitro was sufficient to down-regulate the ß-Gal expression in our
CMVLacZ PC cells. According to these results, we concluded that ß-Gal could be useful to provide some information about the immediate donor cell death (mostly at
6 hours) but not to analyze the later donor cell survival due
to the possibilities of down-regulation (see Discussion). On
the contrary, although [14C]thymidine and Y chromosome
cannot detect donor cell death during the first hours, their
combination give a good approximation of the donor cell
survival dynamics during the first days because they
cannot be affected by factors other than donor cell death
or proliferation.
Dynamics of Early Donor Cell Survival
For the analysis of the early donor cell survival following cell transplantation it is important to understand
its dynamics. Considering that the results reported above
suggested that the analyses that were previously
published were probably distorted by methodological
problems, we wanted to reanalyze the early donor cell
death dynamics following myoblast transplantation. This
was done by using 2 kinds of donor cells: PC cells and
Tag cells.
Time 0 vs 6 Hours (Fig. 2A): The first 6 hours posttransplantation were considered separately, because it
was the only period in which ß-Gal could be useful as a
reference for cell death. Although [14C]thymidine and Y
chromosome were not able to detect the donor cell death
during the first 6 hours following transplantation of dead
cells, the same experiment was repeated with living cells.
Consistent with our previous observations, no significant
differences were observed between time 0 and 6 hours
using those labels. ß-Gal did not show a significant decrease in the case of PC cells, but a significant decrease
of ;50% was observed for Tag cells.
First Days Post-Transplantation (Fig. 2B): Most of the
radiolabel present in the donor cells was lost from the
muscles during the first 3 days following cell transplantation. For PC cells, 69% of the original radiolabel remained at day 1, 38% at day 2, 30% at day 3, and 24%
at day 6. In this experiment, only 6% of the cells were
dead (detected by a trypan blue exclusion test) before
transplantation. For Tag cells, the radiolabel at day 1 was
only 14% of that present at time 0, 6% at day 2, 3% at
day 3, and 1.5% at day 6. Only 9% of the cells were
dead before transplantation.
In separate experiments, the Y chromosome analysis
showed that the whole population of PC cells was preserved or increased in the host muscles during this period
due to a proliferation of the surviving cells. In the case
of Tag cells, the Y chromosome showed that the donor
cell population was preserved at day 3 (due also to proliferation) but disappeared at day 6.
J Neuropathol Exp Neurol, Vol 62, September, 2003
Detection of Cell Death Mechanisms
Considering that the mechanisms of donor cell death
have not, to date, been sufficiently studied, our next step
was to detect 3 potential mechanisms of cell death (apoptosis, necrosis, and autolytic cell death) and to define
their dynamics. This was done by histological analysis of
the muscles injected with cells (Fig. 3).
In the muscle sections, the donor cells were seen as
pockets of mononuclear cells (Fig. 3A, B). Since both
PC cells and Tag cells expressed LacZ genes, ß-Gal detection confirmed that these pockets corresponded to the
transplanted cells (Fig. 3C). Activated caspase-3 (detected by immunohistochemistry as a marker of apoptosis)
was observed in the donor cell pockets (Fig. 3D). The
percentage of the donor cell pocket cross-section that was
positive for active caspase-3 was less than 2% during a
5-day follow-up (Fig. 4). Since inflammatory cells infiltrate the donor cell pockets (discussed later), colocalization of active caspase-3 and desmin (Fig. 3D, E) was
done to distinguish caspase activation in myogenic donor
cells from the apoptosis that can normally occur in infiltrating leukocytes. The percentage of desmin-positive
cells in the donor cell pockets that were active caspase3-positive was less than 4% (Fig. 4).
A TUNEL method was used to morphologically identify DNA fragmentation among the donor cells. TUNEL
is frequently used to detect apoptosis, although it can also
detect necrosis and autolysis. We observed that some nuclei into the donor cell pockets were positive by TUNEL
(Fig. 3F, G). Low levels (less than 1.5%) of TUNELpositive nuclei were observed at most periods, except for
a peak of 8% to 12% at 6 hours (Fig. 4). This peak of
TUNEL-positive cells was similar to the death observed
before transplantation (detected by a trypan-blue exclusion test) that was 9% to 12% in the case of PC cells
and 6% to 7% in Tag cells.
Alizarin red staining was used to identify cells with a
damaged membrane. Membrane damage is an early event
of necrosis; it allows the entry of extracellular calcium
ions and the subsequent intracellular calcium deposits are
stained with alizarin red. We observed alizarin red staining both in myofibers damaged by the injection (Fig. 3H)
and among the Tag cells (Fig. 3H). This technique failed
to demonstrate calcium deposition in PC cells. The percentages of Tag cells that were alizarin red-positive are
shown in Figure 4. There were peaks of 23% at 1 hour
and 19% at 6 hours. These percentages declined later and
only isolated cells were alizarin red-positive from day 2
to day 5.
Detection of acid phosphatase was used to detect lysosomal activity among the donor cells, which is frequently associated with autolytic cell death in many tissues. Surprisingly, acid phosphatase activity was detected
CELL DEATH IN MYOBLAST TRANSPLANTATION
957
Fig. 3. A: Illustration of how donor cell pockets were produced in the muscles. B–I: Muscle cross-sections show these donor
cell pockets under different techniques: hematoxylin and eosin (B), ß-Gal detection (C), immunofluorescent detection of active
caspase-3 (D), immunofluorescent detection of desmin (E), propidium iodide (F), fluorescent TUNEL (G), alizarin red (H), and
histochemical detection of acid phosphatase (I). The boundaries of the donor cell pockets are indicated between arrowheads in
(B) and (H). Donor cell pockets were seen as pockets of mononuclear cells (B) that were ß-Gal-positive (C, arrows). These
pockets showed different densities of desmin-positive cells (E), depending on their cell composition and on the leukocyte infiltration (Fig. 7). Co-detection of desmin and active caspase-3 showed apoptosis in myogenic cells and not in infiltrating leukocytes:
a pocket of donor Tag cells (day 5) is shown after co-detection of active caspase-3 (D) and desmin (E). Examples of desminpositive cells (arrows) and desmin-negative cells (arrowheads) that are positive for active caspase-3 are indicated. The same
section of a pocket of donor PC cells (6 hours after implantation) is shown with propidium iodide (F) and TUNEL (G). Alizarin
red (H) shows calcium deposition both in myofibers damaged by the injection (arrows) and among donor Tag cells (between
arrowheads) 6 hours after transplantation. A strong acid phosphatase reaction is observed in a donor cell pocket, 1 hour after
transplantation (I). Scale bars: C 5 1 mm; B, D, E, H, I 5 100 mm; F, G 5 50 mm.
in all the donor cells as early as 1 hour after transplantation (Fig. 3I). These cells were also positive for acid
phosphatase at time zero.
Nonspecificity of TUNEL
Since we observed a peak of TUNEL-positive nuclei
without similar amounts of previous caspase-3 activation,
we wanted to verify whether this was due to apoptosis
or if it was nonspecific. Indeed, since the percentages
were similar to the cell death observed before transplantation, we wanted to verify whether 1 or 6 hours were
sufficient to detect by TUNEL the donor cell death produced before transplantation. Therefore, cells killed by
freezing/thawing were injected into muscles and the
J Neuropathol Exp Neurol, Vol 62, September, 2003
958
SKUK ET AL
pockets were similar to the density and distribution of
alizarin red-positive cells. This suggested that nuclei with
degraded DNA (TUNEL-positive) corresponded to the
necrotic cells detected by calcium deposition (alizarin
red-positive), offering further evidence of TUNEL-detected necrosis in this case.
Follow-Up of Cell Death During the First 24 Hours
Post-Transplantation (Fig. 6)
At 5 days, follow-up showed that an event such as a
TUNEL-positive peak was detected at 6 hours but not in
the other periods. The importance of this observation is
that a mechanism of cell death may occur in the first
hours after transplantation, but may remain undetected if
the right time period is not analyzed. Consequently, we
decided to observe more accurately the cell death detection by TUNEL, alizarin red, and active-caspase-3 labeling during the first 24 hours post-transplantation.
TUNEL labeling was similar in both kinds of donor
cells: positive nuclei were rare at time 0, they increased
to a maximum at 9 to 12 hours (;15%–20%), and declined later. In contrast, there was a difference for active
caspase-3: the levels were negligible in PC cells but increased progressively in Tag cells, with a peak of ;15%
of the desmin-positive cells at 24 hours and of ;5% of
the donor cell pocket at 18 to 24 hours. Alizarin red was
negative at time 0, 14% and 11% of the donor cells were
positive at 1 and 3 hours, respectively. There was a 35%
peak at 6 hours and the percentages declined thereafter.
Acid phosphatase activity was observed in the donor cells
at all periods, including time 0.
Immune Cellular Infiltration
Fig. 4. Histological follow-up of cell death events during
the first 5 days post-transplantation. Active caspase-3 was detected in the donor cell pockets although in low percentages:
i.e. ,4% of the myogenic cells. Among the nuclei in the donor
cell pockets, TUNEL showed a peak of 12% (PC cells) and 8%
(Tag cells) at 6 hours. In the other time periods examined the
levels were ,1.5%. Alizarin red revealed calcium deposition in
23% and 19% of the Tag cells at 1 and 6 hours respectively,
and declined to negligible levels from day 2 to 5. Abbreviations: H: hours; D: days.
muscles were analyzed by histology after 1 and 6 hours
(Fig. 5A–D). TUNEL showed 0.7% to 7% of positive
nuclei at 1 hour and 60% to 85% at 6 hours. This confirmed the nonspecificity of the mechanism of cell death
detected by TUNEL, and that when donor cells were
killed just before transplantation, few DNA fragmentations were detected by TUNEL after 1 hour, but such
fragmentations were abundant at 6 hours. In addition, we
observed similarities between serial sections stained with
TUNEL or alizarin red (Fig. 5E, F). The density and distribution of TUNEL-positive nuclei in the donor cell
J Neuropathol Exp Neurol, Vol 62, September, 2003
Finally, since some immune cells were suggested as
potential killers of the donor cells, we wanted to correlate
the dynamics of the early donor cell death (described
above) with the dynamics of the immune cell infiltration.
The dynamics of the immune cell infiltration in the donor
cell pockets is illustrated in Figure 7. Gr-1-positive cells
(neutrophils) infiltrated the donor cell pockets at 1 hour,
6 hours, and day 1 (the maximal infiltration was at 6
hours) and were rare from day 2 to day 6. The neutrophil
infiltration was similar in PC cells and Tag cells. F4/80positive cells (macrophages) were observed infiltrating
the donor cell pockets starting at day 1, where they remained until day 6. Macrophages constituted 20% to 30%
of the cells into the pockets of donor PC cells and 60%
to 80% into the pockets of Tag cells. CD8-positive cells
showed a strong infiltration starting at day 2 after Tag
cell transplantation, while the strong infiltration began at
day 6 following PC cell transplantation.
DISCUSSION
The aim of the present study is to introduce a rational
basis to the confusing and contradictory cumulus of data
CELL DEATH IN MYOBLAST TRANSPLANTATION
959
Fig. 5. Cells killed by freezing/thawing were transplanted in muscles and observed with propidium iodide (PI) staining and
TUNEL labeling (A–D). One hour after transplantation, clusters of propidium iodide fluorescent nuclei are visible (A). Within
these clusters a few nuclei are TUNEL-positive (B). Six hours after transplantation, most nuclei in the donor cell pockets stained
with propidium iodide (C) were TUNEL-positive (D). A close similarity in density and distribution is observed between alizarin
red-positive cells (E) and TUNEL-positive nuclei (F), 12 hours after transplantation of living Tag cells. Arrows in (E) indicate
alizarin red labeling in some damaged myofibers. Scale bars: A, B, E, F 5 200 mm; C, D 5 100 mm.
and interpretations about the early survival of donor muscle cells following transplantation. Since previous studies
have shown that muscle cells obtained or manipulated in
different ways may exhibit different post-transplantation
survival (14–16), we used 2 sources of donor cells in our
study. These 2 kinds of cells were those preferentially
used in previous studies: PC cells (4, 8–10, 14, 15) and
Tag cells (6, 7, 9, 14–17).
Properties and Limitations of the Methods to Detect
Donor Cell Survival
To date, 3 labels were used to quantify the donor cells
after intramuscular implantation: ß-Gal (6–8, 14), radiolabeled thymidine (9, 17), and Y chromosome (4, 9–11,
17). To evaluate data obtained with these labels, however,
care must be taken to understand the properties and limitations of each one. This was the first step of our study.
Rapidity to Detect Donor Cell Death: The 3 specific
labels were cleared from the host muscle at different
times when donor cells were killed before transplantation.
ß-Gal was the first to decrease. Since ß-Gal is a soluble
cytoplasmic enzyme, we can suppose that it can diffuse
to the extracellular fluid as soon as the cell membrane is
disrupted (Fig. 8B), and then enters the circulation system. Membrane breakdown is an early event in necrosis,
which explains why ß-Gal was the first label to decrease;
however, the total clearance of ß-Gal took some hours.
Detection of [14C]thymidine and Y chromosome labels
has not evidenced the donor cell death during the first 6
hours post-transplantation. This can be explained by the
mechanisms needed to delete the Y-chromosome sequence and to clear [14C]thymidine following the death
of the cell. The Y-chromosome sequence amplified by the
PCR needs DNA degradation to be lost (Fig. 8C). The
clearance of [ 14 C]thymidine may need, in addition,
phagocytosis followed by exocytosis (Fig. 8D). DNA
degradation can proceed by autolysis (breakdown of endogenous lysosomes) or heterolysis (leukocyte degranulation). In the case where heterolysis is the cause of the
Y-chromosome sequence degradation, the DNase activity
of macrophages is by far superior to that of neutrophils
(18, 19). Therefore, macrophages are the best candidates
both to digest the DNA of the dead cells (deleting the Ychromosome sequence) and to eliminate the [14C]thymidine
by phagocytosis, digestion and exocytosis. The dynamics
of [14C]thymidine and Y-chromosome loss is thus easily
J Neuropathol Exp Neurol, Vol 62, September, 2003
960
SKUK ET AL
Fig. 6. Histological follow-up of cell death events during
the first 24 hours post-transplantation. Active caspase-3 was
negligible in PC cells, but in Tag cells was present in 5% of
the pocket surface and in 13% of the desmin-positive cells.
Among the nuclei in the donor cell pockets, TUNEL showed a
peak of ;15% to 20% between 6 and 12 hours in both PC cells
and Tag cells. Tag cells showed a 35% peak of alizarin red
staining at 6 hours. Abbreviations: H: hours; D: days.
explained by our observation that macrophages start to
infiltrate the donor cell pockets between 6 to 24 hours
post-transplantation.
Correlation of the Different Methods to Detect Donor
Cell Survival: The behavior of each label in detecting cell
survival 3 days post-transplantation was different. The
differences between [14C]thymidine and Y chromosome,
in fact, were explained by the authors who developed the
method of combining both labels (9, 17). As observed by
these authors, the Y chromosome evaluates the result of
a process that involves death and proliferation: it decreases with donor cell death but increases with proliferation
J Neuropathol Exp Neurol, Vol 62, September, 2003
of the surviving cells. On the contrary, radiolabeled DNA
is divided among daughter cells during proliferation and
cannot increase—the radiolabel can only decrease reflecting the donor cell death.
ß-Gal should be expected to have a behavior similar
to Y chromosome, i.e. to increase with donor cell proliferation. However, this was not observed in our study,
when the Y chromosome increased whereas ß-Gal activity decreased. Thereafter, we observed in vitro that after
culturing newborn CMVLacZ cells during a 3-day period, the number of cells increased roughly 3-fold, while
the expression of ß-Gal (in the whole population) decreased to roughly 30% of the original. This demonstrated that the ß-Gal expression can be down-regulated, and
in the case of our CMVLacZ cells, it was sufficient to
proliferate in vitro. We cannot exclude that similar or
other factors down-regulate ß-Gal expression in vivo. Examples of these factors are inflammatory cytokines,
which can down-regulate the expression of transgenes
driven by viral promoters such as CMV (20). In fact, we
observed that pockets of donor cells were the sites of an
intense inflammatory infiltration, and thus, the release of
inflammatory cytokines could be responsible for downregulating ß-Gal during the first days after cell transplantation. Therefore, ß-Gal may not be appropriate for analyzing the early donor cell survival following cell
transplantation, although it can be useful to have a relative idea of whether there is a rapid donor cell death.
Inconvenience of Using the Cell Pellet: Our results
demonstrated that quantifying the amount of label in the
cell pellet to establish the 100% baseline for evaluation
of the donor cell death following cell transplantation, as
done in some studies (4, 10), is not an adequate method.
One source of error may be the fact that many cells remain in the tube and in the instrument used for injection.
Still more important is the specific problem that we observed in the Y chromosome analysis. The band of the
PCR product is 5- to 6-fold more intense in an isolated
cell pellet than in a muscle injected with the same cell
pellet and immediately frozen. This can not be attributed
to immediate DNA degradation caused by cell death, as
previously assumed (4, 10), because we observed that
DNA degradation takes more than 1 hour to be detected
by TUNEL and more than 6 hours to delete the sequences
detected with the Y chromosome PCR. Indeed, the intensity of the Y chromosome band in the cell pellet was
always stronger than the intensity of the Y chromosome
band in the muscles injected with the cells, regardless of
whether the cells had been killed previously. These observations suggest that when the DNA is extracted from
muscles injected with cells, rather than from the cells
alone, some factors due to the presence of the muscle
tissue (such as nucleases from the tissue or a decreased
yield of nucleic acid recovery due to high content in protein) may reduce the Y-chromosome signal. Regardless
CELL DEATH IN MYOBLAST TRANSPLANTATION
961
Fig. 7. The infiltration of pockets of PC cells and or Tag cells by immune cells is illustrated with representative examples of
the histological observations. Immune cells were detected by immunofluorescence. Each figure shows a portion of a donor cell
pocket between the myofibers. Gr-11 cells (neutrophils) were observed at 1 hour, 6 hours (maximal infiltration), and 1 day; they
were rare thereafter. F4/801 cells (macrophages) infiltrated the donor cell pockets starting at day 1 through day 6 (this was more
intense in Tag cells). CD81 cells infiltrated the donor cell pockets starting at day 6 in PC cells, while the infiltration started at
day 2 in Tag cells. H: hours; D: days. Scale bar: 50 mm (the same bar applies to all panels).
Fig. 8. Mechanisms needed to clear the donor cell labels following necrosis. A: A donor cell with the labels is represented.
B: Membrane damage, an early event of necrosis, may allow ß-Gal to diffuse to the extracellular space. C: Y chromosome
sequences may disappear following DNA degradation, which can proceed by autolysis (breakdown of endogenous lysosomes)
and/or heterolysis (leukocyte degranulation). D: [14C]thymidine may need phagocytosis and exocytosis to be cleared from the
host muscle. This dynamics will be different in apoptosis, mainly because membrane defect is a later event.
of the reason, it is important to note that taking the cell
pellet as the 100% baseline for quantifying cell survival
by Y chromosome leads to artifactual misinterpretation
and explains why previous reports interpreted that 80%
of the donor cells were dying as soon as they were placed
in a muscle (4, 10).
Dynamics of Donor Cell Survival
In order to elucidate the factors that could be involved
in producing donor cell death or favoring donor cell proliferation it is important to understand the dynamics of
donor cell survival. If donor cell death is detected at a
J Neuropathol Exp Neurol, Vol 62, September, 2003
962
J Neuropathol Exp Neurol, Vol 62, September, 2003
SKUK ET AL
CELL DEATH IN MYOBLAST TRANSPLANTATION
given period and some factors (such as some specific
immune cells) are suspected to cause this death, it must
be proven that these factors are present at the time that
death occurs.
In the present study, the dynamics of radiolabel loss
revealed that there was a continuous donor cell death
during the first 3 days. In the case of PC cells, 31% of
the radiolabel present at time 0 was cleared at day 1 (only
6% can be attributed to the cell death observed previous
to transplantation), 45% of the radiolabel still present at
day 1 was cleared at day 2, and 23% of the radiolabel
present at day 2 was cleared at day 3. The most significant radiolabel loss in PC cells was between day 1 and
day 2. In the case of Tag cells, 86% of the radiolabel
present at time 0 was cleared at day 1 (only 9% can be
attributed to the cell death observed previous to transplantation), and 78% of the radiolabel still present at day
1 was cleared at day 3. The most significant radiolabel
loss in Tag cells was detected between time 0 and day 1,
and it was 3-fold greater than that observed in PC cells.
The cell death observed during the first 3 days, however,
did not affect the whole population of donor cells. The
Y chromosome content increased (PC cells) or remained
stable (Tag cells). We concluded that cell death was efficiently compensated by cell proliferation during the first
3 days. In the case of PC cells, the cell death reduced the
radiolabel to 30%, but proliferation increased the whole
population over the original. Death and proliferation of
Tag cells were more dramatic: 3% of the radiolabel remained at 3 days, while the whole population was similar
to the original. Correlating these dynamics with the immune cell infiltration, we can presume that the only potential cellular effectors of the donor cell death observed
in the first 2-day period are neutrophils and macrophages.
This is in agreement with the previous hypothesis of
Guerette et al (6, 7) that considered the inflammatory
reaction as the main cause of the early donor cell death.
The donor PC cell population was relatively stable
from day 3 to day 6. On the contrary, donor Tag cells
963
disappeared during the same period. A priori, this difference could be interpreted on the basis of the immune
responses in immunocompetent mice. We observed that
Tag cells triggered a rapid influx of cytotoxic T-lymphocytes starting at day 2, while cytotoxic lymphocyte infiltration after PC cell transplantation started at day 6. This
rapid cytotoxic T-lymphocyte infiltration may be a good
candidate for the rapid disappearance of Tag cells compared to PC cells.
Our present results challenge previous studies which
proposed that the definitive loss of most donor cells occurred immediately after transplantation. Beauchamp et
al, using Y chromosome and radiolabeled thymidine to
quantify the donor cells, have suggested that the death of
donor muscle cells is biphasic, with 60% to 70% of cells
being lost during the first 60 minutes, followed by a second event beginning 8 hours post-transplantation (9). The
studies of another group, using the Y-chromosome label
to quantify the donor cells and the cell pellet as the baseline reference, have proposed that more than 80% of the
donor cells die as soon as they are transplanted (4, 10).
As previously discussed, our observations showed that
immediate donor cell death cannot be detected so rapidly
by the labels used in those studies, and that in some cases
(using the cell pellet as 100% baseline for Y-chromosome
quantification) artifactual misinterpretation is produced.
In the present study, only ß-Gal revealed a significant
decrease at 6 hours, but this was only in the case of donor
Tag cells. Since 6 hours was sufficient to detect ;90%
of the cell death when cells were transplanted, this serves
as evidence that most of the donor cell death revealed by
[14C]thymidine began very early. However, we cannot be
sure that this value was not overestimated, because if
transgenic ß-Gal has a short half-life, rapid down-regulation due to the intense inflammatory infiltration can
contribute to reduced ß-Gal expression.
In addition, our histological evidence was not compatible with a massive and immediate cell death. The observation of 8% to 12% TUNEL-positive nuclei at 6
←
Fig. 9. There are 3 dynamics proposed for the early survival of donor muscle cells. In the figure, the intensity of dark inside
the arrows illustrates the intensity of death or proliferation. 1) A first dynamics proposes a biphasic donor cell death (9). A very
rapid phase not compensated by proliferation (60%–70% of the original cells lost during the first hour) and a second phase,
beginning 8 hours after transplantation, that is efficiently compensated by proliferation. The final donor cell population at day 4
is quite similar to that observed 1-hour post-transplantation. 2) A second dynamics proposes a loss of more than 80% of the
donor cells immediately after transplantation, not compensated by proliferation, and a slowly donor cell death during the following
days (4). 3) The third is the dynamics proposed in the present study. There is not a massive donor cell loss during the first hour.
Only a moderate percentage of donor cells are dead due to the process of cell preparation (and maybe the traumatism of injection),
but this original cell death does not reduce strongly the population of donor cells. The dynamics of death and proliferation are
different in PC cells and Tag cells. PC cells exhibit a moderate rate of death the first 3 days, which is efficiently compensated
by proliferation. From day 3 to day 6 the donor cell population is quite stable (acute rejection starts at day 6). Tag cells exhibit
more intense death, beginning early in the first day and lasting until the almost complete elimination of the donor cells around
day 6. An intense proliferation compensates well the intense death at least until day 3, but either it declines or is not sufficient
to compensate cell death.
J Neuropathol Exp Neurol, Vol 62, September, 2003
964
SKUK ET AL
TABLE
Method
Transgenic bGal
Y-chromosome
Radiolabeled
thymidine
Properties for donor cell
survival detection
1) Decreases with donor cell
death
2) Increases with donor cell
proliferation
1) Decreases with donor cell
death
2) Increases with donor cell
proliferation
1) Only decreases with donor
cell death
Specific reasons of potential
misinterpretation (excluding
technical factors)
1) The promoter of the transgene can be down-regulated
—
—
hours among the donor cells is not consistent with a massive and immediate cell death. When we killed all the
cells immediately before transplantation, 60% to 85%
were TUNEL-positive at 6 hours. The 8% to 12% of
TUNEL-positive cells that we have observed 6 hours after transplanting cells (not previously killed) can be simply explained by the cell death observed before transplantation, an inevitable consequence of collecting and
preparing the cells for transplantation. The alizarin red
staining suggested that 14% to 23% of the donor cells
were necrotic 1 hour post-transplantation, 11% at 3 hours,
and 19% to 35% at 6 hours. The increase in alizarin redpositive cells observed at 6 hours was coincident with
the neutrophil infiltration, a potential effector of donor
cell killing (7). It must be remarked that the similarities
between TUNEL and alizarin red patterns suggested that
both methods detect the same necrotic cells, due to a
coexistence of membrane breakdown (detected by alizarin red) and DNA degradation (detected by TUNEL). The
fact that alizarin red-positive cells were observed earlier
and were later more numerous than TUNEL-positive nuclei can be explained by the kinetics of necrosis: early
membrane breakdown and later DNA degradation. The
decline of TUNEL-positive and alizarin red-positive cells
after 9 to 12 hours can be attributed to the fact that macrophages began to infiltrate the donor cell pockets, since
they can phagocytize dead cells. Indeed, since macrophages are constantly present in the donor cell pockets
from 24 hours to day 5, the dead cells can be rapidly
cleared during this period. The consequence of this clearing is that detection of cell death by histology will be
difficult after 24 hours post-transplantation. This explains
why few TUNEL-positive or alizarin red-positive cells
are observed during the 1- to 5-day period, in spite of
the continuous cell death during days 2 to 3 in PC cells
and the 5 days in Tag cells.
Mechanisms of Donor Cell Death
Apoptosis: Three previous studies briefly mentioned
the analysis of apoptosis in limited time periods after
J Neuropathol Exp Neurol, Vol 62, September, 2003
To disappear after cell death
the following processes
are needed:
1) Diffusion of the enzyme by membrane permeabilization (in necrosis)
or
2) Enzyme proteolysis (in apoptosis)
1) The DNA sequence must be digested by autolysis (endogenous lysosomal breakdown) or heterolysis
(phagocytes)
1) The DNA must be degraded and
eliminated by phagocytes
myoblast transplantation. Two of them did not observe
apoptosis either by electron microscopy (4), electrophoresis (4), or TUNEL (9). Another study reported less than
1% TUNEL-positive cells 2 hours and 1, 2, and 3 days
after myoblast implantation (7). The problem is that these
methods are not appropriate to quantify apoptotic cells in
vivo. A limitation of electron microscopy is the difficulty
in screening single apoptotic cells in tissue sections (21).
On the other hand, TUNEL fails to discriminate in vivo
the selective DNA-cleavage of apoptosis from the nonspecific DNA-damage of necrosis (22, 23). This nonspecificity of TUNEL was observed in the present study.
In tissue sections, apoptosis can be better evidenced by
immunodetection of the active form of caspases (24).
Caspases are intracellular proteases that orderly disassembled cells during apoptosis (25). Caspase activation
provides a common biochemical basis for apoptosis, despite the heterogeneity in control and activation among
different cell types (26). In the present study, we used an
antibody to recognize the active form of caspase-3, the
farthest downstream in the cascade of caspase activation
(27). We observed low levels (maximum mean ;3%) of
active caspase-3 among both types of donor cells during
the 5-day follow-up. At first glance, this seems to indicate
that apoptosis is not the predominant mechanism of the
donor cell death. However, even in tissues where the rate
of apoptosis is expected to be high, few morphologically
apoptotic cells may be seen and this may result in underestimation of the significance of apoptosis (21). The
low abundance of apoptotic cells at any time is due on
one hand to the asynchrony of apoptosis in the cell population (22, 28) and on the other hand to the rapidity of
the execution phase of apoptosis, which lasts 15 to 60
minutes (29) to 100 minutes (28). Although in the 5-day
follow-up, we did not observe important differences for
caspase-3 activation between PC cells and Tag cells, this
changed when the first 24 hours post-transplantation were
analyzed in more detail. In this case, caspase-3 activation
was negligible among PC cells, whereas in Tag-myoblasts it reached a maximum of 13% at 24 hours. The
CELL DEATH IN MYOBLAST TRANSPLANTATION
965
Fig. 10. The dynamics of donor cell death and proliferation will conduct the cell graft into 5 potential fates. A: Disappearance:
Cell death is continuous and predominates over proliferation. B: Reduction: Cell death is predominant but is auto-limited. A small
percentage of the original cell population remains. C: Stabilization: Cell proliferation compensates cell death. The surviving donor
cell population is quantitatively similar to the original. D: Increase: Cell proliferation is predominant but auto-limited. The surviving
donor cell population is quantitatively superior to the original. E: Tumor growth: Proliferation is continuous and death is limited.
The only fates that hamper cell transplantation are disappearance (A) and tumor growth (E). Reduction (B), stabilization (C), and
increase (D) are the conditions in which the number of cells needed for successful transplantation may be different.
actual role of apoptosis remains to be tested and experiments are currently in progress.
Necrosis: Necrosis occurs when a cell suffers irreversible damage that produces metabolic impairments leading
to death. Early membrane damage, leading to the influx
of extracellular molecules and ions, is characteristic of
necrosis. Among these extracellular ions, calcium entry
is a classical fate that can produce calcification of
J Neuropathol Exp Neurol, Vol 62, September, 2003
966
SKUK ET AL
necrotic cells. We previously showed that alizarin red at
pH 5.4, used to detect necrosis in muscle fibers (13),
evidenced intracellular calcium deposits among donor
Tag cells after intramuscular implantation (16). This was
similar to the entry of trypan blue into the same cells,
when this dye was injected in the host mouse (7). In the
present study, calcium deposits were detected in Tag
cells, and the alizarin red-staining pattern corresponded
closely with the pattern of TUNEL labeling, suggesting
that both methods were detecting the same necrotic cells.
In the 24-hour follow-up, up to 35% of the donor cell
pocket was alizarin red-positive at 6 hours. This was
higher than the 6% to 10% of cell death observed before
transplantation, and alizarin red staining was detected
earlier than active caspase-3. Thus, necrosis seems to be
the first mechanism of donor cell death after transplantation. As discussed previously, the fact that the percentage of alizarin red-positive cells decreased after 9 to 12
hours can be attributed to the arrival of macrophages during the 6- to 24-hour period. We can suppose that most
of the later donor cell death that was revealed by
[14C]thymidine quantification was hidden from histological detection because macrophages phagocytized the
dead cell debris. The staining of alizarin red was light in
PC cells, possibly because the 5.4 pH was not appropriate
for these cells (13).
Autolysis: One type of cell suicide is autolysis. Autolysis is detected in many tissues by the presence of acid
phosphatase, a lysosomal hydrolase (30, 31). We were
surprised by the presence of strong acid phosphatase activity in the donor cells at all periods, including time 0.
However, since it was present immediately after implantation, we can suppose that it could not be considered a
cell death mechanism triggered by transplantation. This
may be better attributed to the highly developed lysosomal apparatus reported in myoblasts in vitro (32). This extensive lysosomal apparatus in cultured myoblasts was
attributed to the process of differentiation and fusion,
which needs recycling cellular elements.
Conclusions
Following cell transplantation, donor cell labels disappear from the host muscles as a consequence of donor
cell death, but this is not instantaneous. The time-course
of label loss can be explained by the mechanism needed
to clear each label from the host muscle tissue (Fig. 8).
The Table summarizes the properties and limitations of
each method. We suggest that researchers working on
post-transplantation donor cell death may define the sensibility of their methods for cell detection before submitting their results to interpretation.
We did not observe evidences of a massive donor cell
death immediately after transplantation, as previously
suggested. The hypothesis based on such an interpretation
of the results must be critically reconsidered. Figure 9
J Neuropathol Exp Neurol, Vol 62, September, 2003
shows the dynamics of donor cell survival that we propose, contrasting with the dynamics proposed by others.
The early donor cell survival was very different between the 2 cell types that were studied. Tag cells died
earlier and more massively than PC cells, the surviving
cells proliferated more and seemed to exhibit more apoptosis and they triggered a very rapid infiltration of cytotoxic T-lymphocytes. Therefore, observations made in
a particular cell manipulated in a given way may not
automatically apply to cells from different sources or manipulated differently.
Donor cells died both by necrosis and apoptosis following intramuscular implantation. Necrosis is the earliest mechanism of donor cell death. The actual importance
of apoptosis remains to be determined. Donor cells exhibit high acid-phosphatase activity, but this corresponds
probably to the highly developed lysosomal apparatus developed in culture and not to autolytic cell suicide.
As a corollary, we propose a scheme to systematize
the donor cell survival in any kind of cell transplantation
(Fig. 10). Depending on the prevalence and on the limitation of the processes of donor cell death and proliferation, the cell graft can progress towards 5 different fates.
In fact, muscle cell transplantation can follow any one of
these possibilities. Following allogeneic transplantation
without immunosuppression, acute rejection kills all the
donor cells, leading to disappearance of the donor cells
(Fig. 10A). This fate was observed in Tag cells at day 6.
Transplantation of some cell lines (33) can led to continuous proliferation, producing a neoplasm (Fig. 10E). PC
cells in the present study followed the third to fourth fate:
the ratio between death and proliferation lead to a good
‘‘net’’ survival during the 6-day post-transplantation period, with the whole donor cell population being either
stable or increasing.
REFERENCES
1. Savitz SI, Rosenbaum DM, Dinsmore JH, Wechsler LR, Caplan
LR. Cell transplantation for stroke. Ann Neurol 2002;52:266–75
2. Bjorklund A, Lindvall O. Cell replacement therapies for central
nervous system disorders. Nat Neurosci 2000;3:537–44
3. Skuk D, Tremblay JP. Progress in myoblast transplantation: A potential treatment of dystrophies. Microsc Res Tech 2000;48:213–22
4. Hodgetts SI, Beilharz MW, Scalzo AA, Grounds MD. Why do cultured transplanted myoblasts die in vivo? DNA quantification
shows enhanced survival of donor male myoblasts in host mice
depleted of CD41 and CD81 cells or Nk1.11 cells. Cell Transplant
2000;9:489–502
5. Rando TA, Pavlath GK, Blau HM. The fate of myoblasts following
transplantation into mature muscle. Exp Cell Res 1995;220:383–89
6. Guerette B, Asselin I, Skuk D, Entman M, Tremblay JP. Control of
inflammatory damage by anti-LFA-1: Increase success of myoblast
transplantation. Cell Transplant 1997;6:101–7
7. Guerette B, Skuk D, Celestin F, et al. Prevention by anti-LFA-1 of
acute myoblast death following transplantation. J Immunol 1997;
159:2522–31
CELL DEATH IN MYOBLAST TRANSPLANTATION
8. Merly F, Huard C, Asselin I, Robbins PD, Tremblay JP. Anti-inflammatory effect of transforming growth factor-beta1 in myoblast
transplantation. Transplantation 1998;65:793–99
9. Beauchamp JR, Morgan JE, Pagel CN, Partridge TA. Dynamics of
myoblast transplantation reveal a discrete minority of precursors
with stem cell-like properties as the myogenic source. J Cell Biol
1999;144:1113–22
10. Hodgetts SI, Grounds MD. Complement and myoblast transfer therapy: Donor myoblast survival is enhanced following depletion of
host complement C3 using cobra venom factor, but not in the absence of C5. Immunol Cell Biol 2001;79:231–39
11. Caron NJ, Chapdelaine P, Tremblay JP. Male-specific competitive
PCR for the quantification of transplanted cells in mice. Biotechniques 1999;27:424–26, 28
12. Kinoshita I, Huard J, Tremblay JP. Utilization of myoblasts from
transgenic mice to evaluate the efficacy of myoblast transplantation.
Muscle Nerve 1994;17:975–80
13. Dubowitz V. Muscle biopsy: A practical approach (2nd. Ed). London: Bailliere Tindall, 1985
14. Qu Z, Balkir L, van Deutekom JC, Robbins PD, Pruchnic R, Huard
J. Development of approaches to improve cell survival in myoblast
transfer therapy. J Cell Biol 1998;142:1257–67
15. Skuk D, Caron NJ, Goulet M, Roy B, Espinosa F, Tremblay JP.
Dynamics of the early immune cellular reactions after myogeniccell transplantation. Cell Transplant 2002;11:671–81
16. Skuk D, Tremblay JP. Complement deposition and cell death after
myoblast transplantation. Cell Transplant 1998;7:427–34
17. Beauchamp JR, Pagel CN, Partridge TA. A dual-marker system for
quantitative studies of myoblast transplantation in the mouse.
Transplantation 1997;63:1794–97
18. Lamers MC, De Groot ER, Roos D. Phagocytosis and degradation
of DNA-anti-DNA complexes by human phagocytes. I. Assay conditions, quantitative aspects and differences between human blood
monocytes and neutrophils. Eur J Immunol 1981;11:757–64
19. Rozenberg-Arska M, van Strijp JA, Hoekstra WP, Verhoef J. Effect
of human polymorphonuclear and mononuclear leukocytes on chromosomal and plasmid DNA of Escherichia coli. Role of acid DNase. J Clin Invest 1984;73:1254–62
20. Harms JS, Splitter GA. Interferon-gamma inhibits transgene expression driven by SV40 or CMV promoters but augments expression driven by the mammalian MHC I promoter. Hum Gene Ther
1995;6:1291–97
967
21. Saraste A. Morphologic criteria and detection of apoptosis. Herz
1999;24:189–95
22. Grasl-Kraupp B, Ruttkay-Nedecky B, Koudelka H, Bukowska K,
Bursch W, Schulte-Hermann R. In situ detection of fragmented
DNA (TUNEL assay) fails to discriminate among apoptosis, necrosis, and autolytic cell death: A cautionary note. Hepatology 1995;
21:1465–68
23. Ohno M, Takemura G, Ohno A, et al. ‘‘Apoptotic’’ myocytes in
infarct area in rabbit hearts may be oncotic myocytes with DNA
fragmentation: Analysis by immunogold electron microscopy combined with In situ nick end-labeling. Circulation 1998;98:1422–30
24. Stadelmann C, Lassmann H. Detection of apoptosis in tissue sections. Cell Tissue Res 2000;301:19–31
25. Thornberry NA, Lazebnik Y. Caspases: Enemies within. Science
1998;281:1312–16
26. Samali A, Zhivotovsky B, Jones D, Nagata S, Orrenius S. Apoptosis: Cell death defined by caspase activation. Cell Death Differ
1999;6:495–96
27. Mukasa T, Momoi T, Momoi MY. Activation of caspase-3 apoptotic
pathways in skeletal muscle fibers in laminin alpha2-deficient mice.
Biochem Biophys Res Commun 1999;260:139–42
28. Messam CA, Pittman RN. Asynchrony and commitment to die during apoptosis. Exp Cell Res 1998;238:389–98
29. Earnshaw WC. Nuclear changes in apoptosis. Curr Opin Cell Biol
1995;7:337–43
30. Bowen ID, Lewis GH. Acid phosphatase activity and cell death in
mouse thymus. Histochemistry 1980;65:173–79
31. Bowen ID, Mullarkey K, Morgan SM. Programmed cell death during metamorphosis in the blow-fly Calliphora vomitoria. Microsc
Res Tech 1996;34:202–17
32. Bird JW, Roisen FJ, Yorke G, et al. Lysosomes and proteolytic
enzyme activities in cultured striated muscle cells. J Histochem
Cytochem 1981;29:431–39
33. Reinecke H, Murry CE. Transmural replacement of myocardium
after skeletal myoblast grafting into the heart. Too much of a good
thing? Cardiovasc Pathol 2000;9:337–44
Received February 21, 2003
Revision received June 2, 2003
Accepted June 13, 2003
J Neuropathol Exp Neurol, Vol 62, September, 2003