U.S. patent application number 15/977145 was filed with the patent office on 2019-04-11 for muscle derived cells for the treatment of urinary tract pathologies and methods of making and using the same.
The applicant listed for this patent is University of Pittsburgh - Of the Commonwealth System of Higher Education. Invention is credited to Michael B. Chancellor, Johnny Huard, Ronald Jankowski, Ryan Pruchnic.
Application Number | 20190106681 15/977145 |
Document ID | / |
Family ID | 39155487 |
Filed Date | 2019-04-11 |
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United States Patent
Application |
20190106681 |
Kind Code |
A1 |
Chancellor; Michael B. ; et
al. |
April 11, 2019 |
Muscle Derived Cells for the Treatment of Urinary Tract Pathologies
and Methods of Making and Using the Same
Abstract
The present invention provides muscle-derived progenitor cells
that show long-term survival following transplantation into body
tissues and which can augment soft tissue following introduction
(e.g. via injection, transplantation, or implantation) into a site
of soft tissue. Also provided are methods of isolating
muscle-derived progenitor cells, and methods of genetically
modifying the cells for gene transfer therapy. The invention
further provides methods of using compositions comprising
muscle-derived progenitor cells for the augmentation and bulking of
mammalian, including human, soft tissues in the treatment of
various functional conditions, including malformation, injury,
weakness, disease, or dysfunction. In particular, the present
invention provides treatments and amelioration for urinary
incontinence and other urinary tract pathologies.
Inventors: |
Chancellor; Michael B.;
(Pittsburgh, PA) ; Jankowski; Ronald; (Pittsburgh,
PA) ; Pruchnic; Ryan; (Pittsburgh, PA) ;
Huard; Johnny; (Wexford, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Pittsburgh - Of the Commonwealth System of Higher
Education |
Pittsburgh |
PA |
US |
|
|
Family ID: |
39155487 |
Appl. No.: |
15/977145 |
Filed: |
May 11, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14576326 |
Dec 19, 2014 |
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15977145 |
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13336332 |
Dec 23, 2011 |
8961954 |
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14576326 |
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12013076 |
Jan 11, 2008 |
8105834 |
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13336332 |
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60884478 |
Jan 11, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2501/105 20130101;
C12N 2500/38 20130101; A61K 35/12 20130101; C12N 2501/115 20130101;
C12N 2501/39 20130101; A61P 13/02 20180101; A61P 13/00 20180101;
A61P 13/10 20180101; C12N 2501/91 20130101; C12N 5/0658 20130101;
A61K 35/34 20130101; C12N 2501/11 20130101; C12N 2501/165
20130101 |
International
Class: |
C12N 5/077 20060101
C12N005/077; A61K 35/34 20060101 A61K035/34 |
Goverment Interests
GOVERNMENT INTERESTS
[0002] This invention was made with Government support under Grant
No. DK055387 awarded by the National Institutes of Health. The
Government has certain rights in this invention.
Claims
1. A method for preparing a cell population containing muscle
derived cells (MDCs) useful for administration to treat urinary
tract disease in a mammalian subject, comprising: (a) suspending
cells isolated from human skeletal muscle in a first cell culture
container for a duration sufficient to adhere a first cell
population to the container and to leave a second cell population
remaining unadhered and in a culture medium in the container; (b)
transferring the culture medium and second cell population from the
first cell culture container to a second cell culture container;
(c) allowing cells from the second cell population to attach to the
second cell culture container; and (d) isolating the cells attached
to the second cell culture container to obtain said cell population
containing MDCs.
2. A method for preparing a cell population containing muscle
derived progenitor cells (MDCs), comprising: mincing skeletal
muscle tissue obtained from a human patient to be treated with
muscle derived progenitor cells, wherein the skeletal muscle tissue
has been stored under storage conditions causing stress to the
skeletal muscle tissue; digesting the minced muscle tissue to
obtain a mixed population of cells; and preparing an enriched
population of muscle derived progenitor cells from the mixed
population of cells.
3. The method of claim 2, wherein said storage conditions include
cooling.
4. The method of claim 2, wherein said storage conditions include
storing the skeletal muscle tissue in hypothermic medium.
5. The method of claim 4, wherein the hypothermic medium contains
an antibiotic.
6. The method of claim 4, wherein said storage conditions include
storing the skeletal muscle tissue at a temperature from about
4.degree. C. to room temperature.
7. The method of claim 2, wherein the enriched population of muscle
derived progenitor cells is prepared based on differential
adherence characteristics of cells of the mixture of cells.
8. The method of claim 7, wherein said preparing an enriched
population of muscle derived progenitor cells includes suspending
said mixture of cells in a first cell culture container for a
period sufficient for a first population of cells to adhere to the
container and a second population of cells to remain non-adhered to
the container, and transferring the second population of cells to a
second cell culture container.
9. The method of claim 2, also comprising freezing the enriched
population of muscle derived progenitor cells in the presence of
cryopreservation medium.
10. The method of claim 2, wherein the skeletal muscle tissue has
been stored for 1 to 7 days under storage conditions causing stress
to the skeletal muscle tissue
11. A method of treating a urinary tract disease in a human patient
in need thereof, comprising administering to the urinary tract of
the patient an enriched population of muscle derived progenitor
cells prepared in accordance with claim 2.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 14/576,326, filed on Dec. 19, 2014, which is a divisional of
U.S. application Ser. No. 13/336,332, now U.S. Pat. No. 8,961,954,
filed on Dec. 23, 2011, which is a divisional of U.S. application
Ser. No. 12/013,076, now U.S. Pat. No. 8,105,834, filed on Jan. 11,
2008, which claims benefit of priority from U.S. Provisional
Application No. 60/884,478, filed on Jan. 11, 2007, the contents of
each are incorporated herein in their entireties.
FIELD OF THE INVENTION
[0003] The present invention relates to muscle-derived progenitor
cells (MDC) and compositions of MDCs and their use in the
augmentation of body tissues, particularly soft tissue like
urethral and periurethral muscle. In particular, the present
invention relates to muscle-derived progenitor cells that show
long-term survival following introduction into soft tissues,
methods of isolating MDCs, and methods of using MDC-containing
compositions for the augmentation of human or animal soft tissues,
including epithelial, adipose, nerve, organ, muscle, ligament, and
cartilage tissue. The invention also relates to novel uses of
muscle-derived progenitor cells for the treatment of functional
conditions, such as stress urinary incontinence or urinary
incontinence.
BACKGROUND OF THE INVENTION
[0004] Stress urinary incontinence (SUI) is a common condition and
is characterized as the involuntary leakage of urine on effort,
exertion, sneezing or coughing. (Abrams P, et al. Neurourol Urodyn
2002; 21(2): 167-178). The etiology of SUI is multifactorial,
involving damage and/or functional impairment of muscle and
associated nerves that may occur as a result of advancing age,
hormonal status, and pelvic floor damage resulting from vaginal
child-birth. Due to the multifactorial etiology, a single treatment
option, that is not limited in some fashion, does not currently
exist.
[0005] The use of periurethral injectables as a minimally invasive
treatment option, which may be performed on an outpatient basis
under local anesthesia. This method of treatment is more cost
effective in the near-term, with shorter hospitalization, reduced
operating room time, and generally fewer complications when
compared with invasive surgical approaches such as bladder neck
suspension. (Berman C J, et al. J Urol 1997; 157(1): 122-124).
However, it has disadvantages such as need for multiple injections
due to loss of the long-term bulking effect owing to degradation,
reabsorption, and/or migration, as well as other impediments such
as bladder outlet obstruction and allergic reactions. Thus, there
is a need for other, different urinary 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.
[0006] Muscle derived cells isolated from rats have shown some
success models for urinary incontinence. (Cannon T W, et al.
Urology 2003; 62(5): 958-963 and Lee J Y et al. Int Urogynecol J
Pelvic Floor Dysfunct 2003; 14(1): 31-37; discussion 37). The
instant invention provides the use of human skeletal muscle derived
cells (MDC) as an injectable treatment for SUI, and other urinary
tract pathologies.
SUMMARY OF THE INVENTION
[0007] It is an object of the present invention to provide novel
human muscle-derived progenitor cells (MDCs) and human 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,
such as desmin, M-cadherin, MyoD, myogenin, CD34, and Bcl-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.
[0008] It is another object of the present invention to provide
methods for isolating and enriching human muscle-derived progenitor
cells from a starting muscle cell population. These methods result
in the enrichment of human 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, such as desmin, M-cadherin, MyoD, myogenin, CD34, and
Bcl-2. This MDC population also expresses the Flk-1, Sca-1, MNF,
and c-met cell markers, but does not express the CD45 or c-Kit cell
markers.
[0009] It is yet another object of the present invention to provide
methods of using MDCs and compositions comprising MDCs for the
augmentation of muscle soft tissue, or non-muscle soft tissue,
including smooth muscle, and various organ tissues, without the
need for polymer carriers or special culture media for
transplantation. Such methods include the administration of MDC
compositions by introduction into soft tissue, for example by
direct injection into tissue, or by systemic distribution of the
compositions. Preferably, soft tissue includes non-bone body
tissues. More preferably, soft tissue includes non-striated muscle
and non-bone body tissues. Most preferably, soft tissue includes
non-muscle, non-bone body tissues. As used herein, augmentation
refers to filling, bulking, supporting, enlarging, extending, or
increasing the size or mass of body tissue.
[0010] It is another object of the present invention to provide
methods of augmenting soft tissue, either muscle-derived soft
tissue, or non-muscle-derived soft tissue, following injury,
wounding, surgeries, traumas, non-traumas, or other procedures that
result in fissures, openings, depressions, wounds, and the like, in
the skin or in internal soft tissues or organs.
[0011] It is yet another object of the present invention to provide
human MDC-based treatments for urinary tract disease and associated
symptoms. Pharmaceutical compositions comprising MDCs and
compositions comprising MDCs may be used for the treatment of
urinary tract pathologies. These pharmaceutical compositions
comprise isolated human MDCs. These MDCs may be subsequently
expanded by cell culture after isolation. In one embodiment of the
invention, these MDCs are frozen prior to delivery to a subject in
need of the pharmaceutical composition.
[0012] In one embodiment, when the human MDCs and compositions
thereof are used to treat urinary incontinence they are injected
directly into the urethra. Preferably, they may be injected into
the periurethral muscle In another embodiment, human MDCs and
compositions thereof are used to improve at least one symptom of
urinary tract disease. These symptoms include urinary incontinence,
urinary tract infection, frequent urination, painful urination,
burning sensation when urinating, fatigue, tremor, cloudy urine,
blood in urine, and kidney infection.
[0013] Human 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.degree. C. The MDCs are then
isolated using the pre-plate or the single plate technique.
[0014] Using the pre-plate technique, a suspension of skeletal
muscle cells from skeletal muscle tissue is plated in a first
container to which fibroblast cells of the skeletal muscle cell
suspension adhere. Non-adherent cells are then re-plated in a
second container, wherein the step of replating is after 15-20% of
cells have adhered to the first container. This replating step must
be repeated at least once. The MDCs are thereby isolated and may be
administered to the esophagus of the mammalian subject.
[0015] Using the single plate technique, 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.
[0016] 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.
[0017] 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.degree. C. More preferably,
the MDCs are frozen at about -80.degree. C. These frozen MDCs are
used as a pharmaceutical composition.
[0018] Additional objects and advantages afforded by the present
invention will be apparent from the detailed description and
exemplification hereinbelow.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] The patent or patent application file contains at least one
photographic reproduction executed in color. Copies of this patent
or patent application with color photographic reproduction(s) will
be provided by the U.S. Patent and Trademark Office upon request
and payment of the necessary fee.
[0020] The appended drawings of the figures are presented to
further describe the invention and to assist in its understanding
through clarification of its various aspects.
[0021] FIG. 1 is a bar graph showing higher leak point pressure in
rats treated with human MDCs than control.
[0022] FIG. 2A is a light micrograph of a proximal urethral
sphincter in a control rat.
[0023] FIG. 2B is a light micrograph of a proximal urethral
sphincter in a rat treated with human MDCs.
[0024] FIG. 3 shows immunofluorescent labeling with a
human-specific nuclear antibody (lamins A/C) revealing the presence
of human nuclei incorporated within the striated sphincter muscle
layer in human MDC injected tissue (100.times.). Arrows point to
individual nuclei.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The invention provides human MDCs and methods of using such
cells to generate tissue with bulking properties that have the
potential to improve coaptation and intrinsic sphincter function by
remodeling the damaged tissue. The invention further provides
methods of treating urinary tract disorders including incontinence
and stress urinary incontinence. The isolation of human
muscle-derived cells (MDCs) from adult tissue are capable of
achieving functional success within an established urethral
sphincter injury model.
Muscle-Derived Cells and Compositions
[0026] 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 soft
tissues. To obtain the MDCs of this invention, a muscle explant,
preferably skeletal muscle, is obtained from an animal donor,
preferably from a mammal, including rats, dogs and humans. This
explant serves as a structural and functional syncytium including
"rests" of muscle precursor cells (T. A. Partridge et al., 1978,
Nature 73:306-8; B. H. Lipton et al., 1979, Science 205:12924).
[0027] 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 bone
morphogenic protein 2 (rhBMP-2) In vitro by expressing increased
levels of alkaline phosphatase, parathyroid hormone dependent
3',5'-cAMP, and osteogenic lineage and myogenic lineages (U.S. Pat.
No. 6,866,842 of Chancellor et al.; T. Katagiri et al., 1994, J.
Cell Biol., 127:1755-1766).
[0028] 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 cells (PP1-4) and slowly adhering,
round MDCs (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.
Ser. No. 09/302,896 of Chancellor et al.). The PP6 cells expressed
myogenic markers, including desmin, MyoD, and Myogenin. The PP6
cells also expressed c-met and MNF, two genes which are expressed
at an early stage of myogenesis (J. B. Miller et al., 1999, Curr.
Top. Dev. Biol. 43:191-219). 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 Bcl-2, a marker limited to
cells in the early stages of myogenesis (J. A. Dominov et al.,
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 bone marrow (R. G.
Andrews et al., 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 al., 1991, Blood 78:2848-2853). 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). Similarly, the PP6 cells expressed Sca-1, a marker
present in hematopoietic cells with stem cell-like characteristics
(M. van de Rijn et al., 1989, Proc. Natl. Acad. Sci. USA 86:4634-8;
M. Osawa et al., 1996, J. Immunol. 156:3207-14). 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).
[0029] One embodiment of the present invention is the PP6
population of muscle-derived progenitor cells having the
characteristics described herein. These muscle-derived progenitor
cells express the desmin, CD34, and Bcl-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 such as desmin, CD34,
and Bcl-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, Sca-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.
[0030] 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 can be 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 can be used according to both the
pre-plating and single plating methods of the invention.
[0031] 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.degree. 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, it seems that
using the wait period results in a greater yield of MDCs.
[0032] Tissue from the biopsies is minced and centrifuged. The
pellet is resuspended and digested using a digestion enzyme.
Enzymes that may be used include 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 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.
[0033] 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 by removing the culture media from the flask to
which the rapidly adhering cells are attached.
[0034] 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 so that the MDCs can be
expanded in number. 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.
[0035] 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 to
directed 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.
[0036] Using any of the MDCs isolation methods described above,
MDCs that are to be transported, or are not going to be used for a
period of time may be preserved using methods known in the art.
More specifically, the isolated MDCs may be frozen to a temperature
ranging from about -25 to about -90.degree. C. Preferably, the MDCs
are frozen at about -80.degree. 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
[0037] 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 bone structures. 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 Bcl-2. Furthermore, the muscle-derived
progenitor cells of this invention express the Sca-1, and Flk-1
cell markers, but do not express the CD45 or c-Kit cell
markers.
[0038] 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 muscle or non-muscle soft tissues. In particular,
such compositions can be used as soft-tissue bulking agents for the
treatment of urinary incontinence and other instances of smooth
muscle weakness, disease, injury, or dysfunction. In addition, such
MDCs and compositions thereof can be used for augmenting soft
tissue not associated with injury by adding bulk to a soft tissue
area, opening, depression, or void in the absence of disease or
trauma, such as for "smoothing". Multiple and successive
administrations of MDCs are also embraced by the present
invention.
[0039] 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 MDCs for administering to a subject can
comprise 10.sup.8 to 10.sup.9 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.
[0040] 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 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.
[0041] The MDCs or compositions thereof can be administered by
placement of the MDC suspensions onto absorbent or adherent
material, i.e., 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.
[0042] 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.
[0043] 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 al., 1984, Clin. Exp. Immunol. 55:419; D. Faustman et al., 1991,
Science 252:1701).
[0044] Consistent with the present invention, the MDCs can be
administered to body tissues, including epithelial tissue (i.e.,
skin, lumen, etc.) muscle tissue (i.e. smooth muscle), and various
organ tissues such as those organs that are associated with the
urological system (i.e., bladder, urethra, ureter, kidneys,
etc.).
[0045] The number of cells in an MDC suspension and the mode of
administration may vary depending on the site and condition being
treated. As non-limiting examples, in accordance with the present
invention, about 3-5.times.10.sup.5 MDCs are injected for the
treatment of urinary incontinence (see Example 3). Consistent with
the Examples disclosed herein, a skilled practitioner can modulate
the amounts and methods of MDC-based treatments according to
requirements, limitations, and/or optimizations determined for each
case.
[0046] Conditions of the lumen: In another embodiment, the MDCs and
compositions thereof according to the present invention have
further utility as treatments for conditions of the lumen in an
animal or mammal subject, including humans. Specifically, the
muscle-derived progenitor cells are used for completely or
partially blocking, enhancing, enlarging, sealing, repairing,
bulking, or filling various biological lumens or voids within the
body. Lumens include, without limitation the urethra. Voids may
include, without limitation, various tissue wounds (i.e., loss of
muscle and soft tissue bulk due to trauma; destruction of soft
tissue due to penetrating projectiles such as a stab wound or
bullet wound; loss of soft tissue from disease or tissue death due
to surgical removal of the tissue including loss of breast tissue
following a mastectomy for breast cancer or loss of muscle tissue
following surgery to treat sarcoma, etc.), lesions, fissures,
diverticulae, cysts, fistulae, aneurysms, and other undesirable or
unwanted depressions or openings that may exist within the body of
an animal or mammal, including humans. For the treatment of
conditions of the lumen, the MDCs are prepared as disclosed herein
and then administered, e.g. via injection or intravenous delivery,
to the lumenal tissue to fill or repair the void. The number of
MDCs introduced is modulated to repair large or small voids in a
soft tissue environment, as required.
[0047] Conditions of the sphincter: The MDCs and compositions
thereof according to the present invention can also be used for the
treatment of a sphincter injury, weakness, disease, or dysfunction
in an animal or mammal, including humans. In particular, the MDCs
are used to augment tissues of the urinary sphincters. More
specifically, the present invention provides soft tissue
augmentation treatments for urinary incontinence. For the treatment
of sphincter defects, the MDCs are prepared as described herein and
then administered to the sphincter tissue, e.g. via injection, to
provide additional bulk, filler, or support. The number of MDCs
introduced is modulated to provide varying amounts of bulking
material as required. For example, about 3-5.times.10.sup.5 MDCs
are injected for the treatment of urinary incontinence (see Example
3).
[0048] In addition, the MDCs and compositions thereof can be used
to affect contractility in smooth muscle tissue, such as urinary or
bladder tissue, as example. Thus, the present invention also
embraces the use of MDCs of the invention in restoring muscle
contraction, and/or ameliorating or overcoming smooth muscle
contractility problems.
Genetically Engineered Muscle-Derived Cells
[0049] 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, urinary incontienence.
[0050] 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 MDCs 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.
[0051] 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.
[0052] 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 liposomes, electroporation, precipitation with
DEAE-Dextran or calcium phosphate, particle bombardment
(biolistics) with nucleic acid-coated particles (e.g., gold
particles), microinjection, and the like.
[0053] 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.
[0054] 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, such as 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.
[0055] 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.
[0056] 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 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.
[0057] 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.
[0058] 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.
[0059] 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.
[0060] 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 MDCs, 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 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.
[0061] 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 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.
[0062] 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 bone, epithelial tissue, connective tissue,
muscle tissue, and various organ tissues such as those organs that
are associated with the digestive system, cardiovascular system,
respiratory system, reproductive system, urological system, and
nervous system, 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.
[0063] In animal model studies of myoblast-mediated gene therapy,
implantation of 10.sup.6 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
10.sup.12 MDCs suspended in a physiologically compatible medium can
be implanted into muscle tissue for gene therapy for a 70 kg human.
This number of MDCs 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 MDCs into a given tissue or site of injury
comprises a therapeutically effective amount of cells in solution
or suspension, preferably, about 10.sup.5 to 10.sup.6 cells per
cm.sup.3 of tissue to be treated, in a physiologically acceptable
medium.
EXAMPLES
Example 1. MDC Enrichment, Isolation and Analysis According to the
Pre-Plating Method
[0064] Enrichment and isolation of MDCs: 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.degree. 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.
[0065] 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.
TABLE-US-00001 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 - + + - Bcl-2 (-) + + - Flk-1 na
+ + - Sca-1 na + + - M-cadherin -/+ -/+ -/+ - MyoD -/+ +/- +/- -
myogenin -/+ +/- +/- -
[0066] 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 .about.40-80% of the cells showed
expession; "+" indicates that >95% of the cells showed
expression; "nor" indicates normal cells; "na" indicates that the
immunohistochemical data is not available.
[0067] It is noted that both mdx and normal mice showed identical
distribution of all of 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.
[0068] 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
[0069] Enrichment and Isolation of MDCs
[0070] 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.
[0071] Skeletal muscle biopsy tissue was immediately placed in cold
hypothermic medium (HYPOTHERMOSOL.RTM. (BioLife) supplemented with
gentamicin sulfate (100 ng/ml, Roche)) and stored at 4.degree. C.
After 3 to 7 days, biopsy tissue was removed from storage and
production was initiated. Any connective or non-muscle tissue was
dissected from the biopsy sample. The remaining muscle tissue that
was used for isolation is weighed. The tissue was minced in Hank's
Balanced Salt Solution (HBSS), transferred to a conical tube, and
centrifuged (2,500.times.g, 5 minutes). The pellet was then
resuspended in a Digestion Enzyme solution (Liberase Blendzyme 4
(0.4-1.0 U/mL, Roche)). 2 mL of Digestion Enzyme solution was used
per 100 mg of biopsy tissue and was incubated for 30 minutes at
37.degree. C. on a rotating plate. The sample was then centrifuged
(2,500.times.g, 5 minutes). The pellet was resuspended in culture
medium and passed through a 70 .mu.m 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 was then transferred to a T25 culture flask and incubated
for 30-120 minutes at 37.degree. C. in 5% CO.sub.2. Cells that
attached to this flask were termed the "rapidly-adhering
cells".
[0072] After incubation, the cell culture supernatant was 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 (2,500.times.g, 5 minutes). The pellet
was resuspended in culture medium and transferred to a new T25
culture flask. The flask was incubated for .about.2 days at
37.degree. C. in 5% CO2 (cells that attach to this flask were
termed the "slowly-adhering cells"). After incubation, the cell
culture supernatant was aspirated and new culture medium was added
to the flask. The flask was 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) was used to detach the
adherent cells from the flask during passage. Typical expansion of
the "slowly-adhering cells" took an average of 17 days (starting
from the day production is initiated) to achieve an average total
viable cell number of 37 million cells.
[0073] Once the desired cell number was achieved, the cells were
harvested from the flask using Trypsin-EDTA and centrifuged
(2,500.times.g, 5 minutes). The pellet was resuspended in BSS-P
solution (HBSS supplemented with human serum albumin (2% v/v, Sera
Care Life)) and counted. The cell solution was then centrifuged
again (2,500.times.g, 5 minutes), resuspended with Cryopreservation
Medium (CRYOSTOR.TM. (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 was placed into a freezing container and
placed in the -80.degree. C. freezer. Cells were 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 showed: Myogenic (87.4% CD56+,
89.2% desmin+), Endothelial (0.0% CD31+), Hematopoietic (0.3%
CD45+), and Fibroblast (6.8% CD90+/CD56-).
[0074] Analysis for Characterization of Enriched and Isolated
MDCs
[0075] 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, as described above. The
cells were then expanded in culture with growth medium and then
frozen in cryopreservation medium (3.times.10.sup.5 cells in 15
.mu.l) in a 1.5 ml eppendorf tube, also as described above. For the
control group, 15 .mu.l of cryopreservation medium alone was placed
into the tube. These tubes were stored at -80.degree. C. until
injection. Immediately prior to injection, a tube was removed from
storage, thawed at room temperature, and resuspended with 15 .mu.l
of 0.9% sodium chloride solution. The resulting 30 .mu.l 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.
[0076] 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.times.10.sup.5 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.RTM. kit (Stanbio).
Example 3. Treatment of Urinary Incontinence with Human MDCs in a
Rat Model
[0077] Treatment with human MDCs led to restoration of leak point
pressure (LPP) back to normal levels in an experimental model of
stress urinary incontinence (SUI). Injected human MDCs alleviated
urinary incontinence in a well established rat model.
[0078] These experiments demonstrate proof of concept and
feasibility of using human cell therapy for urologic application.
The human MDCs were harvested from a clinically-obtainable sized
muscle biopsy, and improved physiologic outcomes for up to four
weeks in an immunocompromised rat model of SUI. Histologic
evaluation demonstrated periurethral muscle atrophy in the sham
group only. Human MDCs were present in the nude rat urethral
sphincter 4 weeks after injection. Treatment with human MDCs led to
restoration of LPP back to near-normal levels in an experimental
model of SUI in the nude rat.
[0079] Animals:
[0080] The experiments described below were performed using 6-8
week old female, athymic nude rats (Hsd:RH-rnu, Harlan Laboratory).
Procedural protocols were approved by the Animal Research Care
Committee of Children's Hospital of Pittsburgh. The policies and
procedures of the animal laboratory are in accordance with those
detailed in the guide for the `Care and Use of Laboratory Animals`
published by the US Department of Health and Human Services.
[0081] Denervation of Sciatic Nerve (SUI Model):
[0082] A well-established SUI model was created through bilateral
transection of the sciatic nerve. Rats were given isoflurane
anesthesia (2 L/min) and, after appropriate induction, bilateral
vertical dorsal incisions were performed over the ischiorectal
fossa. Under an operating microscope, the sciatic nerve on each
side was identified and 2 mm of those trunks were excised distal to
its origin from the vertebral column, but proximal to the branching
of the pudendal nerve.
[0083] Human MDC Isolation:
[0084] Human MDCs used in this study were isolated from human
skeletal muscle tissue (.about.250 mg) harvested from the rectus
abdominus of a single donor, and isolated according to the single
plate technique described above. Culture expansion was carried out
in an antibiotic-free proprietary medium supplemented with 10%
fetal bovine serum. Flow cytometric analysis of the MDC suspensions
was performed to evaluate myogenic content through antibody
labeling of CD56 expression (BD Pharmingen). MDCs were
cryopreserved at a concentration of 1.times.10.sup.6 viable
cells/10 .mu.L. Separate aliquots of carrier medium alone were also
prepared for sham injection.
[0085] Injection Procedure:
[0086] Seven days following denervation, under isoflurane
anesthesia (2 L/min), a low midline incision was made to expose the
bladder and urethra. Cryopreserved MDCs or sham suspensions were
thawed with an equal volume of saline just prior to injection. A
3/10-mL insulin syringe was used to inject either 10 .mu.L
(5.times.10.sup.5 cells) of MDC suspension or sham aliquot into
each lateral wall of the mid-urethra with microscopic guidance.
Non-denervated, non-injected, age-matched animals served as
controls.
[0087] In Vivo Cystometry (CMG) and Leak Point Pressure (LPP)
Measurement:
[0088] In vivo functional measurements were performed 4 weeks
following injections. Under urethane anesthesia (1.2 g/kg
subcutaneous injection), a midline abdominal incision was made and
the ureters were ligated. A transvesical catheter with a
fire-flared tip (PE-90) was inserted into the dome of the bladder
for bladder filling and pressure recording, and the abdomen was
closed. A three-way stopcock was connected to the transvesical tube
to monitor the bladder pressure during cystometry (continuous
infusion of normal saline at rate of 0.04 mL/min). The voided
volume, bladder capacity and maximal voiding pressure were
monitored. After cystometry, all rats underwent spinal cord
transection at the T9 level in order to eliminate spontaneous
bladder activity in response to increasing intravesical pressures.
The rats were then mounted on a tilt table and placed in the
vertical position. Intravesical pressure was clamped by connecting
a large 50 mL syringe to the bladder catheter and the pressure
transducer via PE-190 tubing and three-way stopcocks. The reservoir
was mounted on a metered vertical pole for controlled height
adjustment. Intravesical pressure was increased in 1-3 cmH.sub.2O
steps from zero upward until visual identification of leakage; this
pressure was identified as the LPP. Three consecutive readings were
obtained and averaged for each animal and presented as a single
LPP.
[0089] Tissue Harvest and Histology:
[0090] Immediately following the LPP measurement, the entire
urethra-bladder complex was removed. The tissues were snap frozen
using 2-methylbutane precooled in liquid nitrogen. Cryosections of
the urethra were labeled with hematoxylin/eosin (H&E) for
general histology, and also immunofluorescently-labeled with human
specific anti-lamins A/C antibody (Novocastra, U.K.) to follow the
fate of the injected MDC.
[0091] Statistical Analysis:
[0092] Data are presented as means.+-.SE. Overall comparisons
between groups were performed using a one-way analysis of variance
(Tukey's multiple comparison test). A p-value of less than 0.05 was
accepted as significant.
[0093] The injected MDC suspensions contained 87.7% myogenic
(CD56-positive) cells; the remainders of the cells were
fibroblastic. There were no serious adverse effects observed in any
rat in the control, sham and MDC-injected groups. However, partial
obstruction of the external urethral meatus due to infection at the
perineal area was found in one animal each in both the sham and
human MDC-injected groups. Thus, these animals were excluded from
the functional analysis.
[0094] CMG and LPP Measurement:
[0095] No difference in any measured cystometric parameter was
observed between the control, sham and human MDC-injected groups
(Table 2).
TABLE-US-00002 TABLE 2 Cystometric variables in each group.
Cystometric parameters Control Sham MDC P-value Maximal voiding
29.8 .+-. 1.4 29.3 .+-. 2.8 33.7 .+-. 5.8 0.677 pressure
(cmH.sub.2O) Bladder capacity 0.40 .+-. 0.06 0.36 .+-. 0.09 0.34
.+-. 0.03 0.827 (ml)
[0096] Denervation of the urethral sphincter resulted in a
significant decrease in LPP from the control to sham groups (FIG.
1) (43.4.+-.0.6 to 27.8.+-.0.7 cmH.sub.2O, respectively;
p<0.05). LPP was restored to significantly higher levels
following MDC injection (35.7.+-.2.0 cmH.sub.2O) when compared to
the sham group (p<0.05); however, at the 4 week time point, this
level of restoration remained significantly less than that of
control group (p<0.05).
[0097] Histological Analysis:
[0098] In the denervated rats, the proximal urethral sphincter was
atrophic at 4 weeks compared with control (FIG. 2). Human nuclei
present within the rat sphincter tissue was revealed through
immunofluorescent labeling using a human-specific antibody to the
nuclear envelope proteins lamins A and C. Tissues from the
MDC-injected group showed clear positive labeling of numerous human
nuclei incorporated within the external (striated) sphincter muscle
(FIG. 3).
[0099] All patent applications, patents, texts, and literature
references cited in this specification are hereby incorporated
herein by reference in their entirety to more fully describe the
state of the art to which the present invention pertains.
[0100] As various changes can be made in the above methods and
compositions without departing from the scope and spirit of the
invention as described, it is intended that all subject matter
contained in the above description, shown in the accompanying
drawings, or defined in the appended claims be interpreted as
illustrative, and not in a limiting sense.
* * * * *