Note: Descriptions are shown in the official language in which they were submitted.
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MYOGBrTIC DIFFERENTIATION OF HUMAN MES~1~'~nr.
STEM CELLS
This invention relates to the field of muscle
regeneration and, more particularly, to the production of
dystrophin-positive cells in dystrophin-negative subjects.
A mesenchymal stem cell (MSC) is a pluripotential
progenitor cell that has the capacity to divide many times
and whose progeny eventually gives rise to mesodezmal
tissue such as: cartilage, bone, muscle, fat and tendon. A
population of these pluripotential stem cells has been
shown to be present in embryonic limb buds of chicken,
mouse and human (1, 2, 6, 14, 15). Such stem cells have
also been shown to exist in postnatal and adult organisms.
Friedenstein, Owen (3, 11) and others reported that cells
derived from bone marrow have the capability to
differentiate into osteogenic cells when assayed in
diffusion chambers. It has been documented that such cells
when loaded in ceramic cubes and implanted to subcutaneous
or intramuscular sites, possess the ability to foam bone
and cartilage tissue (4, 10). Furthermore, the periosteum
has been reported to contain MSCs with the same ability to
form bone or cartilage in ceramic cubes or diffusion
chambers ( 9 ) .
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With regard to myogenic potential of MSCs, rat and
mouse clonal embryonic cell lines have been shown to
transform into myoblasts and form myotubes of ter exposure
to 5-azacytidine or 5-azadeoxy cytidine. This work is
disclosed in U.S. Patent No. 5 736,396 entitled
"Lineage-Directed Induction of Human Mesenchymal Stem Cell
Differentiation." The same cells also exhibit adipocytic
and chondrogenic phenotypes (5, 7, 10, 17). However, the
expression of myogenic properties of stem cells harvested
from postnatal sources has not been observed in humans.
The mdx strain of mice is well recognized and has been
utilized as an animal model correlated with Duchenne-type
human muscular dystrophy (2)., Although mdx mice do not
exhibit severe clinical features of myopathy, their
skeletal and cardiac muscles are composed of predominantly
dystrophin-negative myofibers and show an extensive
myopathic lesion accompanied with muscle fiber necrosis and
degeneration (4). Injection of myoblasts (satellite cell-
derived), also referred to herein as muscle precursor '
cells(MPCs), has been observed to result in the conversion
of dystrophin-negative myofibers to dystrophin-positive
ones (13, 16), giving hope that this procedure may provide
a useful treatment for the patients affected with inherited
myopathies (8, 12, 14). One major drawback in this
myoblast transfer therapy is that a large number of
myoblasts are needed to bring about a clinically relevant
effect.
The present invention is based on the discovery by the
inventors that suitable environmental conditions induce
human mesenchymal stem cells thMSCs) to expres& a myogenic
phenotype. Such conditions can be established in
developing myogenic cell culture or in regenerating and
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reforming dystrophin-positive muscles. The procedures
described here elucidate the behavior of marrow- or
periosteal- derived hMSCs in the presence of muscle
precursor cells (MPCs) in vitro and in vivo.
Because, inter alia, hMSCs can be mitotically
expanded in culture and their harvest is less destructive
than muscle biopsies used to harvest myoblasts, these cells
have been discovered by the inventors to have significant
advantages over the use of myoblasts in treating myopathies.
Additionally, there are several sources of hMSCs in the
human body, and their harvest is less destructive than
muscle biopsies used to harvest MPCs.
With the above in mind, the inventors studied the
behavior of hMSCs co-cultured with myoblasts, and also
injected syngeneic normal MSCs into muscles of the mdx mouse
to evaluate their capability to produce normal muscle
fibers.
Accordingly, in one aspect the invention provides
a composition of matter for inducing myogenic
differentiation of isolated human mesenchymal stem cells
(hMSCs) comprising isolated hMSCs co-cultured with isolated
human muscle precursor cells (hMPCs). Preferably, the
mesenchymal stem cells are marrow-derived or periosteum-
derived. Also, the composition is preferably a composition
of entirely human cells. The composition can further
include a myoinductive agent, such as 5-azacytidine or
5-azadeoxycytidine.
Another aspect of the invention provides a
composition of matter for inducing myogenic differentiation
of isolated periosteum cells comprising isolated periosteum
cells co-cultured with isolated human muscle precursor cells
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(hMPCs). Here also, the composition is preferably a
composition of entirely human cells. This composition also
can further include a myoinductive agent, such as
5-azacytidine or 5-azadeoxycytidine.
Another aspect of the invention provides a method
for inducing isolated human mesenchymal stem cells to
differentiate into myogenic cells by maintaining the cells
in the presence of muscle precursor cells. The isolated
human mesenchymal stem cells can be maintained in the
presence of muscle precursor cells in vitro or in vivo.
Another aspect of the invention provides a method
for producing dystrophin-positive myogenic cells in an
individual by administering to the individual a myogenic
cell producing amount of isolated human mesenchymal stem
cells or of any of the compositions of the invention.
Another aspect of the invention provides a method
for effecting muscle regeneration in an individual in need
thereof by administering to an individual in need of muscle
regeneration a muscle regenerative amount of isolated human
mesenchymal stem cells or of any of the compositions of the
invention.
Another aspect of the invention provides a method
for treating muscular dystrophy in an individual so
afflicted by administering to that individual an amount of
isolated human mesenchymal stem cells or of any of the
compositions of the invention effective to produce
dystrophin-positive myogenic cells in said individual.
Another aspect of the invention provides use of
muscle precursor cells for inducing isolated human
mesenchymal stem cells to differentiate into myogenic cells.
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Another aspect of the invention provides use of a
therapeutically effective amount of isolated mesenchymal
stem cells for producing dystrophin-positive myogenic cells
in a mammal.
Another aspect of the invention provides use of a
therapeutically effective amount of the compositions for
producing dystrophin-positive myogenic cells.
Another aspect of the invention provides use of a
therapeutically effective amount of isolated mesenchymal
stem cells for effecting muscle regeneration in a mammal.
Another aspect of the invention provides use of a
therapeutically effective amount of the compositions for
effecting muscle regeneration.
Another aspect of the invention provides use of a
therapeutically effective amount of isolated mesenchymal
stem cells effective to produce dystrophin-positive myogenic
cells for treating muscular dystrophy in a mammal.
Another aspect of the invention provides use of a
therapeutically effective amount of the compositions
effective to produce dystrophin-positive myogenic cells for
treating muscular dystrophy.
The following is a brief description of the
drawings which are presented only for the purposes of
further illustrating the invention and not for the purposes
of limiting the same.
Figures lA-1C are photomicrographs showing the
histology of the right tibialis anterior muscle of mdx mouse
at 6 weeks after injection (A). There is a large
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area of a typical myopathic lesion accompanied with
cellular infiltration. Inside of the lesion, swollen and
vacuolated myotubes are found. At a weeks post-injection
(B), the regenerating area contains clustered muscle fibers
of small diameter, surrounded by large closely packed
normal muscle fibers. At a higher magnification (C), a
typical regenerating fibers are shown having large
centrally located nuclei. Bar =.100 ~Cm.
Figures 2A-2C are photomicrographs showing dystrophin
expression in the muscle of normal mouse (A). The entire
circumference of muscle fibers are clearly stained with
anti-dystrophin antibody. Anti-dystrophin antibody
localization in the muscle of mdx mouse 8 weeks after
injection of myoblasts (B). Immunostaining clearly
demonstrates multiple dystrophin-positive fibers surrounded
by dystrophin-negative myotubes. The shape and size of the
positive fibers are more varied than those of a normal
muscle. There are few dystrophin-negative fibers
intermingled with the positive fibers (*). Dystrophin
expression in the muscle of an mdx mouse 10 weeks post-
injection with MSCs (C). The intensity of staining is
almost same as that in the muscle injected with myoblasts.
There are contiguous dystrophin-positive muscle fibers,
forming a cluster.
Skeletal muscles develop in a specific sequence of
cellular differentiation, which includes: the commitment
of progenitor cells into myoblasts, the proliferation of
myoblasts, their fusion to form multinucleated myotubes and
the sequential expression of muscle specific proteins.
Each of these steps is controlled by a complex spectrum of
intrinsic and extrinsic biological factors (18).
Particularly, in the process of myoblast fusion, there is a
clear molecular recognition mechanism which allows
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myoblasts to fuse only to cells of their own lineage,
prohibiting their fusion to other cell types such as
osteogenic, chondrogenic and fibroblastic cells (18, 19).
Mesenchymal stem cells are the formative
pluripotential blast cells found inter alia in bone marrow,
blood, dezmis and periosteum that are capable of
differentiating into any of the specific types of
mesenchymal or connective tissues (i.e. the tissues of the
body that support the specialized elements; particularly
adipose, osseous, cartilaginous, elastic, and fibrous
connective tissues) depending upon various influences from
bioactive factors, such as cytokines. Although these cells
are normally present at very low frequencies in bone
marrow, a process for isolating, purifying, and greatly
replicating these cells in culture, i.e. in vitro, is
disclosed in PCT Published Application No. WO
92/22584(published 23 December 1992).
Homogeneous human mesenchymal stem cell compositions
are provided which serve as the progenitors for all
mesenchymal cell lineages. MSCs are identified~by specific
cell surface markers which are identified with unique
monoclonal antibodies. The homogeneous MSC compositions
are obtained ~by positive selection of adherent marrow or
periosteal cells which are free of markers associated with
either hematopoietic cell or differentiated mesenchymal
cells. These isolated mesenchymal cell populations display
epitopic characteristics associated with only mesenchymal
stem cells, have the ability to regenerate in culture
without differentiating, and have the ability to
differentiate into specific mesenchymal lineages when
either induced in vitro or placed in vivo at the site of
damaged tissue.
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In order to obtain human mesenchymal stem cells, it is
necessary to isolate rare pluripotent mesenchymal stem
cells from other cells in the bone marrow or other hMSC
source. Bone marrow cells may be obtained from iliac
crest, femora, tibiae, spine, rib or other medullary
spaces. Other sources of human mesenchymal stem cells
include embryonic yolk sac, placenta, umbilical cord, fetal
and adolescent skin, and blood.
The method of their isolation comprises the steps of
providing a tissue specimen containing mesenchymal stem
cells, adding cells from the tissue specimen to a medium
which contains factors that stimulate mesenchymal stem cell
growth without differentiation and allows, when cultured,
for the selective adherence cf only the mesenchymal stem
cells to a substrate surface, culturing the specimen-medium
mixture, and removing the non-adherent matter from the
substrate surface.
Compositions having greater than 95%, usually greater
than 98% of human mesenchymal stem cells can be achieved
using the previously described technique for isolation,
purification and culture expansion of MSCs. The desired
cells in such compositions are identified as SH2+, SH3+,
SH4+ and CD- and are able to provide for both self renewal
and differentiation into the various mesenchymal lineages.
Ultimately, repair and regeneration of various mesenchymal
tissue defects could be accomplished starting from a single
mesenchymal stem cell.
Examflle 1
D~rstroahin Expressioa is mdx Mice Usina hMSCs
. Mdx mice have been utilized as an animal model of
Duchenne-type human muscular dystrophy. Although mdx mice
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do not exhibit severe clinical features of myopathy, their
skeletal and cardiac muscles are composed of predominantly
dystrophin-negative myofibers and show an extensive
myopathic lesion accompanied with muscle fiber necrosis and
degeneration. Injection of myoblasts (satellite cell-
derived) has been observed to result in the conversion of
dystrophin-negative myofibers to dystrophin-positive ones,
which gives hope that this procedure may provide a useful
treatment for patients affected with inherited myopathies.
Therefore, the mdx mouse was adopted as an experimental
animal model in these studies. One major drawback in this
myoblast transfer therapy is that a large number of
myoblasts are needed to bring about a clinically relevant
effect. Because MSCs can be mitotically expanded in
culture and their harvest is less destructive than muscle
biopsies used to harvest myoblasts, these cells may be an
alternative source of cells for treating myopathies. With
the above in mind, we have studied the behavior of MSCs co-
cultured with myoblasts or injected into either syngeneic
normal muscle or into the tibialis anterior muscle of mdx
mice, in order to evaluate the capability of MSCs to
produce normal muscle fibers.
bIATERIALS AND bD3TSODS
Bone Marrow Cell Preparation
As sources of bone marrow MSCs, femora of male mice
(C57B1/10 SNJ, Jackson Laboratories, Bar Harbor, MB) of 6
to 10 weeks of age were used. After the soft tissue was
completely removed to avoid contamination by myogenic
precursors, the distal and proximal ends of the femora were
cut with a rongeur and the marrow plugs were removed from
the shafts of the bones by expulsion with a syringe filled
with complete medium and fitted with, for rats, an 18-gauge
needle and, for mice, a 20-gauge needle. Complete medium
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consisted of BGJb (Fitton-Jackson Modification; Sigma; St.
Louis, MO) with antibiotics (100 u/ml sodium penicillin G,
100 ~g/ml streptomycin sulfate and 0.25 ~g/ml amphotericin
B: Gibco HRL, Grand Island, NY), and 10% fetal calf serum
(selected batches; JRH Biosciences, Lenexa, KS). Marrow
cells were dispersed several times through 20- and 22-gauge
needles. The cells were gently centrifuged, resuspended in
complete medium, counted and plated at a density of 5.0 x
10~ cells/100-mm dish. At 3 days of culture, the medium
was removed, the dishes were washed twice with Tyrode's
salt solution (Sigma, St. Louis, MO) and fresh complete
medium added; the medium was changed twice a week.
Preparatioa of Mouse ~yoblasts
Muscle tissue was obtained from the tihialis anterior
and quadracepts muscles of three-week-old mice. The
dissected muscle was digested with 0.2% trypsin for 35 min
and the reaction terminated by the addition of fetal bovine
serum at one half the sample volume. The cell suspension
was centrifuged for 5 min at 300 xg, resuspended in 10 ml
of DMBM-LG containing 20% fetal calf serum, triturated at
least 20 times with a 10-ml disposable pipette,~and then
passed through a 100-um Nitex filter (to remove cell
aggregates and debris). Prior to seeding, the cells were
preplated into a 100-mm culture dish and incubated for 30
min to reduce the number of fibroblasts. The nonadherent
muscle precursor cells were counted in a hemocytometer and
the cell density was adjusted to 1.0 x 105 cells/ml with
DM$M-LG supplemented with 20% fetal calf serum. Cells were
seeded onto 35 mm plates at 1.0 x 105 cells per plate, in 1
ml, and the medium changed every 2 days. At the onset of
cell fusion (4-5 days), the medium was changed to DMBM-LG
with 5% fetal calf serum. The cultures were maintained
until the overt formation of myotubes has ceased; usually
by 11 days following seeding.
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In Vivo Injections
Breeding pairs of both mdx mice (C57b1/l0mdx) a~~-d
normal mice (C57b1/lOsnj) were purchased from Jackson
Laboratories. Newborn mdx mice were raised for 3 weeks,
separated from their parents and immediately used as
injection hosts. Normal newborn mice were sacrificed
within 3 days of birth for collection of syngeneic
myoblasts. Normal adult male mice were utilized as a
source of bone marrow-derived MSCs.
Myoblasts were obtained from normal syngeneic newborn
muscle tissue as described above, then centrifuged, washed
with Tyrode's salt solution and counted. MSCs were
released with trypsin-EDTA after 10 to 14 days of primary
culture, washed with Tyrode's salt solution and counted.
The densities of both cell suspensions were adjusted with
serum-free BGJb to 5.0 x 105 cells/5 ~,1.
Mdx mice were anesthetized by limited exposure to C02
gas. The hindlimbs were sterilized with 70% ethanol, and a
longitudinal skin incision was made along the anterior edge
of the tibia with a No. 11 scalpel. The tibialis anterior
muscle was identified and a needle connected to a
microliter syr-inge (Hamilton Company, Reno, NV) containing
~1 of cells was inserted into the interior of the muscle
at a low angle, and the full 5 ~1 of cells were injected
into the middle of the muscle. The right tibialis anterior
was injected with cells and the left side injected with
BGJb medium only. The skin was sutured in an ordinary
manner and closed.
Tissue Section Immunohiatochemistry
At 6, 8 and 10 weeks after injection, mice were
sacrificed, and the muscle mass located on the anterior
surface of the tibia was excised. The mass of each muscle
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sample was measured prior to cutting the muscle in half.
The proximal half was immersed in OCT compound (Tissue-Tek,
Miles Laboratories, Inc., Napperville, IL) and then snap-
frozen in liquid nitrogen-cooled in 2-methylbutane for
cryosectioning and immunohistochemical staining; the distal
half was fixed with 10% neutral formalin for routine
histology.
The frozen samples were sectioned with a cryostat, and
8 ~m sections were placed on glass slides coated with poly-
L-lysine. Prior to staining, sections were blocked in
phosphate-buffered saline containing 20% horse serum (JRH
Biosciences, Lenexa, KS; PBS+HS, pH 7.4). The sections
were immunostained with anti-dystrophin antibody as
described above. Following incubation in the first and
second antibodies, they were washed and incubated for 15
min with Texas red conjugated avidin (Organon Teknika
Corp., West Chester, PA) diluted 1 to 6000 with PBS+HS and
washed 4 times in PBS+HS for 5 min. The slides were
mounted in glycerol: PBS (9:1), pH 8.5 containing 0.01 M p-
phenylenediamine (PPD) (Eastman Kodak, Rochester, NY) and
observed with a fluorescent microscope (BH-2, Olympus,
Tokyo ) .
Histologic Evaluation
For analysis of the dystrophin content in the
transverse section of the muscle, the section was
subdivided into 6 areas and a micrograph of the region
containing the largest number of dystrophin-positive
myofibers in each area was recorded with Kodak TMAX 400
film. From full frame prints, the number of dystrophin-
positive and -negative myofibers was tabulated and the
percentage of positive to total number of myofibers in the
6 areas was calculated. The average values of the right
muscle (cell-injected) were compared with that of .the left
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(cell medium-injected). Sections of the other half of the
muscle samples were stained with hematoxylin and eason for
histologic evaluation.
RESULTS
Zn vivo Muscle Injections
A total of 52 mdx mice were used for the injections in
this study. Muscles were harvested at 6, 8 and 10 weeks
after injection; 29 mice were injected with MSCs and 23
with myoblasts. During harvest, muscles were dissected in
toto and immediately weighed. Table II contains the mass
ratios between right and left muscles harvested at 6, 8 and
weeks after injection. The data were then analyzed both
in total and according to sex; this data is summarized in
Table 1.
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Table 1
Sugary of 6, 8 and 10 Week Post-Injection Muscle Mass
mg muscle (standard deviation)
Total Female Total Male Total F +
M
Myoblast-injected
Cell-injected 93.2 (8.5)f 100.6 (10.1) 97.5 (10.0)
Medium- 85.7 (12.6) 88.9 (26.0) 87.6 (21.1)
injected
Percent 8.8 13.1 13
increase
Number 10 13 23
Mesenchymal em l-injected
St cel
Cell-injected 78.2 (12.2) 97.9 (12.2) 85.6 (15.9)
Medium- 74.7 (10.5) 96.8 (9.5) 83.1 (14.7)
injected
Percent 5.4 0.9 3.0
increase
Number 18 11 29
All cell-injected muscle groups showed statistically
significant increases in muscle mass compared to
contralateral controls except for the male MSC-injected
group. There was an increase in muscle mass of this group
but not statistically significant (p value = 0.7). The
myoblast-injected muscle showed nearly a 4-times greater
percent change in muscle mass (11.1%) than that of MSC-
injected muscle (3.0%).
The histology of the muscle samples showed various
degrees of degeneration and/or regeneration in both MSC-
and periosteal cell-injected groups. At 6 weeks after
injection, large foci of cellular infiltration were found
(Fig. 1A). However, at 8 to 10 weeks, these areas were
reduced in size and were surrounded by myotubes (Fig. 1B).
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The transverse sections appeared normal, except that the
nuclei were centrally located as compared to the normal
peripheral location (Fig. 1C). The frequency of small,
basophilic, centrally nucleated and vacuolated myotubes
increased in number with time. Although there were areas
of fat cell accumulation in some few samples, calcified
myofibers and massive fibrosis were not observed. There
was no evidence of bacterial infection in any of the muscle
specimens.
Immunohistochemical staining for dystrophin served as
a marker for normal muscle differentiation. In the normal
mouse (snj), dystrophin is localized to the entire
circumference of the plasmalemma on every myotube (Fig.
2A). IN the myoblast-injected group, a wide variation of
the extent of dystrophin-positive myofibers was observed.
The shape and diameter of teh positive fibers were more
irregular than those of normal syngeneic mice (Fig. 2B).
Of interest was the finding that some of the fibers with
the centrally located nuclei were also dystrophin-positive.
Quantitation of immunoreactivity far samples harvested
at 6, 8, and 10 weeks after injection showed a consistent
pattern for the percentage of dystrophin-positive fibers
within each experimental group (Table 2).
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Table 2
Incidence of Dystrophin-Positive Muscle Fibers
Proportion of Positive Fibers (%)
6 wk 8 aik 10 wk
Injected
MSC (R) 2.411.7 (9) 2.611.6 (9) 5.719.4 (11)
Sham (L) 0.310.5 (9) 0.510.6 (9) 0.310.5 (11)
MPC (R) 15.4119.1 (9) 35.8116.7 (7) 26.6123.2 (8)
Sham (L) 0.810.8 (8) 0.310.6 (7) 3.012.0 (8)
At each harvest time, the myoblast-injected samples
had, by far, the largest regions of dytrophin-positive
myotubes, followed by the MSC-injected group, and then by
the medium-injected control group. The percentage of
positive myofibers for the myoblast-injected group was from
to 10 times greater than that for the MSC-injected group.
However, the MSC-injected group had 8 to 19 times more
dystrophin-positive myofibers than the medium-injected
muscles. There was a significant difference in'the average
values between the MSC-injected and medium-injected muscles
at 6 and 8 weeks (Pc0.05, two-way ANOVA) (Table II). The
intensity of staining among the test groups was
indistinguishable from that of the myoblast-injected group
(Fig. 2C) .
At 6 weeks after injection, in the medium-injected
group, four samples showed 1 to 8 positive fibers and the
other 5 were completely negative. Most of the positive
fibers were scattered except in one animal where a cluster
containing 8 adjacent positive fibers was found. In MSC-
injected muscles, clusters with more than 3 positive fibers
were more frequently found. However, the number of the
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positive fibers in a cluster was limited to under 8. At S
weeks, the distribution pattern of the positive fibers was
similar to that observed in the 6-week samples. At 10
weeks, there were no dystrophin-positive fibers in 8 of 11
medium-injected muscles. On the other hand, in MSC-
injected muscles, 7 muscles showed a cluster containing
more than 6 contiguous positive fibers and in one case a
marked formation of positive fibers was found.
DISCITSSION
Skeletal muscles develop in a specific sequence of
cellular differentiation, which includes: the commitment of
progenitor cells into myoblasts, the proliferation of
myoblasts, their fusion to form multinucleated myotubes and
the sequential expression of muscle-specific proteins.
Bach of these steps is controlled by a complex spectrum of
intrinsic and extrinsic biological factors. Particularly,
in the process of myoblast fusion, there is a clear
molecular recognition mechanism which allows myoblasts to
fuse only to cells of their own lineage and prohibits their
fusion to other cell types such as osteogenic, chondrogenic
and fibroblastic cells.
In order to account for the spontaneous dystrophin-
expression by~somatic cell mutation in mdx mice with
advancing age, the proportion of dystrophin-positive fibers
was deteimi.ned for the MSC-injected right and medium-
injected left muscles. Although the incidence of positive
fibers was lower in MSC-injected muscles than that of
muscles injected with myoblasts, it was significantly
higher than that of the controls somatic mutation at each
sampling time. Several investigators have proposed that
the mdx mouse can be used as an animal model to study
muscular regenration. It has been shown that myopathic
lesions similar to Duchenne-type human muscular dystrophy
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are manifest in young mdx mice at age 3 to 6 weeks, since
no apparent sex-related difference in histology was
detected. Injections of reparative cells were done at age
3 weeks into both male and female MDX mice. Our results
show that the average rate of conversion was highest at 8
weeks after injection (36%). In comparison, the muscles
injected with MSCs showed a cluster of dystrophin-positive
myofibers at a significantly lower frequency than the
myoblast-injected muscles.
The in vivo experiments presented here indicate that
MSCs are able to fuse with normal and mdx myoblasts and,
when injected, can contribute to increased muscle mass, and
increased numbers of dystrophin-positive fibers as compared
to controls. From these data, taken together, it is
reasonable to interpret that some of the increase in muscle
mass can be attributed to myoblast- or MSC-induced
conversion of dystrophin-negative fibers to dystropin-
positive fibers. We postulate that the host provides the
signals to cause the MSCs to exhibit a myogenic potential.
The result of in vivo injectin experiments indicate that
implanted bone marrow-derived MSCs differentiate into
myogenic cells in regnerating muscles of mdx mice.
However, the frequency for MSC myogenic conversion is low,
so we cannot rule out co-fusion as a possible mechanism for
our observations. In summary. MSC's are able to fuse with
myotubes and express dystrophin, and are a reservoir which
can be utilized for dystrophy-related cell therapies.
We hypothesize that the differentiation efficiency may
be increased by the regeneration conditions inherent in mdx
mice. Therefore, implantation of MSCs in combination with
myoblasts or inductive reagents to promote muscle
regeneration result in more efficient conversion of
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dystrophin-negative to -positive myofibers in the muscle of
mdx mice.
Example 2
Induction of Mvoaeaisis in Suman Volunteers
Bone Marrow Cell Preparatioa
Hone marrow cells aspirated from the iliac crests of
healthy adult human volunteers are used. After soft tissue
is completely removed to avoid contamination of myogenic
precursors, the marrow is triturated with a syringe filled
with Complete Medium and fitted with an 18-gauge needle.
The Complete Medium was made up of DMBM-LG (Dulbecco's
Modified Fagle's Medium - Low Glucose, Gibco HRL, Grand
Island, NY) as more fully described in
U.S. Patent No. 5,811,094. The
Medium contains antibiotics (100 ~/ml sodium penicillin G,
100 ~g/ml streptomycin sulfate and 0.25 ~g/ml amphotericin
H: Gibco BRL, Grand Island, NY) and is supplemented with
10% fetal calf serum (selected batches). Marrow cells are
dispersed several times through 18- and 20-gauge needles.
The cells are gently centrifuged, resuspended in Complete
Medium, counted and plated at a density of. 5.0 x 10~
cells/100 mm-dish. At 3 days of culture, the medium is
removed, the dishes are washed twice with Tyrode's salts
solution (Sigma, St. Louis, MO) and fresh complete medium
is added. The medium is changed twice a week.
Periosteal-Derived Cell Preparatioa
Periosteal cells are obtained from the proximal
tibiae of volunteers. The tibial periostea are digested
with 0.3% collagenase for 2 hours in a 37°C water bath. At
the end of the digestion period, a volume of fetal bovine
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serum equal to half the volume of the sample is added.
Periosteum-derived cells (periosteal cells) are collected
by centrifugation. The pellet of cells and debris are
resuspended in 8 ml of Complete Medium and seeded onto
10o-pan culture dishes. The plated cells are cultured at
37°C in 95% humidified air plus 5% C02, and the medium is
changed every 3 days.
Preparation of myoblaats
Muscle tissue is obtained from muscle biopsies. The
dissected muscle is digested with 0.2% trypsin for 35 min.
and the reaction is terminated by adding fetal bovine serum
at one half the sample volume. The cell suspension is
centrifuged for 5 min. at 300 xg, resuspended in 10 ml of
DMEM-LG containing 20% fetal calf serum, triturated least
20 times with a 10 ml disposable pipette and then passed
through 110 ~m Nitex filter (to remove cell aggregates and
debris). Before seeding, the cells are preplated in a
100-mm culture dish and incubated for 30 min. to reduce the
number of fibroblasts. The nonadherent muscle precursor
cells are counted in a hemocytometer and the cell density
is adjusted to 1.0 x 105 cells/ml with DMBM-LG supplemented
with 20% fetal calf serum. Cells are seeded onto 35 cmn
plates at 1.0 x 105 cells per plate, in 1 ml, and the
medium is changed every 2 days. At the onset of cell
fusion (4-5 days), the medium is changed to DNIEM-LG with 5%
fetal calf serum. The dishes are observed until myotubes
form, usually 11 days following initial seeding.
Alternate Preparation of ldyobla'ta
Myoblasts are also obtained as above. The bone and
cartilage are cleared with fine forceps under a dissecting
microscope. The dissected muscle tissue is transferred to
a sterile tube, 2 ml of Eagle's medium with 8arle's salts
is added and the tissue is vortexed for 30-60 seconds.
*Trade-mark
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Subsequently, 8-9 ml of Complete Medium [Eagle's Minimum
Essential Medium with Earle's salts containing 10% horse
serum, 5% embryo extract and antibiotics] are added. The
cell suspension is passed through 2 thicknesses of sterile
cheesecloth two times and the cells are counted in a
hemocytometer. The cell density is adjusted with Complete
Medium to 2.0 x 105/m1 and one ml is aliquoted onto each 35
mm dish.
Method of Co-culture
A 1 ml of suspension of MPCs is dispersed on
gelatin-coated 35-mm culture dishes. Periosteal-derived
cells (PCs) labeled with [3H]-thymidine are trypsinized for
min. at 37°C with 0.25% trypsin/1 mM ethylenediamine
tetra-acetic acid (Gibco BRL, Grand.Island, NY), the
reaction is terminated by the addition of half volume of
calf serum and cells are collected by centrifugation.
Subsequently, three different cell suspensions are made at
cell densities of 1.0 x 105, 2.0 x 105 and 3.0 x 105
cells/ml separately with Complete Eagle's and 8ar1's salts
Medium. Then, 1 ml of each cell suspension is added to the
dishes in which MPCs are seeded previously and mixed with
each other. Cells are cultured at 37°C in C02 incubator.
The medium is-changed twice a week.
Activity of Creatine Rinase
Every 2 days after the initiation of the co-culture
until day 11, cells are harvested from the dishes by
washing twice With cold Tyrode's salt solution twice and
stored at -70°C until needed. After thawing, 1 ml of 0.05
M glycylglycine (pH 6.75, Sigma, St. Louis, MO) is added
into each dish, and cells are scraped off with a
polypropylene policeman, suspended, sonicated for 30 sec.
in a Sonifier Cell Disrupter (Model W140D, Branson Sonic
Power Co., Plainview, NY) and centrifuged at 12,000 xg for
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WO 96/39035 PCTNS96/08722
min. The activity of creative kinase in the supernatant
is determined from an assay Which couples ATP fozmation
from ADP and creative phosphate with the formation of DADPH
(measured by absorbance at 340 nm), according to Shainberg,
et a1. (34). Briefly, 100 ~,1 of the supernatant are mixed
with 890 ~1 of reaction mixture and the reaction is
initiated by adding 10 ~.1 of creative phosphate (15 mM).
The change in adsorbance at 340 nm is measured in a W160
spectrophotometer (Shimidazu, Tokyo) and the slope of the
change in absorbance per min. is used to determine creative
kinase activity.
Autoradiography
First-passage human MSCs and periosteal cells are
dispersed into 100-van dishes at a density of 4.0 x 105
cells in 5 ml of complete medium. After 24 hours, 2.5 ~CCi
of [methyl-3H]thymidine (Amersham, Arlington Heights, IL;
0..5 ~Ci/ml) are added to each dish. The cells are
incubated for 48 hours, washed twice with Tyrode's salts
solution and Complete Medium is added to chase
unincozporated radioisotope for 48 hours. Subsequently,
cells were harvested by digestion With trypsin and counted
in a hemocytometer. The cell density is adjusted to 5.0 x
104 cells/ml for MSCs, and 1.0 x 105 cell/ml for
periosteum-derived cells. Aliquots of 1.0 ml of each cell
suspension are added to the dishes into which twice the
number of myoblasts are plated.
At 2-day intervals up to 11 days, co-culture plates
are harvested for autoradiography. Randomly chosen dishes
are washed twice with cold Tyrode's salts solution, and the
cells are fixed with 10% neutral fonnalin. After the cells
are dehydrated in graded ethanol and air-dried, the dishes
, are coated with NTB-2 autoradiography emulsion ($astman
Kodak, Rochester, NY) and stored in the dark at -20°C for 7
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WO 96/39035 PCT/US96/08722
days. Subsequently, the dishes are developed and
air-dried, and the cells are observed under a microscope
(BH-2, Olympus, Tokyo).
Autoradiography with Immunohistochemistry
Co-cultures of MSCs with myoblasts are immunostained
with anti-dystrophin antibody prior to autoradiography. At
9 days, the dishes are washed twice with Tyrode's salts
solution and the cells are fixed with cold methanol for 15
min. After washing with Tyrode's salts solution, PBS+HS
(phosphate buffered saline pH 7.4, containing 10% horse
serum; JRH Biosciences, Lenesa, KS) is added onto the
cells. The cells are incubated for 2.5 hours in a humid
chamber with anti-dystrophin antibody diluted to 1 to 250
with PHS+HS. After incubation, the cells are washed 4
times with PBS+HS for 5 min. each and incubated with
biotinylated sheep anti-sheep IgG (Vector Laboratories,
Inc., Burlingame, CA) and diluted (1:500) in PBS +HS.
After washing 4 times, the cells are exposed to
streptavidin-horseradish peroxidase conjugate (Bethesda
Research Laboratories, Life Technologies Inc.,
Gaithersburg, MD) for 30 min. To develop color; the cells
are immersed in the solution of diaminobenzidine (DAB) (0.2
mg/ml) in 20 mM (tris)hydroxyethyl aminoethane, 150 mM
sodium chloride, pH 7.6 containing hydrogen peroxide
(0.01%) at room temperature for 15 min. in the dark. The
cells are washed once with PBS and twice with distilled
water and then exposed to 10% copper nitrate for 1 min. to
intensify the DAB product. After washing again with
distilled water, cells are then dehydrated in graded
ethanol and air-dried, and autoradiography is performed as
described above.
The invention has been described and illustrated with
reference to the preferred embodiment. Modifications and
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WO 96/39035 PCT/US96108722
alterations will occur to others upon reading and
understanding the preceding detailed description. It is
intended that the invention be construed as including all
such modifications and alterations insofar as they come
within the scope of the appended claims or the equivalents
thereof .
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16. Partridge TA, Sloper JC: Evidence of fusion
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