Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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SKELETAL MUSCLE AUGMENTATION UTILIZING MUSCLE-DERIVED
PROGENITOR COMPOSITIONS, AND TREATMENTS THEREOF
FIELD OF THE INVENTION
The present invention relates to muscle-derived progenitor cells (MDCs) and
compositions of MDCs and their use in the augmentation of body tissues,
particularly skeletal
muscle. In particular, the present invention relates to muscle-derived
progenitor cells that
show long-term survival following introduction into skeletal muscle, methods
of isolating
MDCs and methods of using MDC-containing compositions for the augmentation of
human
or animal skeletal muscle. The invention also relates to novel uses of muscle-
derived
progenitor cells for the treatment of cosmetic or functional conditions,
including, but not
limited to skeletal muscle weakness, muscular dystrophy, muscle atrophy,
spasticity,
myoclonus and myalgia. The invention also relates to the novel use of MDCs for
the increase
of skeletal muscle mass in athletes or other organisms in need of greater than
average skeletal
muscle mass.
BACKGROUND OF THE INVENTION
Myoblasts, the precursors of muscle fibers, are mononucleated muscle cells
that fuse
to form post-mitotic multinucleated myotubes, which can provide long-term
expression and
delivery of bioactive proteins (T. A. Partridge and K. E. Davies, 1995, Brit.
Med. Bulletin
51:123 137; J. Dhawan et al., 1992, Science 254: 1509 12; A. D. Grinnell,
1994, Myology Ed
2, A. G. Engel and C. F. Armstrong, McGraw-Hill, Inc., 303 304; S. Jiao and J.
A. Wolff,
1992, Brain Research 575:143 7; H. Vandenburgh, 1996, Human Gene Therapy
7:2195
2200).
Cultured myoblasts contain a subpopulation of cells that show some of the self-
renewal properties of stem cells (A. Baroffio et al., 1996, Differentiation
60:47 57). Such
cells fail to fuse to form myotubes, and do not divide unless cultured
separately (A. Baroffio
et al., supra). Studies of myoblast transplantation (see below) have shown
that the majority of
transplanted cells quickly die, while a minority survive and mediate new
muscle formation (J.
R. Beuchamp et al., 1999, J. Cell Biol. 144:1113 1122). This minority of cells
shows
distinctive behavior, including slow growth in tissue culture and rapid growth
following
transplantation, suggesting that these cells may represent myoblast stem cells
(J. R.
Beuchamp et al., supra).
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Myoblasts have been used as vehicles for gene therapy in the treatment of
various
muscle- and non-muscle-related disorders. For example, transplantation of
genetically
modified or unmodified myoblasts has been used for the treatment of Duchenne
muscular
dystrophy (E. Gussoni et al., 1992, Nature, 356:435 8; J. Huard et al., 1992,
Muscle & Nerve,
15:550 60; G. ICarpati et a/., 1993, Ann. Neurol., 34:8 17; J. P. Tremblay et
aL, 1993, Cell
Transplantation, 2:99 112; P. A. Moisset et al., 1998, Biochem. Biophys. Res.
Conunun.
247:949; P. A. Moisset eral., 1998, Gene Ther. 5:134046). In addition,
myoblasts have
been genetically engineered to produce proinsulin for the treatment of Type 1
diabetes (L.
Gros et al., 1999, Hum. Gen. Ther. 10:1207 17); Factor IX for the treatment of
hemophilia B
(M. Roman etal., 1992, Somat. Cell. Mol. Genet. 18:247 58; S. N. Yao et al.,
1994, Gen.
Ther. 1:99 107; J. M. Wang etal., 1997, Blood 90:1075 82; G. Hortelano etal.,
1999, Hum.
Gene Ther. 10:1281 8); adenosine deaminase for the treatment of adenosine
deaminase
deficiency syndrome (C. M. Lyncher al., 1992, Proc. Natl. Acad. Sci. USA,
89:113842);
erythropoietin for the treatment of chronic anemia (E. Regulier et al., 1998,
Gene Ther.
5:1014 22; B. Dalle eta!, 1999, Gene Then 6:157 61), and human growth hormone
for the
treatment of growth retardation (K. Anwer et al., 1998, Hum. Gen. Ther. 9:659
70).
Myoblasts have also been used to treat muscle tissue damage or disease, as
disclosed
in U.S. Pat. No. 5,130,141 to Law et al.,U.S. Pat. No. 5,538,722 to Blau
etal., and
application U.S. Patent No. 6,866,842 by Chancellor et al.
In addition, myoblast transplantation has been employed for the repair of
myocardial
dysfunction (C. E. Murry etal., 1996, J. Clin. Invest. 98:2512 23; B. Z.
Atkins et al., 1999,
Ann. Thorac. Surg. 67:124 129; B. Z. Atkins etal., 1999, J. Heart Lung
Transplant. 18:1173
80).
In spite of the above, in most cases, primary myoblast-derived treatments have
been
associated with low survival rates of the cells following transplantation due
to migration
and/or phagocytosis. To circumvent this problem, U.S. Pat. No. 5,667,778 to
Atala,
discloses the use of myoblasts suspended in a liquid
polymer, such as alginate. The polymer solution acts as a matrix to prevent
the myoblasts
from migrating and/or undergoing phagocytosis after injection. However, the
polymer
solution presents the same problems as the biopolymers discussed above.
Furthermore, the
Atala patent is limited to uses of myoblasts in only muscle tissue, but no
other tissue.
Thus, there is a need for other, different tissue augmentation materials that
are long-
lasting, compatible with a wide range of host tissues, and which cause minimal
inflammation,
scarring, and/or stiffening of the tissues surrounding the implant site.
Accordingly, the
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muscle-derived progenitor cell (MDC)-containing compositions of the present
invention are
provided as improved and novel materials for augmenting skeletal muscle.
Further provided
are methods of producing muscle-derived progenitor cell compositions that show
long-term
survival following transplantation, and methods of utilizing MDCs and
compositions
containing MDCs to treat various aesthetic and/or functional defects,
including, but not
limited to, skeletal muscle weakness, muscular dystrophy, muscle atrophy,
spasticity,
myoclonus and myalgia. Also provided are methods of using MDCs and
compositions
containing MDCs for the increase of skeletal muscle mass in athletes or other
organisms in
need of greater than average skeletal muscle mass.
It is notable that prior attempts to use myoblasts for non-muscle tissue
augmentation
were unsuccessful (U.S. Pat. No. 5,667,778 to Atala). Therefore, the findings
disclosed
herein are unexpected, as they show that the muscle-derived progenitor cells
according to the
present invention can be successfully transplanted into non-muscle tissue,
including skeletal
muscle tissue, and exhibit long-term survival. As a result, MDCs and
compositions
comprising MDCs can be used as a general augmentation material for skeletal
muscle
production. Moreover, since the muscle-derived progenitor cells and
compositions of the
present invention can be derived from autologous sources, they carry a reduced
risk of
immunological complications in the host, including the reabsorption of
augmentation
materials, and the inflammation and/or scarring of the tissues surrounding the
implant site.
Although mesenchymal stem cells can be found in various connective tissues of
the
body including muscle, skeletal muscle, cartilage, etc. (H. E. Young et al.,
1993, In vitro Cell
Dev. Biol. 29A:723 736; H. E. Young, et al., 1995, Dev. Dynam. 202:137 144),
the term
mesenchymal has been used historically to refer to a class of stem cells
purified from skeletal
muscle marrow, and not from muscle. Thus, mesenchymal stem cells are
distinguished from
the muscle-derived progenitor cells of the present invention. Moreover,
mesenchymal cells
do not express the CD34 cell marker (M. F. Pittenger et al., 1999, Science
284:143 147),
which is expressed by the muscle-derived progenitor cells described herein.
The description herein of disadvantages and problems associated with known
compositions, and methods is in no way intended to limit the scope of the
embodiments
described in this document to their exclusion. Indeed, certain embodiments may
include one
or more known compositions, compounds, or methods without suffering from the
so-noted
disadvantages or problems.
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SUMMARY OF THE INVENTION
It is an object of the present invention to provide novel muscle-derived
progenitor
cells (MDCs) and MDC compositions exhibiting long-term survival following
transplantation. The MDCs of this invention and compositions containing the
MDCs
comprise early progenitor muscle cells, i.e., muscle-derived stem cells that
express progenitor
cell markers, including, but not limited to desmin, M-cadherin, MyoD,
myogenin, CD34, and
Bc1-2. In addition, these early progenitor muscle cells express the Flk-1, Sca-
1, MNF, and c-
met cell markers, but do not express the CD45 or c-Kit cell markers.
It is another object of the present invention to provide methods for isolating
and
enriching muscle-derived progenitor cells from a starting muscle cell
population. These
methods result in the enrichment of MDCs that have long-term survivability
after
transplantation or introduction into a site of soft tissue. The MDC population
according to
the present invention is particularly enriched with cells that express
progenitor cell markers,
including, but not limited to desmin, M-cadherin, MyoD, myogenin, CD34, and
Bc1-2. This
MDC population also expresses the Flk-1, Sea-1, MNF, and c-met cell markers,
but does not
express the CD45 or c-Kit cell markers.
It is yet another object of the present invention to provide methods of using
MDCs
and compositions comprising MDCs for the augmentation of muscle tissue,
including skeletal
muscle, without the need for polymer carriers or special culture media for
transplantation.
Such methods include the administration of MDC compositions by introduction
into skeletal
muscle, for example by direct injection into or on the surface of the tissue,
or by systemic
distribution of the compositions.
It is yet another object of the present invention to provide methods of
augmenting
skeletal muscle, following injury, wounding, surgeries, traumas, non-traumas,
or other
procedures that result in fissures, openings, depressions, wounds, and the
like.
It is a further object of the present invention to provide MDCs and
compositions
comprising MDCs that are modified through the use of chemicals, growth media,
and/or
genetic manipulation. Such MDCs and compositions thereof comprise chemically
or
genetically modified cells useful for the production and delivery of
biological compounds,
and the treatment of various diseases, conditions, injuries, or illnesses.
It is a further object of the present invention to provide MDCs and
compositions
comprising MDCs that are modified through the use of chemicals, growth media,
and/or
genetic manipulation. Such MDCs and compositions thereof comprise chemically
or
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genetically modified cells useful for the production and delivery of
biological compounds,
and the treatment of various diseases, conditions, injuries, or illnesses.
It is yet another embodiment of the invention to provide pharmaceutical
compositions
comprising MDCs and compositions comprising MDCs. These pharmaceutical
compositions
comprise isolated MDCs. These MDCs may be subsequently expanded by cell
culture after
isolation. In one aspect of this embodiment, these MDCs are frozen prior to
delivery to a
subject in need of the pharmaceutical composition.
The invention also provides compositions and methods involving the isolation
of
MDCs using a single plating technique. MDCs are isolated from a biopsy of
skeletal muscle.
In one embodiment, the skeletal muscle from the biopsy may be stored for 1-6
days. In one
aspect of this embodiment, the skeletal muscle from the biopsy is stored at 4
C. The cells
are minced, and digested using a collagenase, dispase, another enzyme or a
combination of
enzymes. After washing the enzyme from the cells, the cells are cultured in a
flask in culture
medium for between about 30 and about 120 minutes. During this period of time,
the
"rapidly adhering cells" stick to the walls of the flask or container, while
the "slowly
adhering cells" or MDCs remain in suspension. The "slowly adhering cells" are
transferred
to a second flask or container and cultured therein for a period of 1-3 days.
During this
second period of time the "slowly adhering cells" or MDCs stick to the walls
of the second
flask or container.
In another embodiment of the invention, these MDCs are expanded to any number
of
cells. In a preferred aspect of this embodiment, the cells are expanded in new
culture media
for between about 10 and 20 days. More preferably, the cells are expanded for
17 days.
The MDCs, whether expanded or not expanded, may be preserved in order to be
transported or stored for a period of time before use. In one embodiment, the
MDCs are
frozen. Preferably, the MDCs are frozen at between about -20 and -90 C. More
preferably,
the MDCs are frozen at about -80 C. These frozen MDCs are used as a
pharmaceutical
composition.
MDCs, whether frozen or preserved as a pharmaceutical composition, or used
fresh,
may be used to treat a number of skeletal muscle degenerative pathologies.
These conditions
include but are not limited to skeletal muscle weakness, muscular dystrophy,
muscle atrophy,
spasticity, myoclonus and myalgia. MDCs, whether frozen or preserved as a
pharmaceutical
composition, or used fresh, may also be used for the increase of skeletal
muscle mass in
athletes or other organisms in need of greater than average skeletal muscle
mass.
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Additional objects and advantages afforded by the present invention will be
apparent
from the detailed description and exemplification herein below.
BRIEF DESCRIPTION OF THE DRAWINGS
The patent or patent application file contains at least one photographic
reproduction
executed in color.
The appended figures are presented to further describe the invention and to
assist in
its understanding through clarification of its various aspects.
Figures IA and 1B show micrographs of human MDCs (hMDCs) injected into mouse
muscle with human dystrophin and mouse Y-chromosome stained to show chimerism
in the
human and mouse muscle cells that have fused.
Figure 2 is a bar graph showing the percentage of dystrophin positive fibers
in mice
injected with hMDCs.
DETAILED DESCRIPTION OF THE INVENTION
The invention provides human MDCs and methods of using such cells to generate
skeletal muscle tissue to repair damaged skeletal muscle or to increase
skeletal muscle
volume and/or strength to above wild type levels. The invention further
provides methods of
treating skeletal muscle disorders including but not limited to skeletal
muscle weakness,
muscular dystrophy, muscle atrophy, spasticity, myoclonus and myalgia. The
isolation of
human muscle-derived cells (MDCs) from adult tissue are capable of achieving
increased
skeletal muscle density and skeletal muscle volume within human subjects
administered these
cells.
Muscle-Derived Cells and Compositions
The present invention provides MDCs comprised of early progenitor cells (also
termed muscle-derived progenitor cells or muscle-derived stem cells herein)
that show long-
term survival rates following transplantation into body tissues, preferably
skeletal muscle. To
obtain the MDCs of this invention, a muscle explant, preferably skeletal
muscle, is obtained
from an animal donor, preferably from a mammal, including humans. This explant
serves as
a structural and functional syncytium including "rests" of muscle precursor
cells (T. A.
Partridge etal., 1978, Nature 73:3068; B. H. Lipton etal., 1979, Science
205:12924).
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Cells isolated from primary muscle tissue contain mixture of fibroblasts,
myoblasts,
adipocytes, hematopoietic, and muscle-derived progenitor cells. The progenitor
cells of a
muscle-derived population can be enriched using differential adherence
characteristics of
primary muscle cells on collagen coated tissue flasks, such as described in
U.S. Pat. No.
6,866,842 of Chancellor et al. Cells that are slow to adhere
tend to be morphologically round, express high levels of desmin, and have the
ability to fuse
and differentiate into multinucleated myotubes U.S. Pat. No. 6,866,842 of
Chancellor et al.).
A subpopulation of these cells was shown to respond to recombinant human
skeletal muscle
morphogenic protein 2 (rhBMP-2) in vitro by expressing increased levels of
alkaline
phosphatase, parathyroid hormone dependent 3',5'-cAMP, and other markers of
osteogenic
and myogenic lineages (U.S. Pat. No. 6,866,842 of Chancellor et al.; T.
Katagiri etal., 1994,
J. Cell Biol., 127:1755 1766).
In one embodiment of the invention, a preplating procedure may be used to
differentiate rapidly adhering cells from slowly adhering cells (MDCs). In
accordance with
the present invention, populations of rapidly adhering MDC (PP1-4) and slowly
adhering,
round MDC (PP6) were isolated and enriched from skeletal muscle explants and
tested for
the expression of various markers using immunohistochemistry to determine the
presence of
pluripotent cells among the slowly adhering cells (Example 1; patent
application U.S. Patent
No. 6,866,842 to Chancellor etal.). As shown in Table 1, Example 1 herein, the
PP6 cells
expressed myogenic markers, including desmin, MyoD, and Myogenin. The PP6
cells also
expressed c-met and MNF, two genes that are expressed at an early stage of
myogenesis (J.
B. Miller etal., 1999, Curr. Top. Dev, Biol. 43:191 219; see Table 3). The PP6
showed a
lower percentage of cells expressing M-cadherin, a satellite cell-specific
marker (A. Irintchev
et al., 1994, Development Dynamics 199:326 337), but a higher percentage of
cells
expressing Bc1-2, a marker limited to cells in the early stages of myogenesis
(J. A. Dominov
etal., 1998, J. Cell Biol. 142:537 544). The PP6 cells also expressed CD34, a
marker
identified with human hematopoietic progenitor cells, as well as stromal cell
precursors in
skeletal muscle marrow (R. G. Andrews etal., 1986, Blood 67:842 845; C. I.
Civin et al.,
1984, J. Immunol. 133:157 165; L. Fina et al, 1990, Blood 75:2417 2426; P. J.
Simmons et
a/., 1991, Blood 78:2848 2853; see Table 3). The PP6 cells also expressed Flk-
1, a mouse
homologue of human KDR gene which was recently identified as a marker of
hematopoietic
cells with stem cell-like characteristics (B. L. Ziegler et al., 1999, Science
285:1553 1558;
see Table 3). Similarly, the PP6 cells expressed Sca-1, a marker present in
hematopoietic
cells with stem cell-like characteristics (M. van de Rijn et at., 1989, Proc.
Natl. Acad. Sci.
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USA 86:4634 8; M. Osawa et al., 1996, J. Irnmunol. 156:3207 14; see Table 3).
However,
the PP6 cells did not express the CD45 or c-Kit hematopoietic stem cell
markers (reviewed in
L K. Ashman, 1999, Int. J. Biochem. Cell. Biol. 31:1037 51; G. A. Koretzky,
1993, FASEB
J. 7:420 426; see Table 3).
In one embodiment of the present invention, the PP6 population of muscle-
derived
progenitor cells having the characteristics described herein are provided.
These muscle-
derived progenitor cells express the desmin, CD34, and Bc1-2 cell markers. In
accordance
with the present invention, the PP6 cells are isolated by the techniques
described herein
(Example 1) to obtain a population of muscle-derived progenitor cells that
have long-term
survivability following transplantation. The PP6 muscle-derived progenitor
cell population
comprises a significant percentage of cells that express progenitor cell
markers, including, but
not limited to desmin, CD34, and Bc1-2. In addition, PP6 cells express the Flk-
1 and Sca-1
cell markers, but do not express the CD45 or c-Kit markers. Preferably,
greater than 95% of
the PP6 cells express the desmin, Sea-1, and Flk-1 markers, but do not express
the CD45 or
c-Kit markers. It is preferred that the PP6 cells are utilized within about 1
day or about 24
hours after the last plating.
In a preferred embodiment, the rapidly adhering cells and slowly adhering
cells
(MDCs) are separated from each other using a single plating technique. One
such technique
is described in Example 2. First, cells are provided from a skeletal muscle
biopsy. The
biopsy need only contain about 100 mg of cells. Biopsies ranging in size from
about 50 mg
to about 500 mg are used according to both the pre-plating and single plating
methods of the
invention. Further biopsies of 50, 100, 110, 120, 130, 140, 150, 200, 250,
300, 400 and 500
mg are used according to both the pre-plating and single plating methods of
the invention.
In a preferred embodiment of the invention, the tissue from the biopsy is then
stored
for 1 to 7 days. This storage is at a temperature from about room temperature
to about 4 C.
This waiting period causes the biopsied skeletal muscle tissue to undergo
stress. While this
stress is not necessary for the isolation of MDCs using this single plate
technique, using the
wait period generally results in a greater yield of MDCs.
According to preferred embodiments, tissue from the biopsies is minced and
centrifuged. The pellet is resuspended and digested using a digestion enzyme.
Enzymes that
may be used include, but are not limited to, collagenase, dispase or
combinations of these
enzymes. After digestion, the enzyme is washed off of the cells. The cells are
transferred to
a flask in culture media for the isolation of the rapidly adhering cells. Many
culture media
may be used. Particularly preferred culture media include those that are
designed for culture
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of endothelial cells including Cambrex Endothelial Growth Medium. This medium
may be
supplemented with other components including fetal bovine serum, IGF-1, bFGF,
VEGF,
EGF, hydrocortisone, heparin, and/or ascorbic acid. Other media that may be
used in the
single plating technique include InCell M310F medium. This medium may be
supplemented
as described above, or used unsupplemented.
The step for isolation of the rapidly adhering cells may require culture in
flask for a
period of time from about 30 to about 120 minutes. The rapidly adhering cells
adhere to the
flask in 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120 minutes. After they
adhere, the slowly
adhering cells are separated from the rapidly adhering cells from removing the
culture media
from the flask to which the rapidly adhering cells are attached to.
The culture medium removed from this flask is then transferred to a second
flask.
The cells may be centrifuged and resuspended in culture medium before being
transferred to
the second flask. The cells are cultured in this second flask for between 1
and 3 days.
Preferably, the cells are cultured for two days. During this period of time,
the slowly
adhering cells (MDCs) adhere to the flask. After the MDCs have adhered, the
culture media
is removed and new culture media is added in order to promote expansion of the
MDCs. The
MDCs may be expanded in number by culturing them for from about 10 to about 20
days.
The MDCs may be expanded in number by culturing them for 10, 11, 12, 13, 14,
15, 16, 17,
18, 19 or 20 days. Preferably, the MDCs are subject to expansion culture for
17 days.
As an alternative to the pre-plating and single plating methods, the MDCs of
the
present invention can be isolated by fluorescence-activated cell sorting
(FACS) analysis
using labeled antibodies against one or more of the cell surface markers
expressed by the
MDCs (C. Webster et al., 1988, Exp. Cell. Res. 174:252 65; J. R. Blanton et
al., 1999,
Muscle Nerve 22:43 50). For example, FACS analysis can be performed using
labeled
antibodies that specifically bind to CD34, Flk-1, Sca-1, and/or the other cell-
surface markers
described herein to select a population of PP6-like cells that exhibit long-
term survivability
when introduced into the host tissue. Also encompassed by the present
invention is the use of
one or more fluorescence-detection labels, for example, fluorescein or
rhodamine, for
antibody detection of different cell marker proteins.
Using any of the MDCs isolation methods described above, or otherwise known in
the
art, MDCs that are to be transported, or are not going to be used for a period
of time may be
preserved using any method known in the art. For example, the isolated MDCs
may be
frozen to a temperature ranging from about -25 to about -90 C. Preferably,
the MDCs are
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frozen at about -80 C on dry ice for delayed use or transport. The freezing
may be done with
any cryopreservation medium known in the art.
Muscle-Derived Cell-Based Treatments
In one embodiment of the present invention, the MDCs are isolated from a
skeletal
muscle source and introduced or transplanted into a muscle or non-muscle soft
tissue site of
interest, or into skeletal muscle. Advantageously, the MDCs of the present
invention are
isolated and enriched to contain a large number of progenitor cells showing
long-term
survival following transplantation. In addition, the muscle-derived progenitor
cells of this
invention express a number of characteristic cell markers, such desmin, CD34,
and Bc1-2.
Furthermore, the muscle-derived progenitor cells of this invention express the
Sca-1, and F1k-
1 cell markers, but do not express the CD45 or c-Kit cell markers (see Example
1).
MDCs and compositions comprising MDCs of the present invention can be used to
repair, treat, or ameliorate various aesthetic or functional conditions (e.g.
defects) through the
augmentation of skeletal muscle. In particular, such compositions can be used
for the
treatment of skeletal muscle disorders. Multiple and successive
administrations of MDC are
also embraced by the present invention.
For MDC-based treatments, a skeletal muscle explant is preferably obtained
from an
autologous or heterologous human or animal source. An autologous animal or
human source
is more preferred. MDC compositions are then prepared and isolated as
described herein. To
introduce or transplant the MDCs and/or compositions comprising the MDCs
according to
the present invention into a human or animal recipient, a suspension of
mononucleated
muscle cells is prepared. Such suspensions contain concentrations of the
muscle-derived
progenitor cells of the invention in a physiologically-acceptable carrier,
excipient, or diluent.
For example, suspensions of MDC for administering to a subject can comprise
108 to 109
cells/ml in a sterile solution of complete medium modified to contain the
subject's serum, as
an alternative to fetal bovine serum. Alternatively, MDC suspensions can be in
serum-free,
sterile solutions, such as cryopreservation solutions (Celox Laboratories, St.
Paul, Minn.).
The MDC suspensions can then be introduced e.g., via injection, into one or
more sites of the
donor tissue.
The described cells can be administered as a pharmaceutically or
physiologically
acceptable preparation or composition containing a physiologically acceptable
carrier,
excipient, or diluent, and administered to the tissues of the recipient
organism of interest,
including humans and non-human animals. The MDC-containing composition can be
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prepared by resuspending the cells in a suitable liquid or solution such as
sterile physiological
saline or other physiologically acceptable injectable aqueous liquids. The
amounts of the
components to be used in such compositions can be routinely determined by
those having
skill in the art.
The MDCs or compositions thereof can be administered by placement of the MDC
suspensions onto absorbent or adherent material, e.g., a collagen sponge
matrix, and insertion
of the MDC-containing material into or onto the site of interest.
Alternatively, the MDCs can
be administered by parenteral routes of injection, including subcutaneous,
intravenous,
intramuscular, and intrasternal. Other modes of administration include, but
are not limited to,
intranasal, intrathecal, intracutaneous, percutaneous, enteral, and
sublingual. In one
embodiment of the present invention, administration of the MDCs can be
mediated by
endoscopic surgery.
For injectable administration, the composition is in sterile solution or
suspension or
can be resuspended in pharmaceutically- and physiologically-acceptable aqueous
or
oleaginous vehicles, which may contain preservatives, stabilizers, and
material for rendering
the solution or suspension isotonic with body fluids (i.e. blood) of the
recipient. Non-limiting
examples of excipients suitable for use include water, phosphate buffered
saline, pH 7.4, 0.15
M aqueous sodium chloride solution, dextrose, glycerol, dilute ethanol, and
the like, and
mixtures thereof. Illustrative stabilizers are polyethylene glycol, proteins,
saccharides, amino
acids, inorganic acids, and organic acids, which may be used either on their
own or as
admixtures. The amounts or quantities, as well as the routes of administration
used, are
determined on an individual basis, and correspond to the amounts used in
similar types of
applications or indications known to those of skill in the art.
To optimize transplant success, the closest possible immunological match
between
donor and recipient is desired. If an autologous source is not available,
donor and recipient
Class I and Class II histocompatibility antigens can be analyzed to determine
the closest
match available. This minimizes or eliminates immune rejection and reduces the
need for
immunosuppressive or immunomodulatory therapy. If required, immunosuppressive
or
immunomodulatory therapy can be started before, during, and/or after the
transplant
procedure. For example, cyclosporin A or other immunosuppressive drugs can be
administered to the transplant recipient. Immunological tolerance may also be
induced prior
to transplantation by alternative methods known in the art (D. J. Watt et at.,
1984, Clin. Exp.
Inununol. 55:419; D. Faustman et al., 1991, Science 252:1701).
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Consistent with the present invention, the MDCs can be administered to body
tissues,
including skeletal muscle. The number of cells in an MDC suspension and the
mode of
administration may vary depending on the site and condition being treated.
From about
1.0x105 to about 1x108 MDCsmay be administered according to the invention. As
a non-
limiting example, in accordance with the present invention, about 0.5-2.0x106
MDCs are
administered via a collagen sponge matrix for the treatment of an
approximately 5 mm region
of skull defect (see Example 3).
For skeletal muscle augmentation or treatment of skeletal muscle disorders,
the MDCs
are prepared as described above and are administered, e.g. via injection,
onto, into or around
skeletal muscle tissue to provide additional skeletal muscle strength and/or
volume. As is
appreciated by the skilled practitioner, the number of MDC introduced is
modulated to
provide varying amounts of skeletal muscle density and/or skeletal muscle
volume, as needed
or required. Thus, the present invention also embraces the use of MDC of the
invention in
treating skeletal muscle disorders or enhancing skeletal muscle density and/or
skeletal muscle
volume. Skeletal muscle disorders include but are not limited to skeletal
muscle weakness,
muscular dystrophy, muscle atrophy, spasticity, myoclonus and myalgia. The
invention also
relates to the novel use of MDCs for the increase of skeletal muscle mass in
athletes or other
organisms in need of greater than average skeletal muscle mass.
12
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Genetically Engineered Muscle-Derived Cells
In another aspect of the present invention, the MDCs of this invention may be
genetically engineered to contain a nucleic acid sequence(s) encoding one or
more active
biomolecules, and to express these biomolecules, including proteins,
polypeptides, peptides,
hormones, metabolites, drugs, enzymes, and the like. Such MDCs may be
histocompatible
(autologous) or nonhistocompatible (allogeneic) to the recipient, including
humans. These
cells can serve as long-term local delivery systems for a variety of
treatments, for example,
for the treatment of skeletal muscle diseases and pathologies, including, but
not limited to
skeletal muscle weakness, muscular dystrophy, muscle atrophy, spasticity,
myoclonus and
myalgia.
Preferred in the present invention are autologous muscle-derived progenitor
cells,
which will not be recognized as foreign to the recipient. In this regard, the
MDC used for
cell-mediated gene transfer or delivery will desirably be matched vis-a-vis
the major
histocompatibility locus (MHC or HLA in humans). Such MHC or HLA matched cells
may
be autologous. Alternatively, the cells may be from a person having the same
or a similar
MHC or HLA antigen profile. The patient may also be tolerized to the
allogeneic MHC
antigens. The present invention also encompasses the use of cells lacking MHC
Class I
and/or II antigens, such as described in U.S. Pat. No. 5,538,722.
The MDCs may be genetically engineered by a variety of molecular techniques
and
methods known to those having skill in the art, for example, transfection,
infection, or
transduction. Transduction as used herein commonly refers to cells that have
been
genetically engineered to contain a foreign or heterologous gene via the
introduction of a
viral or non-viral vector into the cells. Transfection more commonly refers to
cells that have
been genetically engineered to contain a foreign gene harbored in a plasmid,
or non-viral
vector. MDCs can be transfected or transduced by different vectors and thus
can serve as
gene delivery vehicles to transfer the expressed products into muscle.
Although viral vectors are preferred, those having skill in the art will
appreciate that
the genetic engineering of cells to contain nucleic acid sequences encoding
desired proteins
or polypeptides, cytokines, and the like, may be carried out by methods known
in the art, for
example, as described in U.S. Pat. No. 5,538,722, including fusion,
transfection, lipofection
mediated by the use of liposornes, electroporation, precipitation with DEAE-
Dextran or
calcium phosphate, particle bombardment (biolistics) with nucleic acid-coated
particles (e.g.,
gold particles), microinjection, and the like.
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Vectors for introducing heterologous (i.e., foreign) nucleic acid (DNA or RNA)
into
muscle cells for the expression of bioactive products are well known in the
art. Such vectors
possess a promoter sequence, preferably, a promoter that is cell-specific and
placed upstream
of the sequence to be expressed. The vectors may also contain, optionally, one
or more
expressible marker genes for expression as an indication of successful
transfection and
expression of the nucleic acid sequences contained in the vector.
Illustrative examples of vehicles or vector constructs for transfection or
infection of
the muscle-derived cells of the present invention include replication-
defective viral vectors,
DNA virus or RNA virus (retrovirus) vectors, including, but not limited to
adenovirus, herpes
simplex virus and adeno-associated viral vectors. Adeno-associated virus
vectors are single
stranded and allow the efficient delivery of multiple copies of nucleic acid
to the cell's
nucleus. Preferred are adenovirus vectors. The vectors will normally be
substantially free of
any prokaryotic DNA and may comprise a number of different functional nucleic
acid
sequences. Examples of such functional sequences include polynucleotide, e.g.,
DNA or
RNA, sequences comprising transcriptional and translational initiation and
termination
regulatory sequences, including promoters (e.g., strong promoters, inducible
promoters, and
the like) and enhancers which are active in muscle cells.
Also included as part of the functional sequences is an open reading frame
(polynucleotide sequence) encoding a protein of interest; flanking sequences
may also be
included for site-directed integration. In some situations, the 5'-flanking
sequence will allow
homologous recombination, thus changing the nature of the transcriptional
initiation region,
so as to provide for inducible or noninducible transcription to increase or
decrease the level
of transcription, as an example.
In general, the nucleic acid sequence desired to be expressed by the muscle-
derived
progenitor cell is that of a structural gene, or a functional fragment,
segment or portion of the
gene, that is heterologous to the muscle-derived progenitor cell and encodes a
desired protein
or polypeptide product, for example. The encoded and expressed product may be
intracellular, i.e., retained in the cytoplasm, nucleus, or an organelle of a
cell, or may be
secreted by the cell. For secretion, the natural signal sequence present in
the structural gene
may be retained, or a signal sequence that is not naturally present in the
structural gene may
be used. When the polypeptide or peptide is a fragment of a protein that is
larger, a signal
sequence may be provided so that, upon secretion and processing at the
processing site, the
desired protein will have the natural sequence. Examples of genes of interest
for use in
accordance with the present invention include genes encoding cell growth
factors, cell
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differentiation factors, cell signaling factors and programmed cell death
factors. Specific
examples include, but are not limited to, genes encoding BMP-2 (rhBMP-2), IL-
1Ra, Factor
IX, and connexin 43.
As mentioned above, a marker may be present for selection of cells containing
the
vector construct. The marker may be an inducible or non-inducible gene and
will generally
allow for positive selection under induction, or without induction,
respectively. Examples of
commonly-used marker genes include neomycin, dihydrofolate reductase,
glutamine
synthetase, and the like.
The vector employed will generally also include an origin of replication and
other
genes that are necessary for replication in the host cells, as routinely
employed by those
having skill in the art. As an example, the replication system comprising the
origin of
replication and any proteins associated with replication encoded by a
particular virus may be
included as part of the construct. The replication system must be selected so
that the genes
encoding products necessary for replication do not ultimately transform the
muscle-derived
cells. Such replication systems are represented by replication-defective
adenovirus
constructed as described, for example, by G. Acsadi et al., 1994, Hum. Mol.
Genet 3:579
584, and by Epstein-Barr virus. Examples of replication defective vectors,
particularly,
retroviral vectors that are replication defective, are BAG, described by Price
et al., 1987,
Proc. Natl. Acad. Sci. USA, 84:156; and Sanes et al., 1986, EMBO J., 5:3133.
It will be
understood that the final gene construct may contain one or more genes of
interest, for
example, a gene encoding a bioactive metabolic molecule. In addition, cDNA,
synthetically
produced DNA or chromosomal DNA may be employed utilizing methods and
protocols
known and practiced by those having skill in the art.
If desired, infectious replication-defective viral vectors may be used to
genetically
engineer the cells prior to in vivo injection of the cells. In this regard,
the vectors may be
introduced into retroviral producer cells for amphotrophic packaging. The
natural expansion
of muscle-derived progenitor cells into adjacent regions obviates a large
number of injections
into or at the site(s) of interest.
In another aspect, the present invention provides ex vivo gene delivery to
cells and
tissues of a recipient mammalian host, including humans, through the use of
MDC, e.g., early
progenitor muscle cells, that have been virally transduced using an adenoviral
vector
engineered to contain a heterologous gene encoding a desired gene product.
Such an ex vivo
approach provides the advantage of efficient viral gene transfer, which is
superior to direct
gene transfer approaches. The ex vivo procedure involves the use of the muscle-
derived
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progenitor cells from isolated cells of muscle tissue. The muscle biopsy that
will serve as the
source of muscle-derived progenitor cells can be obtained from an injury site
or from another
area that may be more easily obtainable from the clinical surgeon.
It will be appreciated that in accordance with the present invention, clonal
isolates can
be derived from the population of muscle-derived progenitor cells (i.e., PP6
cells or "slowly
adhering" cells using the single plate procedure) using various procedures
known in the art,
for example, limiting dilution plating in tissue culture medium. Clonal
isolates comprise
genetically identical cells that originate from a single, solitary cell. In
addition, clonal
isolates can be derived using FACS analysis as described above, followed by
limiting dilution
to achieve a single cell per well to establish a clonally isolated cell line.
An example of a
clonal isolate derived from the PP6 cell population is mc13, which is
described in Example 1.
Preferably, MDC clonal isolates are utilized in the present methods, as well
as for genetic
engineering for the expression of one or more bioactive molecules, or in gene
replacement
therapies.
The MDCs are first infected with engineered viral vectors containing at least
one
heterologous gene encoding a desired gene product, suspended in a
physiologically
acceptable carrier or excipient, such as saline or phosphate buffered saline,
and then
administered to an appropriate site in the host. Consistent with the present
invention, the
MDCs can be administered to body tissues, including skeletal muscle, as
described above.
The desired gene product is expressed by the injected cells, which thus
introduce the gene
product into the host. The introduced and expressed gene products can thereby
be utilized to
treat, repair, or ameliorate the injury, dysfunction, or disease, due to their
being expressed
over long time periods by the MDCs of the invention, having long-term survival
in the host.
In animal model studies of myoblast-mediated gene therapy, implantation of 106
myoblasts per 100 mg muscle was required for partial correction of muscle
enzyme defects
(see, J. E. Morgan et al., 1988, J. Neural. Sci. 86:137; T. A. Partridge et
al., 1989, Nature
337:176). Extrapolating from this data, approximately 1012 MDCs suspended in a
physiologically compatible medium can be implanted into muscle tissue for gene
therapy for
a 70 kg human. This number of MDC of the invention can be produced from a
single 100 mg
skeletal muscle biopsy from a human source (see below). For the treatment of a
specific
injury site, an injection of genetically engineered MDC into a given tissue or
site of injury
comprises a therapeutically effective amount of cells in solution or
suspension, preferably,
about 105 to 106 cells per cm3 of tissue to be treated, in a physiologically
acceptable medium.
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EXAMPLES
Example 1. MDC Enrichment, Isolation and Analysis According to the Pre-Plating
Method.
MDCs were prepared as described (U.S. Pat. No. 6,866,842 of Chancellor et
al.).
Muscle explants were obtained from the hind limbs of a number of sources,
namely from 3-
week-old mdx (dystrophic) mice (C57BL/10ScSn mdx/mdx, Jackson Laboratories), 4-
6
week-old normal female SD (Sprague Dawley) rats, or SCID (severe combined
immunodeficiency) mice. The muscle tissue from each of the animal sources was
dissected
to remove any bones and minced into a slurry. The slurry was then digested by
1 hour serial
incubations with 0.2% type XI collagenase, dispase (grade II, 240 unit), and
0.1% trypsin at
37 C. The resulting cell suspension was passed through 18, 20, and 22 gauge
needles and
centrifuged at 3000 rpm for 5 minutes. Subsequently, cells were suspended in
growth
medium (DMEM supplemented with 10% fetal bovine serum, 10% horse serum, 0.5%
chick
embryo extract, and 2% penicillin/streptomycin). Cells were then preplated in
collagen-
coated flasks (U.S. Pat. No. 6,866,842 of Chancellor et al.). After
approximately 1 hour, the
supernatant was removed from the flask and re-plated into a fresh collagen-
coated flask. The
cells which adhered rapidly within this 1 hour incubation were mostly
fibroblasts (Z. Qu et
al., supra; U.S. Pat. No. 6,866,842 of Chancellor et al.). The supernatant was
removed and
re-plated after 30-40% of the cells had adhered to each flask. After
approximately 5-6 serial
platings, the culture was enriched with small, round cells, designated as PP6
cells, which
were isolated from the starting cell population and used in further studies.
The adherent cells
isolated in the early platings were pooled together and designated as PP1-4
cells.
The mdx PP1-4, mdx PP6, normal PP6, and fibroblast cell populations were
examined
by immunohistochemical analysis for the expression of cell markers. The
results of this
analysis are shown in Table 1.
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TABLE 1
Cell markers expressed in PP1-4 and PP6 cell populations.
mdx PP1-4 mdx PP6 nor PP6
cells cells cells fibroblasts
desmin +/-
CD34
Bc1-2 (-)
Flk-1 na
Sca-1 na
M-cadherin -/+ -/+ -/+
MyoD -/+ +/- +/-
myogenin -/+ +/- +/-
Mdx PP1-4, mdx PP6, normal PP6, and fibroblast cells were derived by
preplating
technique and examined by immunohistochemical analysis. "-" indicates less
than 2% of the
cells showed expression; "(-)"; "-/+" indicates 5-50% of the cells showed
expression;
indicates -40-80% of the cells showed expression; "+" indicates that >95% of
the cells
showed expression; "nor" indicates normal cells; "na" indicates that the
inununohistochemical data is not available.
It is noted that both mdx and normal mice showed identical distribution of all
the cell
markers tested in this assay. Thus, the presence of the mdx mutation does not
affect the cell
marker expression of the isolated PP6 muscle-cell derived population.
MDCs were grown in proliferation medium containing DMEM (Dulbecco's Modified
Eagle Medium) with 10% FBS (fetal bovine serum), 10% HS (horse serum), 0.5%
chick
embryo extract, and 1% penicillin/streptomycin, or fusion medium containing
DMEM
supplemented with 2% fetal bovine serum and 1% antibiotic solution. All media
supplies
were purchased through Gibco Laboratories (Grand Island, N.Y.).
Example 2. MDC Enrichment, Isolation and Analysis According to the Single
Plate
Method.
Populations of rapidly- and slowly-adhering MDCs were isolated from skeletal
muscle of a mammalian subject. The subject may be a human, rat, dog or other
mammal.
Biopsy size ranged from 42 to 247 mg.
Skeletal muscle biopsy tissue is immediately placed in cold hypothermic medium
(HYPOTHERMOSOL (BioLife) supplemented with gentamicin sulfate (100 ng/ml,
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Roche)) and stored at 4 C. After 3 to 7 days, biopsy tissue is removed from
storage and
production is initiated. Any connective or non-muscle tissue is dissected from
the biopsy
sample. The remaining muscle tissue that is used for isolation is weighed. The
tissue is
minced in Hank's Balanced Salt Solution (HESS), transferred to a conical tube,
and
centrifuged (2,500xg, 5 minutes). The pellet is then resuspended in a
Digestion Enzyme
solution (Liberase Blendzyme 4 (0.4-1.0 U/mL, Roche)). 2 mL of Digestion
Enzyme
solution is used per 100 mg of biopsy tissue and is incubated for 30 minutes
at 37 C on a
rotating plate. The sample is then centrifuged (2,500xg, 5 minutes). The
pellet is
resuspended in culture medium and passed through a 70 gm cell strainer. The
culture media
used for the procedures described in this Example was Cambrex Endothelial
Growth Medium
EGM-2 basal medium supplemented with the following components: i. 10% (v/v)
fetal
bovine serum, and ii. Cambrex EGM-2 SingleQuot Kit, which contains: Insulin
Growth
Factor-1 (IGF-1), Basic Fibroblast Growth Factor (bFGF), Vascular Endothelial
Growth
Factor (VEGF), Epidermal Growth Factor (EGF), Hydrocortisone, Heparin, and
Ascorbic
Acid. The filtered cell solution is then transferred to a T25 culture flask
and incubated for
30-120 minutes at 37 C in 5% CO2. Cells that attach to this flask are the
"rapidly-adhering
cells".
After incubation, the cell culture supernatant is removed from the T25 flask
and
placed into a 15 mL conical tube. The T25 culture flask is rinsed with 2 mL of
warmed
culture medium and transferred to the aforementioned 15 mL conical tube. The
15 mL
conical tube is centrifuged (2,500xg, 5 minutes). The pellet is resuspended in
culture
medium and transferred to a new T25 culture flask. The flask is incubated for
¨2 days at
37 C in 5% CO2 (cells that attach to this flask are the "slowly-adhering
cells"). After
incubation, the cell culture supernatant is aspirated and new culture medium
is added to the
flask. The flask is then returned to the incubator for expansion. Standard
culture passaging is
carried out from here on to maintain the cell confluency in the culture flask
at less than 50%.
Trypsin-EDTA (0.25%, Invitrogen) is used to detach the adherent cells from the
flask during
passage. Typical expansion of the "slowly-adhering cells" takes an average of
17 days
(starting from the day production is initiated) to achieve an average total
viable cell number
of 37 million cells.
Once the desired cell number is achieved, the cells are harvested from the
flask using
Trypsin-EDTA and centrifuged (2,500xg, 5 minutes). The pellet is resuspended
in BSS-P
solution (HESS supplemented with human serum albumin (2% v/v, Sera Care Life))
and
counted. The cell solution is then centrifuged again (2,500xg, 5 minutes),
resuspended with
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Cryopreservation Medium (CryoStor (Biolife) supplemented with human serum
albumin (2%
v/v, Sera Care Life Sciences)) to the desired cell concentration, and packaged
in the
appropriate vial for cryogenic storage. The cryovial is placed into a freezing
container and
placed in the -80 C freezer. Cells are administered by thawing the frozen
cell suspension
at room temperature with an equal volume of physiologic saline and injected
directly
(without additional manipulation). The lineage characterization of the slowly
adhering cell
populations shows: Myogenic (87.4% CD56+, 89.2% desmin+), Endothelial (0.0%
CD31+),
Hematopoietic (0.3% CD45+), and Fibroblast (6.8% CD90+/CD56-).
Following disassociation of the skeletal muscle biopsy tissue, two fractions
of cells
were collected based on their rapid or slow adhesion to the culture flasks.
The cells were then
expanded in culture with growth medium and then frozen in cryopreservation
medium (3 x
105 cells in 15 I) in a 1.5 ml eppendorf tube. For the control group, 15 I
of
cryopreservation medium alone was placed into the tube. These tubes were
stored at -80 C
until injection. Immediately prior to injection, a tube was removed from
storage, thawed at
room temperature, and resuspended with 15 1 of 0.9% sodium chloride solution.
The
resulting 30 I solution was then drawn into a 0.5 cc insulin syringe with a
30 gauge needle.
The investigator performing the surgery and injection was blinded to the
contents of the
tubes.
Cell count and viability was measured using a Guava flow cytometer and
Viacount
assay kit (Guava). CD56 was measured by flow cytometry (Guava) using PE-
conjugated
anti-CD56 antibody (1:50, BD Pharmingen) and PE-conjugated isotype control
monoclonal
antibody (1:50, BD Pharmingen). Desmin was measured by flow cytometry (Guava)
on
paraformaldehyde-fixed cells (BD Pharmingen) using a monoclonal desmin
antibody (1:100,
Dako) and an isotype control monoclonal antibody (1:200, BD Pharmingen).
Fluorescent
labeling was performed using a Cy3-conjugated anti-mouse IgG antibody (1:250,
Sigma). In
between steps, the cells were washed with permeabilization buffer (BD
Pharmingen). For
creatine kinase (CK) assay, 1 x 105 cells were plated per well into a 12 well
plate in
differentiation-inducing medium. Four to 6 days later, the cells were
harvested by
trypsinization and centrifuged into a pellet. The cell lysis supernatant was
assayed for CK
activity using the CK Liqui-UV kit (Stanbio).
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Example 3. Augmentation of Skeletal Muscle with MDCs.
Populations of human muscle derived cells (hMDCs) isolated from human muscle
biopsies by way of the preplate technique were tested to show that hMDCs had
similar
myogenic and regenerative characteristics to their murine counterparts.
Methods.
Pre-Plate Technique: This technique is disclosed throughout the application
and
specifically, above in Example 1.
Isolation and Cell Culture: Candidate populations were obtained using the pre-
plate
technique. These cells were grown in EGMTm-2 media (Cambrex) at a density of
600
cells/cm2 and passaged every 72-96 hours before confluence under standard
conditions (5.0%
CO2, 37 C). Flow Cytometry: hMDC were analyzed for the presence of the cell
surface
cluster of differentiation markers CD34, CD56, CD144, and CD146.
Immunochemistry: hMDC were stained for desmin, myosin heavy chain, and
dystrophin.
Bioinformatic Live Cell Imaging: Cells were grown in a cell culture system
with
dynamic imaging. Images were taken at 10-min. intervals. We measured numerous
parameters such as doubling rate, growth rate, total cell number, elongation,
area, and perimeter for
the various populations at early passage and late passage. We compared human
MDC phenotypic
profiles among the different preplate fractions to identify both molecular and
behavioral
characteristics that might predict in vivo regeneration efficiency.
In vivo regeneration: We transplanted early passage human populations into the
gastrocnemius muscles of nubc/SOD mice. We harvested the muscles 2 weeks after
transplantation. The muscles were frozen sectioned into l0- m sections. For
immunohistochemical analysis, we used mouse anti-human or anti-mouse
dystrophin
(Novocastra, DYS3/2, 1:50), biotinylated goat anti-mouse secondary Ab (Vector,
1:500) and
streptavidin-Cy3 (Sigma, 1:500). We used a human nuclear antigen antibody to
label human
nuclei in the skeletal muscle sections.
Results.
We examined 3 preplate fractions ¨ preplates 1 and 2 (pp1-2), preplates 2-3
(pp2-3)
and preplates 5-6). All populations were negative for CD34 and CD144, and
positive for
CD56 and CD146. Over time, a decrease in CD56 and CD146 was observed.
Imunostaining
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revealed the myogenic potential of the cells, as they displayed the ability to
fuse into multi-
nucleated myotubes, and showed desmin, myosin, and dystrophin expression.
Time-elapsed imaging showed much variability in parameters such as cellular
division time, population doubling time, cellular motility behavior, and
morphological
parameters. We observed media-specific changes in morphology. Cells grown in
EGM2
showed a decrease in proliferation rates as cells were expanded. Our analysis
to date has no
shown any differences in these behaviors which are related to preplate
fraction.
We injected several preplate populations; pp2 (n=11 muscles), pp4 (n=19), pp6
(n=20). Sex-crossed transplantation of the hMDC to host mdx-scid muscle
resulted in fusion
of the donor cells to the host skeletal muscle fibers and subsequent delivery
and expression of
dystrophin in the host. Chimerism was determined by use of the human specific
antibody,
and donor specific Y-chromosome (Figure 1). The number of regenerating
dystrophin
positive fibers was significantly higher in transplantations using pp6
fraction as compared to
pp2 fraction (P = 0.037, 2-tailed 2-test, Figure2). However, there was no
significant
difference in the level of regeneration between pp2 and pp4 transplantations.
All groups had
dystrophin levels greater than the PBS sham controls (Figure 2).
This study shows that hMDCs are similarly obtained from the pp6 which appears
to
be distinct from pp2 obtained from human muscle biopsy. In the transplantation
study, we
observe human dystrophin expression, although the number of dystrophin
positive
regenerating muscle fibers was lower than what has been observed with mouse
MDCs.
Initial results show that culture in SkGM yields greater proliferation of
hMDCs than culture
in either EGM-2 or DMEM. Ongoing imaging studies will determine whether this
increase
in growth is coupled with greater therapeutic efficacy and differing cellular
phenotypes.
22
