U.S. patent application number 10/430100 was filed with the patent office on 2004-04-22 for methods and compositions for the repair and/or regeneration of damaged myocardium.
This patent application is currently assigned to The Government of the United States of America as represented by the Department of Health and. Invention is credited to Anversa, Piero, Orlic, Donald.
Application Number | 20040076619 10/430100 |
Document ID | / |
Family ID | 27575213 |
Filed Date | 2004-04-22 |
United States Patent
Application |
20040076619 |
Kind Code |
A1 |
Anversa, Piero ; et
al. |
April 22, 2004 |
Methods and compositions for the repair and/or regeneration of
damaged myocardium
Abstract
Methods, compositions, and kits for repairing damaged myocardium
and/or myocardial cells including the administration of stem cells,
such as adult stem cells, optionally with cytokines are disclosed
and claimed.
Inventors: |
Anversa, Piero; (New York,
NY) ; Orlic, Donald; (Bethesda, MD) |
Correspondence
Address: |
KLARQUIST SPARKMAN, LLP
121 S.W. SALMON STREET, SUITE #1600
ONE WORLD TRADE CENTER
PORTLAND
OR
97204-2988
US
|
Assignee: |
The Government of the United States
of America as represented by the Department of Health and
Human Services
|
Family ID: |
27575213 |
Appl. No.: |
10/430100 |
Filed: |
May 5, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10430100 |
May 5, 2003 |
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09919979 |
Jul 31, 2001 |
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60221902 |
Jul 31, 2000 |
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60258564 |
Dec 29, 2000 |
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60258805 |
Jan 2, 2001 |
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60295807 |
Jun 6, 2001 |
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60295806 |
Jun 6, 2001 |
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60295805 |
Jun 6, 2001 |
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60295804 |
Jun 6, 2001 |
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60295803 |
Jun 6, 2001 |
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Current U.S.
Class: |
424/93.7 |
Current CPC
Class: |
A61K 38/18 20130101;
A01K 67/0271 20130101; A61K 35/34 20130101; A61K 38/193 20130101;
A61K 35/28 20130101; A61P 43/00 20180101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 38/193 20130101; A61K 38/18 20130101; A61K 35/34
20130101; C12N 2501/12 20130101; A61K 35/28 20130101; C12N 5/0647
20130101; A61K 2035/124 20130101 |
Class at
Publication: |
424/093.7 |
International
Class: |
A61K 045/00 |
Goverment Interests
[0003] This work was in part supported by the government, by grants
from the National Institutes of Health, Grant No's: HL-38132,
HL-39902, HL-43023, AG-15756, AG-17042, HL-66923, and HL-65577.
[0004] Without any admission, prejudice, waiver, or estoppel, the
government may have certain rights.
Claims
We claim:
1. A method for repairing and/or generating and/or regenerating
myocardium and/or myocardial cells comprising the administration of
hematopoietic stem cells.
2. The method of claim 1, wherein the administered hematopoietic
stem cells are lineage negative.
3. The method of claim 2, wherein the administered lineage negative
hematopoietic stem cells are c-kit.sup.POS.
4. The method of claim 1, wherein a therapeutically effective dose
of hematopoietic stem cells are delivered to the heart.
5. The method of claim 4, wherein the therapeutically effective
dose of the hematopoietic stem cells is
2.times.10.sup.4-1.times.10.sup.5 cells.
6. The method of claim 4, wherein the therapeutically effective
dose is delivered to the border area of the damaged myocardium
and/or myocardial cells.
7. The method of claim 4, wherein the therapeutically effective
dose is administered by injection.
8. The method of claim 7, wherein the therapeutically effective
dose is injected intramyocardially.
9. The method of claim 8, wherein the therapeutically effective
dose is injected trans-epicardially or transendocardially.
10. The method of claim 9, wherein with the trans-endocardial
approach a catheter-based approach is used.
11. The method of claim 4, wherein the administered hematopoietic
stem cells migrate into the damaged myocardium and/or myocardial
cells.
12. The method of claim 11, wherein the delivered hematopoietic
stem cells differentiate into one or more of the following types of
cells selected from the group consisting of: a. myocyctes; b.
smooth muscle cells; and c. endothelial cells.
13. The method of claim 12, wherein the differentiated
hematopoietic stem cells proliferate.
14. The method of claim 12, wherein the differentiated
hematopoietic stem cells assemble into myocardium and/or myocardial
cells.
15. The method of claim 12, wherein the differentiated
hematopoietic stem cells assemble into a coronary artery.
16. The method of claim 12, wherein the differentiated
hematopoietic stem cells assemble into an arteriole.
17. The method of claim 12, wherein the differentiated
hematopoietic stem cells assemble into a capillary.
18. The method of claim 12, wherein the differentiated
hematopoietic stem cells at least partially restore structural and
functional integrity to the damaged myocardium and/or myocardial
cells.
19. A pharmaceutical composition comprising a therapeutically
effective amount of hematopoietic stem cells.
20. The pharmaceutical composition of claim 19, wherein the
therapeutically effective dose of the hematopoietic stem cells is
2.times.10.sup.4-1.times.10.sup.5 cells.
Description
RELATED APPLICATIONS/PATENT & INCORPORATION BY REFERENCE
[0001] This application claims priority from Provisional U.S.
Patent Applications Serial Numbers 60/221,902 filed Jul. 31, 2000,
60/258,564 filed Dec. 29, 2000 and 60/258,805 filed Jan. 2, 2001,
and 60/295,807, 60/295,806, 60/295,805, 60/295,804, and 60/295,803
filed Jun. 5, 2001.
[0002] Each of the applications and patents cited in this text,
including each of the foregoing cited applications, as well as each
document or reference cited in each of the applications and patents
(including during the prosecution of each issued patent;
"application cited documents"), and each of the PCT and foreign
applications or patents corresponding to and/or claiming priority
from any of these applications and patents, and each of the
documents cited or referenced in each of the application cited
documents, are hereby expressly incorporated herein by reference.
More generally, various documents or references are cited in this
text, either in a Reference List before the claims or in the text
itself; and, each of the documents or references ("herein cited
documents") and all of the documents cited in this text (also
"herein cited documents"), as well as each document or reference
cited in each of the herein cited documents (including any
manufacturer's specifications, instructions, etc. for products
mentioned herein and in any document incorporated herein by
reference), is hereby expressly incorporated herein by reference.
There is no admission that any of the various documents cited in
this text are prior art as to the present invention. Any document
having as an author or inventor person or persons named as an
inventor herein is a document that is not by another as to the
inventive entity herein. Also, teachings of herein cited documents
and documents cited in herein cited documents and more generally in
all documents incorporated herein by reference can be employed in
the practice and utilities of the present invention.
FIELD OF THE INVENTION
[0005] The present invention relates generally to the field of
cardiology, and more particularly relates to methods and cellular
compositions for treatment of a patient suffering from a
cardiovascular disease, including, but not limited to,
atherosclerosis, ischemia, hypertension, restenosis, angina
pectoris, rheumatic heart disease, congenital cardiovascular
defects and arterial inflammation and other disease of the
arteries, arterioles and capillaries.
[0006] Moreover, the present invention relates to any one or more
of:
[0007] Methods and/or pharmaceutical composition comprising a
therapeutically effective amount of somatic stem cells alone or in
combination with a cytokine such as a cytokine selected from the
group consisting of stem cell factor (SCF), granulocyte-colony
stimulating factor (G-CSF), granulocyte-macrophage colony
stimulating factor (GM-CSF), stromal cell-derived factor-1, steel
factor, vascular endothelial growth factor, macrophage colony
stimulating factor, granulocyte-macrophage stimulating factor or
Interleukin-3 or any cytokine capable of the stimulating and/or
mobilizing stem cells. Cytokines may be administered alone or in
combination of with any other cytokine capable of: the stimulation
and/or mobilization of stem cells; the maintenance of early and
late hematopoiesis (see below); the activation of monocytes (see
below), macrophage/monocyte proliferation; differentiation,
motility and survival (see below) and a pharmaceutically acceptable
carrier, diluent or excipient (including combinations thereof). The
stem cells are advantageously adult stem cells, such as
hematopoietic or cardiac stem cells or a combination thereof or a
combination of cardiac stem cells and any other type of stem
cells.
[0008] The implanting, depositing, administering or causing of
implanting or depositing or administering of stem cells, such as
adult stem cells, for instance hematopoietic or cardiac stem cells
or a combination thereof or any combination of cardiac stem cells
(e.g., adult cardiac stem cells) and stem cells of another type of
(e.g., adult stem cells of another type), alone or with a cytokine
such as a cytokine selected from the group consisting of stem cell
factor (SCF), granulocyte-colony stimulating factor (G-CSF),
granulocyte-macrophage colony stimulating factor (GM-CSF), stromal
cell-derived factor-1, steel factor, vascular endothelial growth
factor, macrophage colony stimulating factor,
granulocyte-macrophage stimulating factor or Interleukin-3 or any
cytokine capable of the stimulating and/or mobilizing stem cells
(wherein "with a cytokine . . . " can include sequential
implanting, depositing administering or causing of implanting or
depositing or administering of the stem cells and the cytokine or
the co-implanting co-depositing or co-administering or causing of
co-implanting or co-depositing or co-administering or the
simultaneous implanting, depositing administering or causing of
implanting or depositing or administering of the stem cells and the
cytokine), in circulatory tissue or muscle tissue or circulatory
muscle tissue, e.g., cardiac tissue, such as the heart or blood
vessels--e.g., veins, arteries, that go to or come from the heart
such as veins and arteries directly connected or attached or
flowing into the heart, for instance the aorta. This implanting,
depositing, or administering or causing of implanting, depositing
or administering can be in conjunction with grafts. Such
implanting, depositing or administering or causing of implanting,
depositing or administering is advantageously employed in the
treatment or therapy or prevention of cardiac conditions, such as
to treat areas of weakness or scarring in the heart or prevent the
occurrence or further occurrence of such areas or to treat
conditions which cause or irritate such areas, for instance
myocardial infarction or ischemia or other e.g., genetic,
conditions that impart weakness or scarring to the heart (see also
cardiac conditions mentioned infra).
[0009] The use of such stem cells alone or in combination with said
cytokine(s), in the formulation of medicaments for such treatment,
therapy or prevention.
[0010] Medicaments for use in such treatment, therapy or prevention
comprising the stem cells and optionally the cytokine(s).
[0011] Kits comprising the stem cells and optionally the
cytokine(s) for formulations for use in such treatment, therapy or
prevention.
[0012] Compositions comprising such stem cells and optionally at
least one cytokine and kits for preparing such compositions.
[0013] Methods of making the kits and compositions described
herein.
[0014] Methods of implanting or depositing stem cells or causing
the implanting or depositing of stem cells.
BACKGROUND OF THE INVENTION
[0015] Cardiovascular disease is a major health risk throughout the
industrialized world. Atherosclerosis, the most prevalent of
cardiovascular diseases, is the principal cause of heart attack,
stroke, and gangrene of the extremities, and thereby the principal
cause of death in the United States. Atherosclerosis is a complex
disease involving many cell types and molecular factors (for a
detailed review, see Ross, 1993, Nature 362: 801-809).
[0016] Ischemia is a condition characterized by a lack of oxygen
supply in tissues of organs due to inadequate perfusion. Such
inadequate perfusion can have number of natural causes, including
atherosclerotic or restenotic lesions, anemia, or stroke, to name a
few. Many medical interventions, such as the interruption of the
flow of blood during bypass surgery, for example, also lead to
ischemia. In addition to sometimes being caused by diseased
cardiovascular tissue, ischemia may sometimes affect cardiovascular
tissue, such as in ischemic heart disease. Ischemia may occur in
any organ, however, that is suffering a lack of oxygen supply.
[0017] The most common cause of ischemia in the heart is myocardial
infarction (MI), commonly known as a heart attack, is one of the
most well-known types of cardiovascular disease. 1998 estimates
show 7.3 million people in the United States suffer from MI, with
over one million experiencing an MI in a given year (American Heart
Association, 2000). Of these individuals, 25% of men, and 38% of
females will die within a year of their first recognized MI
(American Heart Association, 2000). MI is caused by a sudden and
sustained lack of blood flow to an area of the heart, commonly
caused by narrowing of a coronary artery. Without adequate blood
supply, the tissue becomes ischemic, leading to the death of
myocytes and vascular structures. This area of necrotic tissue is
referred to as the infarct site, and will eventually become scar
tissue.
[0018] Current treatments for MI focus on reperfusion therapy,
which attempts to start the flow of blood to the affected area to
prevent the further loss of tissue. The main choices for
reperfusion therapy include the use of anti-thrombolytic agents, or
performing balloon angioplasty, or a coronary artery bypass graft.
Anti-thrombolytic agents solubilize blood clots that may be
blocking the artery, while balloon angioplasty threads a catheter
into the artery to the site of the occlusion, where the tip of the
catheter is inflated, pushing open the artery. Still more invasive
procedures include the bypass, where surgeons remove a section of a
vein from the patient, and use it to create a new artery in the
heart, which bypasses the blockage, and continues the supply of
blood to the affected area. In 1998, there were an estimated
553,000 coronary artery bypass graft surgeries and 539,000
percutaneous transluminal coronary angioplastys. These procedures
average $27,091 and $8,982 per patient, respectively (American
Heart Association, 2000).
[0019] These treatments may succeed in reestablishing the blood
supply, however tissue damage that occurred before the reperfusion
treatment began has been thought to be irreversible. For this
reason, eligible MI patients are started on reperfusion therapy as
soon as possible to limit the area of the infarct.
[0020] As such, most studies on MI have also focused on reducing
infarct size. There have been a few attempts to regenerate the
necrotic tissue by transplanting cardiomyocytes or skeletal
myoblasts (Leor et al., 1996; Murray, et al., 1996; Taylor, et al.,
1998; Tomita et al., 1999; Menasche et al., 2000). While the cells
may survive after transplantation, they fail to reconstitute
healthy myocardium and coronary vessels that are both functionally
and structurally sound.
[0021] All of the cells in the normal adult originate as precursor
cells which reside in various sections of the body. These cells, in
turn, derive from very immature cells, called progenitors, which
are assayed by their development into contiguous colonies of cells
in 1-3 week cultures in semisolid media such as methylcellulose or
agar. Progenitor cells themselves derive from a class of progenitor
cells called stem cells. Stem cells have the capacity, upon
division, for both self-renewal and differentiation into
progenitors. Thus, dividing stem cells generate both additional
primitive stem cells and somewhat more differentiated progenitor
cells. In addition to the well-known role of stem cells in the
development of blood cells, stem cells also give rise to cells
found in other tissues, including but not limited to the liver,
brain, and heart.
[0022] Stem cells have the ability to divide indefinitely, and to
specialize into specific types of cells. Totipotent stem cells,
which exist after an egg is fertilized and begins dividing, have
total potential, and are able to become any type of cell. Once the
cells have reached the blastula stage, the potential of the cells
has lessened, with the cells still able to develop into any cell
within the body, however they are unable to develop into the
support tissues needed for development of an embryo. The cells are
considered pluripotent, as they may still develop into many types
of cells. During development, these cells become more specialized,
committing to give rise to cells with a specific function. These
cells, considered multipotent, are found in human adults and
referred to as adult stem cells. It is well known that stem cells
are located in the bone marrow, and that there is a small amount of
peripheral blood stem cells that circulate throughout the blood
stream (National Institutes of Health, 2000).
[0023] Due to the regenerative properties of stem cells, they have
been considered an untapped resource for potential engineering of
tissues and organs. It would be an advance to provide uses of stem
cells with respect to addressing cardiac conditions.
[0024] Mention is made of:
[0025] U.S. Pat. No. 6,117,675 which relates to the differentiation
of retinal stem cells into retinal cells in vivo or in vitro, which
can be used as a therapy to restore vision.
[0026] U.S. Pat. No. 6,001,934 involving the development of
functional islets from islets of Langerhans stem cells.
[0027] U.S. Pat. Nos. 5,906,934 and 6,174,333 pertaining to the use
of mesenchymal stem cells for cartilage repair, and the use of
mesenchymal stem cells for regneration of ligaments; for instance,
wherein the stem cells are embedded in a gel matrix, which is
contracted and then implanted to replace the desired soft
tissue.
[0028] U.S. Pat. Nos. 6,099,832, and 6,110,459 involving grafts
with cell transplantation.
[0029] PCT Application Nos. PCT/US00/08353 (WO 00/57922) and
PCT/US99/17326 WO 00/06701) involving intramyocardial injection of
autologous bone marrow and mesenchymal stem cells which fails to
teach or suggest administering, implanting, depositing or the use
of hematopoietic stem cells as in the present invention, especially
as hematopoietic stem cells as in the present invention are
advantageously isolated and/or purified adult hematopoietic stem
cells.
[0030] Furthermore, at least certain of these patent documents fail
to teach or suggest the present invention for additional reasons.
The source of the stem cells of interest is limited to the known
precursors of the type of tissue for which regeneration is
required. Obtaining and purifying these specific cells can be
extremely difficult, as there are often very few stem cells in a
given tissue. In contrast, a benefit of the present invention
results from the ability of various lineages of stem cells to home
to the myocardium damage and differentiate into the appropriate
cell types--an approach that does not require that the stem cells
are recovered directly from myocardium, and, a variety of types of
stem cells may be used without compromising the functionality of
the regenerated tissue. And, other of these patent documents
utilize stem cells as the source of various chemical compositions,
without utilizing their proliferative capabilities, and thereby
fail to teach or suggest the invention.
[0031] Only recent literature has started to investigate the
potentials for stem cells to aid in the repair of tissues other
than that of known specialization. This plasticity of stem cells,
the ability to cross the border of germ layers, is a concept only
in its infancy (Kempermann et al, 2000, Temple, 2001). Kocher et al
(2001) discusses the use of adult bone marrow to induce
neovascularization after infarction as an alternative therapy for
left ventricle remodeling (reviewed in Rosenthal and Tsao, 2001).
Other studies have focused on coaxing specific types of stem cells
to differentiate into myocardial cells, i.e. liver stem cells as
shown in Malour et al (2001). Still other work focuses on the
possibilities of bone-marrow derived stem cells (Krause, et al.,
2001).
[0032] One of the oldest uses of stem cells in medicine is for the
treatment of cancer. In these treatments, bone marrow is
transplanted into a patient whose own marrow has been destroyed by
radiation, allowing the stem cells in the transplanted bone marrow
to produce new, healthy, white blood cells.
[0033] In these treatments, the stem cells are transplanted into
their normal environment, where they continue to function as
normal. Until recently, it was thought that any particular stem
cell line was only capable of producing three or four types of
cells, and as such, they were only utilized in treatments where the
stem cell was required to become one of the types of cells for
which their ability was already proven. Researchers are beginning
to explore other options for treatments of myriad disorders, where
the role of the stem cell is not well defined. Examples of such
work will be presented in support of the present invention.
[0034] Organ transplantation has been widely used to replace
diseased, nonfunctional tissue. More recently, cellular
transplantation to augment deficiencies in host tissue function has
emerged as a potential therapeutic paradigm. One example of this
approach is the well publicized use of fetal tissue in individuals
with Parkinsonism (reviewed in Tompson, 1992), where dopamine
secretion from transplanted cells alleviates the deficiency in
patients. In other studies, transplanted myoblasts from uneffected
siblings fused with endogenous myotubes in Duchenne's patients;
importantly the grafted myotubes expressed wild-type dystrophin
(Gussoni et al., 1992).
[0035] Despite their relevance in other areas, these earlier
studies do not describe any cellular transplantation technology
that can be successfully applied to the heart, where the ability to
replace damaged myocardium would have obvious clinical relevance.
Additionally, the use of intra-cardiac grafts to target the
long-term expression of angiogenic factors and ionotropic peptides
would be of therapeutic value for individuals with myocardial
ischemia or congestive heart failure, respectively.
[0036] In light of this background there is a need for the
improvement of myocardial regeneration technology in the heart.
Desirably, such technology would not only result in tissue
regeneration in the heart but also enable the delivery of useful
compositions directly to the heart. The present invention addresses
these needs.
[0037] It is therefore believed that heretofore the administration,
implanting, depositing, causing to be deposited, implanted or
administered of stem cells, alone or in combination with at least
one cytokine, as well as the use of such stem cells alone or in
combination with said cytokine(s), in the formulation of
medicaments for treatment, therapy or prevention, as in this
disclosure and as in the present invention, has not been taught, or
suggested in the art and that herein methods, compositions, kits
and uses are novel, nonobvious and inventive, i.e., that the
present invention has not been taught or suggested in the art and
that the present invention is novel, nonobvious and inventive.
OBJECT AND SUMMARY OF THE INVENTION
[0038] It has surprisingly been found that the implantation of
hematopoietic stem cells into the myocardium surrounding an infarct
following a myocardial infarction, migrate into the damaged area,
where they differentiate into myocytes, endothelial cells and
smooth muscle cells and then proliferate and form structures
including myocardium, coronary arteries, arterioles, and
capillaries, restoring the structural and functional integrity of
the infarct.
[0039] In this disclosure, "comprises," "comprising," "containing"
and "having" and the like can have the meaning ascribed to them in
U.S. patent law and can mean "includes," "including," and the like;
"consisting essentially of" or "consists essentially" likewise has
the meaning ascribed in U.S. patent law and the term is open-ended,
allowing for the presence of more than that which is recited so
long as basic or novel characteristics of that which is recited is
not changed by the presence of more than that which is recited, but
excludes prior art embodiments.
[0040] These and other embodiments are disclosed or are obvious
from and encompassed by, the following Detailed Description.
BRIEF DESCRIPTION OF FIGURES
[0041] The following Detailed Description, given to describe the
invention by way of example, but not intended to limit the
invention to specific embodiments described, may be understood in
conjunction with the accompanying Figures, incorporated herein by
reference, in which:
[0042] FIG. 1 shows a log-log plot showing Lin.sup.- bone marrow
cells from EGFP transgenic mice sorted by FACS based on c-kit
expression (The fraction of c-kit.sup.POS cells (upper gate) was
6.4%. c-kit.sup.NEG cells are shown in the lower gate.
c-kit.sup.POS cells were 1-2 logs brighter than c-kit.sup.NEG
cells)
[0043] FIG. 2A shows a photograph of a tissue section from a MI
induced mouse (The photograph shows the area of myocardial infarct
(MI) injected with Lin.sup.-c-kit.sup.POS cells from bone marrow
(arrows), the remaining viable myocardium (VM), and the
regenerating myocardium (arrowheads). Magnification is
12.times.);
[0044] FIG. 2B shows a photograph of the same tissue section of
FIG. 2A at a higher magnification, centering on the area of the MI
with magnification being 50.times.;
[0045] FIGS. 2C, D show photographs of a tissue section at low and
high magnifications of the area of MI, injected with
Lin.sup.-c-kit.sup.POS cells, with the magnification of 2C being
25.times., and the magnification of 2D being 50.times.;
[0046] FIG. 2E shows a photograph of a tissue section of the area
of MI injected with Lin.sup.-c-kit.sup.NEG cells wherein only
healing is apparent and the magnification is 50.times. (*Necrotic
myocytes. Red=cardiac myosin; green=PI labeling of nuclei);
[0047] FIGS. 3A-C show photographs of a section of tissue from a MI
induced mouse, showing the area of MI injected with
Lin.sup.-c-kit.sup.POS cells (Visible is a section of regenerating
myocardium from endocardium (EN) to epicardium (EP). All
photographs are labeled to show the presence of infarcted tissue in
the subendocardium (IT) and spared myocytes in the subendocardium
(SM). FIG. 3A is stained to show the presence of EGFP (green).
Magnification is 250.times.. FIG. 3B is stained to show the
presence of cardiac myosin (red). Magnification is 250.times.. FIG.
3C is stained to show the presence of both EGFP and myosin
(red-green), as well as PI-stained nuclei (blue). Magnification is
250.times.);
[0048] FIG. 4A shows of grafts depicting the effects of myocardial
infarction on left ventricular end-diastolic pressure (LVEDP),
developed pressure (LVDP), LV+rate of pressure rise (dP/dt), and
LV-rate of pressure decay (dP/dt) (From left to right, bars
indicate: sham-operated mice (SO, n--11); mice non-injected with
Lin.sup.-c-kit.sup.POS cells (MI, n=5 injected with
Lin.sup.-c-kit.sup.NEG cells; n=6 non-injected); mice injected with
Lin.sup.-c-kit.sup.POS cells (MI+BM, n=9). Error bars are the
standard deviation. *.dagger.p<0.05 vs SO and MI);
[0049] FIG. 4B shows a drawing of a proposed scheme for
Lin.sup.-c-kit.sup.POS cell differentiation in cardiac muscle and
functional implications;
[0050] FIGS. 5A-I show photographs of a tissue sections from a MI
induced mouse depicting regenerating myocardium in the area of the
MI which has been injected with Lin.sup.-c-kit.sup.POS cells (FIG.
5A is stained to show the presence of EGFP (green). Magnification
is 300.times.. FIG. 5B is stained to show the presence of
.alpha.-smooth muscle actin in arterioles (red). Magnification is
300.times.. FIG. 5C is stained to show the presence of both EGFP
and .alpha.-smooth muscle actin (yellow-red), as well as PI-stained
nuclei (blue). Magnification is 300.times.. FIGS. 5D-F and G-I
depict the presence of MEF2 and Csx/Nkx2.5 in cardiac myosin
positive cells. FIG. 5D shows PI-stained nuclei (blue).
Magnification is 300.times.. FIG. 5E is stained to show MEF2 and
Csx/Nkx2.5 labeling (green). Magnification is 300.times.. FIG. 5F
is stained to show cardiac myosin (red), as well as MEF2 or
Csx/Nkx2.5 with PI (bright fluorescence in nuclei). Magnification
is 300.times.. FIG. 5G shows PI-stained nuclei (blue).
Magnification is 300.times.. FIG. 5H is stained to show MEF2 and
Csx/Nkx2.5 labeling (green). Magnification is 300.times.. FIG. 5I
is stained to show cardiac myosin (red), as well as MEF2 or
Csx/Nkx2.5 with PI (bright fluorescence in nuclei). Magnification
is 300.times.);
[0051] FIG. 6 (FIGS. 6A-F) shows photographs of tissue sections
from MI induced mice, showing regenerating myocardium in the area
of the MI injected with Lin.sup.-c-kit.sup.POS cells (FIGS. 6A-C
show tissue which has been incubated in the presence of antibodies
to BrdU. FIG. 6A has been stained to show PI-labeled nuclei (blue).
Magnification is 900.times.. FIG. 6B has been stained to show BrdU-
and Ki67-labeled nuclei (green). Magnification is 900.times.. FIG.
6C has been stained to show the presence of .alpha.-sarcomeric
actin (red). Magnification is 900.times.. FIGS. 6D-F shows tissue
that has been incubated in the presence of antibodies to Ki67. FIG.
6D has been stained to show PI-labeled nuclei (blue). Magnification
is 500.times.. FIG. 6E has been stained to show BrdU- and
Ki67-labeled nuclei (green). Magnification is 500.times.. FIG. 6F
has been stained to show the presence of a-smooth muscle actin
(red). Magnification is 500.times.. Bright fluorescence:
combination of PI with BrdU (C) or Ki67 (F));
[0052] FIG. 7 (FIGS. 7A-C) shows photographs of tissue sections
from MI induced mice, showing the area of MI injected with
Lin.sup.-c-kit.sup.POS cells (Depicted are the border zone, viable
myocardium (VM) and the new band (NB) of myocardium separated by an
area of infarcted non-repairing tissue (arrows). FIG. 7A is stained
to show the presence of EGFP (green). Magnification is 280.times..
FIG. 7B is stained to show the presence of cardiac myosin (red).
Magnification is 280.times.. FIG. 7C is stained to show the
presence of both EGFP and myosin (red-green), as well as PI-stained
nuclei (blue). Magnification is 280.times.);
[0053] FIG. 8 (FIGS. 8A-F) shows photographs of tissue sections
from MI induced mice, showing regenerating myocardium in the area
of MI injected with Lin.sup.-c-kit.sup.POS cells (FIG. 8A is
stained to show the presence of EGFP (green). Magnification is
650.times.. FIG. 8B is stained to show the presence of cardiac
myosin (red). Magnification is 650.times.. FIG. 8C is stained to
show both the presence of EGFP and myosin (yellow), as well as
PI-stained nuclei (blue). Magnification is 650.times.. FIG. 8D is
stained to show the presence of EGFP (green). Magnification is
650.times.. FIG. 8E is stained to show the presence of
.alpha.-smooth muscle actin in arterioles (red). Magnification is
650.times.. FIG. 8F is stained to show the presence of both EGFP
and .alpha.-smooth muscle actin (yellow-red) as well as PI-stained
nuclei (blue). Magnification is 650.times.);
[0054] FIG. 9 (FIGS. 9A-C) shows photographs of tissue sections
from MI induced mice, showing the area of MI injected with
Lin.sup.-c-kit.sup.POS cells and showing regenerating myocardium
(arrowheads). (FIG. 9A is stained to show the presence of cardiac
myosin (red) Magnification is 400.times.. FIG. 9B is stained to
show the presence of the Y chromosome (green). Magnification is
400.times.. FIG. 9C is stained to show both the presence of the Y
chromosome (light blue) and PI-labeled nuclei (dark blue). Note the
lack of Y chromosome in infarcted tissue (IT) in subendocardium and
spared myocytes (SM) in subepicardium. Magnification is
400.times.);
[0055] FIG. 10 (FIGS. 10A-C) shows photographs of tissue sections
from MI induced mice, showing GATA-4 in cardiac myosin positive
cells (FIG. 10A shows PI-stained nuclei (blue). Magnification is
650.times.. FIG. 10B shows the presence of GATA-4 labeling (green).
Magnification is 650.times.. FIG. 10C is stained to show cardiac
myosin (red) in combination with GATA-4 and PI (bright fluorescence
in nuclei). Magnification is 650.times.);
[0056] FIG. 11 (FIGS. 11A-D) shows photograph of tissue sections
from a MI induced mouse (FIG. 11A shows the border zone between the
infarcted tissue and the surviving tissue. Magnification is
500.times.. FIG. 11B shows regenerating myocardium. Magnification
is 800.times.. FIG. 11C is stained to show the presence of connexin
43 (yellow-green), and the contacts between myocytes are shown by
arrows. Magnification is 800.times.. FIG. 11D is stained to show
both .alpha.-sarcomeric actin (red) and PI-stained nuclei (blue).
Magnification is 800.times.);
[0057] FIG. 12 (FIGS. 12A-B) shows photographs of tissue sections
from a MI induced mouse showing the area of MI that was injected
with Lin.sup.-c-kit.sup.POS cells and now shows regenerating
myocytes (FIG. 12A is stained to show the presence of cardiac
myosin (red) and PI-labeled nuclei (yellow-green). Magnification is
1,000. FIG. 12B is the same as FIG. 12A at a magnification of
700.times.);
[0058] FIGS. 13A-B show photographs of tissue sections from MI
induced mice (FIG. 13A shows a large infarct (MI) in a
cytokine-treated mouse with forming myocardium (arrowheads)
(Magnification is 50.times.) at higher magnification
(80.times.--adjacent panel). FIG. 13B shows a MI in a non-treated
mouse. Healing comprises the entire infarct (arrowheads)
(Magnification is 50.times.). Scarring is seen at higher
magnification (80.times.--adjacent panel). Red=cardiac myosin;
yellow-green=propidium iodide (PI) labeling of nuclei;
blue-magenta=collagen types I and III);
[0059] FIG. 13C shows a graph showing the mortality and myocardial
regeneration in treated and untreated MI induced mice
(Cytokine-treated infarcted mice, n=15; untreated infarcted mice,
n=52 Log-rank test: p<0.0001);
[0060] FIG. 14 shows a graph showing quantitative measurement of
infarct size (Total number of myocytes in the left ventricular free
wall (LVFW) of sham-operated (SO, n=9), infarcted non-treated (MI,
n=9) and cytokine-treated (MI-C, n=11) mice at sacrifice, 27 days
after infarction or sham operation. The percentage of myocytes lost
equals infarct size. X.+-.SD, *p<0.05 vs SO);
[0061] FIGS. 15A-C show graphs comparing aspects of myocardial
infarction, cardiac anatomy and function (FIGS. 15A-C depict LV
dimensions at sacrifice, 27 days after surgery; sham-operated (SO,
n=9), non-treated infarcted (MI, n=9) and cytokine-treated
infarcted (MI-C, n=10));
[0062] FIG. 15D shows EF by echocardiography; (SO, n=9; MI, n=9;
and MI-C, n=9);
[0063] FIGS. 15E-M show M-mode echocardiograms of SO (e-g), MI
(h-j) and MI-C (k-m) (Newly formed contracting myocardium
(arrows));
[0064] FIG. 15N shows a graph showing wall stress; SO (n=9), MI
(n=8) and MI-C (n=9) (Results are mean.+-.SD. *.sup.,**p<0.05 vs
SO and MI, respectively);
[0065] FIGS. 16A-G show grafts depicting aspects of myocardial
infarction, cardiac anatomy and ventricular function (FIGS. 16A-D
show echocardiographic LVESD (a), LVEDD (b), PWST (c) and PWDT (d)
in SO (n=9), MI (n=9) and MI-C (n=9). FIGS. 16E-G show mural
thickness (e), chamber diameter (f) and longitudinal axis (g)
measured anatomically at sacrifice in SO (n=9), MI (n=9) and MI-C
(n=10). ***p<0.05 vs SO and MI, respectively;
[0066] FIGS. 16H-P show two dimensional (2D) images and M-mode
tracings of SO (h-j), MI (k-m) and MI-C (n-p);
[0067] FIG. 17 (FIGS. 17A-D) shows graphs depicting aspects of
ventricular function (FIGS. 17A-D show LV hemodynamics in
anesthetized mice at sacrifice, 27 days after infarction or sham
operation; SO (n=9), MI (n=9) and MI-C (n=10). For symbols and
statistics, see also FIG. 13);
[0068] FIGS. 18A-E shows graphs of aspects of myocardial
regeneration (FIG. 18A classifies the cells in the tissue as
remaining viable (Re), lost (Lo) and newly formed (Fo) myocardium
in LVFW at 27 days in MI and MI-C; SO, myocardium without infarct.
FIG. 18B shows the amount of cellular hypertrophy in spared
myocardium. FIG. 18C shows cell proliferation in the regenerating
myocardium. Myocytes (M), EC and SMC labeled by BrdU and Ki67;
n=11. *.sup.,**p<0.05 vs M and EC. FIGS. 18D-E depict the
volume, number (n=11) and class distribution (bucket size, 100
.mu.m.sup.3; n=4,400) of myocytes within the formed myocardium;
[0069] FIGS. 18F-H show photographs of tissue sections from MI
induced mice depicting arterioles with TER-119 labeled erythrocyte
membrane (green fluorescence); blue fluorescence=PI staining of
nuclei; red fluorescence=.alpha.-smooth muscle actin in SMC (FIG.
18F is magnified at 800.times.. FIGS. 18G-H are magnified at
1,200.times.);
[0070] FIG. 19 (FIGS. 19A-D) shows photographs of tissue sections
from MI induced mice that were incubated with antibodies to Ki67
(A,B) and BrdU (C,D) (FIG. 19A shows labeling of myocytes by
cardiac myosin. Bright fluorescence of nuclei reflects the
combination of PI and Ki67. Magnification is 800.times.. FIG. 19B
shows labeling of SMC by a-smooth muscle actin. Bright fluorescence
of nuclei reflects the combination of PI and Ki67. Magnification is
1,200.times.. FIG. 19C shows labeling of SMC by .alpha.-smooth
muscle actin. Bright fluorescence of nuclei reflects the
combination of PI and BrdU. Magnification is 1,200.times.. FIG. 19D
shows labeling of EC in the forming myocardium by factor VIII.
Bright fluorescence of nuclei reflects the combination of PI and
BrdU. Magnification is 1,600.times.;
[0071] FIG. 20 (FIGS. 20A-F) shows photographs of tissue sections
from MI induced mice showing markers of differentiating cardiac
cells (FIG. 20A is stained to show labeling of myocytes by nestin
(yellow)). Red fluorescence indicates cardiac myosin. Magnification
is 1,200.times.. FIG. 20B is stained to show labeling of desmin
(red). Magnification is 800.times.. FIG. 20C is stained to show
labeling of connexin 43 (green). Red fluorescence indicates cardiac
myosin. Magnification is 1,400.times.. FIG. 20D shows VE-cadherin
and yellow-green fluorescence reflects labeling of EC by flk-1
(arrows). Magnification is 1,800.times.. FIG. 20E shows red
fluorescence indicating factor VIII in EC and and yellow-green
fluorescence reflects labeling of EC by flk-1 (arrows).
Magnification is 1,200.times.. FIG. 20F shows green fluorescence
labeling of SMC cytoplasms by flk-1 and endothelial lining labeled
by flk-1. Red fluorescence indicates .alpha.-smooth muscle actin.
Blue fluorescence indicates PI labeling of nuclei. Magnification is
800.times.; and
[0072] FIGS. 21A-C show tissue sections from MI induced mice (FIG.
21A uses bright fluorescence to depict the combination of PI
labeling of nuclei with Csx/Nkx2.5. Magnification is 1,400.times..
FIG. 21B uses bright fluorescence to depict the combination of PI
labeling of nuclei with GATA-4. Magnification is 1,200.times.. FIG.
21C uses bright fluorescence to depict the combination of PI
labeling of nuclei with MEF2. Magnification is 1,200.times. (Red
fluorescence shows cardiac myosin antibody staining and blue
fluorescence depicts PI labeling of nuclei. The fraction of myocyte
nuclei labeled by Csx/Nkx2.5, GATA-4 and MEF2 was 63.+-.5% (nuclei
sampled=2,790; n=11), 94.+-.9% (nuclei sampled=2,810; n=11) and
85.+-.14% (nuclei sampled=3,090; n=11), respectively).
DETAILED DESCRIPTION
[0073] The present invention provides methods and/or pharmaceutical
composition comprising a therapeutically effective amount of
hematopoietic stem cells
[0074] In a preferred aspect, the pharmaceutical composition of the
present invention is delivered via injection. These routes for
administration (delivery) include, but are not limited to
subcutaneous or parenteral including intravenous, intraarterial,
intramuscular, intraperitoneal, intramyocardial, transendocardial,
trans-epicardial, intranasal administration as well as intrathecal,
and infusion techniques. Hence, preferably the pharmaceutical
composition is in a form that is suitable for injection.
[0075] When administering a therapeutic of the present invention
parenterally, it will generally be formulated in a unit dosage
injectable form (solution, suspension, emulsion). The
pharmaceutical formulations suitable for injection include sterile
aqueous solutions or dispersions and sterile powders for
reconstitution into sterile injectable solutions or dispersions.
The carrier can be a solvent or dispersing medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, liquid polyethylene glycol, and the like), suitable
mixtures thereof, and vegetable oils.
[0076] Proper fluidity can be maintained, for example, by the use
of a coating such as lecithin, by the maintenance of the required
particle size in the case of dispersion and by the use of
surfactants. Nonaqueous vehicles such a cottonseed oil, sesame oil,
olive oil, soybean oil, corn oil, sunflower oil, or peanut oil and
esters, such as isopropyl myristate, may also be used as solvent
systems for compound compositions
[0077] Additionally, various additives which enhance the stability,
sterility, and isotonicity of the compositions, including
antimicrobial preservatives, antioxidants, chelating agents, and
buffers, can be added. Prevention of the action of microorganisms
can be ensured by various antibacterial and antifungal agents, for
example, parabens, chlorobutanol, phenol, sorbic acid, and the
like. In many cases, it will be desirable to include isotonic
agents, for example, sugars, sodium chloride, and the like.
Prolonged absorption of the injectable pharmaceutical form can be
brought about by the use of agents delaying absorption, for
example, aluminum monostearate and gelatin. According to the
present invention, however, any vehicle, diluent, or additive used
would have to be compatible with the compounds.
[0078] Sterile injectable solutions can be prepared by
incorporating the compounds utilized in practicing the present
invention in the required amount of the appropriate solvent with
various amounts of the other ingredients, as desired.
[0079] The pharmaceutical composition of the present invention,
e.g., comprising a therapeutic compound, can be administered to the
patient in an injectable formulation containing any compatible
carrier, such as various vehicles, adjuvants, additives, and
diluents; or the compounds utilized in the present invention can be
administered parenterally to the patient in the form of
slow-release subcutaneous implants or targeted delivery systems
such as monoclonal antibodies, iontophoretic, polymer matrices,
liposomes, and microspheres.
[0080] The pharmaceutical composition utilized in the present
invention can be administered orally to the patient. Conventional
methods such as administering the compounds in tablets,
suspensions, solutions, emulsions, capsules, powders, syrups and
the like are usable. Known techniques which deliver the compound
orally or intravenously and retain the biological activity are
preferred.
[0081] In one embodiment, a composition of the present invention
can be administered initially, and thereafter maintained by further
administration. For instance, a composition of the invention can be
administered in one type of composition and thereafter further
administered in a different or the same type of composition. For
example, a composition of the invention can be administered by
intravenous injection to bring blood levels to a suitable level.
The patient's levels are then maintained by an oral dosage form,
although other forms of administration, dependent upon the
patient's condition, can be used.
[0082] It is noted that humans are treated generally longer than
the mice or other experimental animals which treatment has a length
proportional to the length of the disease process and drug
effectiveness. The doses may be single doses or multiple doses over
a period of several days, but single doses are preferred. Thus, one
can scale up from animal experiments, e.g., rats, mice, and the
like, to humans, by techniques from this disclosure and documents
cited herein and the knowledge in the art, without undue
experimentation.
[0083] The treatment generally has a length proportional to the
length of the disease process and drug effectiveness and the
patient being treated.
[0084] The quantity of the pharmaceutical composition to be
administered will vary for the patient being treated. In a
preferred embodiment, 2.times.10.sup.4-1.times.10.sup.5 stem were
administered to the patient. While there would be an obvious size
difference between the hearts of a mouse and a human, it is
possible that 2.times.10.sup.4-1.times.10.sup.5 stem cells would be
sufficient in a human as well. However, the precise determination
of what would be considered an effective dose may be based on
factors individual to each patient, including their size, age, size
of the infarct, and amount of time since damage. Therefore, dosages
can be readily ascertained by those skilled in the art from this
disclosure and the knowledge in the art. Thus, the skilled artisan
can readily determine the amount of compound and optional
additives, vehicles, and/or carrier in compositions and to be
administered in methods of the invention. Typically, any additives
(in addition to the active stem cell(s)) are present in an amount
of 0.001 to 50 wt % solution in phosphate buffered saline, and the
active ingredient is present in the order of micrograms to
milligrams, such as about 0.0001 to about 5 wt %, preferably about
0.0001 to about 1 wt %, most preferably about 0.0001 to about 0.05
wt % or about 0.001 to about 20 wt %, preferably about 0.01 to
about 10 wt %, and most preferably about 0.05 to about 5 wt %. Of
course, for any composition to be administered to an animal or
human, and for any particular method of administration, it is
preferred to determine therefore: toxicity, such as by determining
the lethal dose (LD) and LD.sub.50 in a suitable animal model e.g.,
rodent such as mouse; and, the dosage of the composition(s),
concentration of components therein and timing of administering the
composition(s), which elicit a suitable response. Such
determinations do not require undue experimentation from the
knowledge of the skilled artisan, this disclosure and the documents
cited herein. And, the time for sequential administrations can be
ascertained without undue experimentation.
[0085] Examples of compositions comprising a therapeutic of the
invention include liquid preparations for orifice, e.g., oral,
nasal, anal, vaginal, peroral, intragastric, mucosal (e.g.,
perlingual, alveolar, gingival, olfactory or respiratory mucosa)
etc., administration such as suspensions, syrups or elixirs; and,
preparations for parenteral, subcutaneous, intradermal,
intramuscular or intravenous administration (e.g., injectable
administration), such as sterile suspensions or emulsions. Such
compositions may be in admixture with a suitable carrier, diluent,
or excipient such as sterile water, physiological saline, glucose
or the like. The compositions can also be lyophilized. The
compositions can contain auxiliary substances such as wetting or
emulsifying agents, pH buffering agents, gelling or viscosity
enhancing additives, preservatives, flavoring agents, colors, and
the like, depending upon the route of administration and the
preparation desired. Standard texts, such as "REMINGTON'S
PHARMACEUTICAL SCIENCE", 17th edition, 1985, incorporated herein by
reference, may be consulted to prepare suitable preparations,
without undue experimentation.
[0086] Compositions of the invention, are conveniently provided as
liquid preparations, e.g., isotonic aqueous solutions, suspensions,
emulsions or viscous compositions which may be buffered to a
selected pH. If digestive tract absorption is preferred,
compositions of the invention can be in the "solid" form of pills,
tablets, capsules, caplets and the like, including "solid"
preparations which are time-released or which have a liquid
filling, e.g., gelatin covered liquid, whereby the gelatin is
dissolved in the stomach for delivery to the gut. If nasal or
respiratory (mucosal) administration is desired, compositions may
be in a form and dispensed by a squeeze spray dispenser, pump
dispenser or aerosol dispenser. Aerosols are usually under pressure
by means of a hydrocarbon. Pump dispensers can preferably dispense
a metered dose or, a dose having a particular particle size.
[0087] Compositions of the invention can contain pharmaceutically
acceptable flavors and/or colors for rendering them more appealing,
especially if they are administered orally. The viscous
compositions may be in the form of gels, lotions, ointments, creams
and the like (e.g., for transdermal administration) and will
typically contain a sufficient amount of a thickening agent so that
the viscosity is from about 2500 to 6500 cps, although more viscous
compositions, even up to 10,000 cps may be employed. Viscous
compositions have a viscosity preferably of 2500 to 5000 cps, since
above that range they become more difficult to administer. However,
above that range, the compositions can approach solid or gelatin
forms which are then easily administered as a swallowed pill for
oral ingestion.
[0088] Liquid preparations are normally easier to prepare than
gels, other viscous compositions, and solid compositions.
Additionally, liquid compositions are somewhat more convenient to
administer, especially by injection or orally. Viscous
compositions, on the other hand, can be formulated within the
appropriate viscosity range to provide longer contact periods with
mucosa, such as the lining of the stomach or nasal mucosa.
[0089] Obviously, the choice of suitable carriers and other
additives will depend on the exact route of administration and the
nature of the particular dosage form, e.g., liquid dosage form
(e.g., whether the composition is to be formulated into a solution,
a suspension, gel or another liquid form), or solid dosage form
(e.g., whether the composition is to be formulated into a pill,
tablet, capsule, caplet, time release form or liquid-filled
form).
[0090] Solutions, suspensions and gels normally contain a major
amount of water (preferably purified water) in addition to the
active compound. Minor amounts of other ingredients such as pH
adjusters (e.g., a base such as NaOH), emulsifiers or dispersing
agents, buffering agents, preservatives, wetting agents, jelling
agents, (e.g., methylcellulose), colors and/or flavors may also be
present. The compositions can be isotonic, i.e., they can have the
same osmotic pressure as blood and lacrimal fluid.
[0091] The desired isotonicity of the compositions of this
invention may be accomplished using sodium chloride, or other
pharmaceutically acceptable agents such as dextrose, boric acid,
sodium tartrate, propylene glycol or other inorganic or organic
solutes. Sodium chloride is preferred particularly for buffers
containing sodium ions.
[0092] Viscosity of the compositions may be maintained at the
selected level using a pharmaceutically acceptable thickening
agent. Methylcellulose is preferred because it is readily and
economically available and is easy to work with. Other suitable
thickening agents include, for example, xanthan gum, carboxymethyl
cellulose, hydroxypropyl cellulose, carbomer, and the like. The
preferred concentration of the thickener will depend upon the agent
selected. The important point is to use an amount which will
achieve the selected viscosity. Viscous compositions are normally
prepared from solutions by the addition of such thickening
agents.
[0093] A pharmaceutically acceptable preservative can be employed
to increase the shelf-life of the compositions. Benzyl alcohol may
be suitable, although a variety of preservatives including, for
example, parabens, thimerosal, chlorobutanol, or benzalkonium
chloride may also be employed. A suitable concentration of the
preservative will be from 0.02% to 2% based on the total weight
although there may be appreciable variation depending upon the
agent selected.
[0094] Those skilled in the art will recognize that the components
of the compositions should be selected to be chemically inert with
respect to the active compound. This will present no problem to
those skilled in chemical and pharmaceutical principles, or
problems can be readily avoided by reference to standard texts or
by simple experiments (not involving undue experimentation), from
this disclosure and the documents cited herein.
[0095] The inventive compositions of this invention are prepared by
mixing the ingredients following generally accepted procedures. For
example the selected components may be simply mixed in a blender,
or other standard device to produce a concentrated mixture which
may then be adjusted to the final concentration and viscosity by
the addition of water or thickening agent and possibly a buffer to
control pH or an additional solute to control tonicity. Generally
the pH may be from about 3 to 7.5. Compositions can be administered
in dosages and by techniques well known to those skilled in the
medical and veterinary arts taking into consideration such factors
as the age, sex, weight, and condition of the particular patient,
and the composition form used for administration (e.g., solid vs.
liquid). Dosages for humans or other mammals can be determined
without undue experimentation by the skilled artisan, from this
disclosure, the documents cited herein, and the knowledge in the
art.
[0096] Suitable regimes for initial administration and further
doses or for sequential administrations also are variable, may
include an initial administration followed by subsequent
administrations; but nonetheless, may be ascertained by the skilled
artisan, from this disclosure, the documents cited herein, and the
knowledge in the art.
[0097] The pharmaceutical compositions of the present invention are
used to treat cardiovascular diseases, including, but not limited
to, atherosclerosis, ischemia, hypertension, restenosis, angina
pectoris, rheumatic heart disease, congenital cardiovascular
defects and arterial inflammation and other diseases of the
arteries, arterioles and capillaries or related complaint.
Accordingly, the invention involves the administration of stem
cells as herein discussed, alone or in combination with one or more
cytokine, as herein discussed, for the treatment or prevention of
any one or more of these conditions or other conditions involving
weakness in the heart, as well as compositions for such treatment
or prevention, use of stem cells as herein discussed, alone or in
combination with one or more cytokine, as herein discussed, for
formulating such compositions, and kits involving stem cells as
herein discussed, alone or in combination with one or more
cytokine, as herein discussed, for preparing such compositions
and/or for such treatment, or prevention. And, advantageous routes
of administration involves those best suited for treating these
conditions, such as via injection, including, but are not limited
to subcutaneous or parenteral including intravenous, intraarterial,
intramuscular, intraperitoneal, intramyocardial, transendocardial,
trans-epicardial, intranasal administration as well as intrathecal,
and infusion techniques.
[0098] The pharmaceutical compositions of the present invention may
be used as therapeutic agents--i.e. in therapy applications. As
herein, the terms "treatment" and "therapy" include curative
effects, alleviation effects, and prophylactic effects.
[0099] As used herein, "patient" may encompass any vertebrate
including but not limited to humans, mammals, reptiles, amphibians
and fish. However, advantageously, the patient is a mammal such as
a human, or an animal mammal such as a domesticated mammal, e.g.,
dog, cat, horse, and the like, or production mammal, e.g., cow,
sheep, pig, and the like.
[0100] As used herein "somatic stem cell" or "stem cell" or
"hematopoietic cell" refers to either autologous or allogenic stem
cells, which may be obtained from the bone marrow, peripheral
blood, or other source.
[0101] As used herein, "adult" stem cells refers to stem cells that
are not embryonic in origin nor derived from embryos or fetal
tissue.
[0102] As used herein "recently damaged myocardium" refers to
myocardium which has been damaged within one week of treatment
being started. In a preferred embodiment, the myocardium has been
damaged within three days of the start of treatment. In a further
preferred embodiment, the myocardium has been damaged within 12
hours of the start of treatment. It is advantageous to employ stem
cells alone or in combination with cytokine(s) as herein disclosed
to a recently damaged myocardium.
[0103] As used herein "damaged myocardium" refers to myocardial
cells which have been exposed to ischemic conditions. These
ischemic conditions may be caused by a myocardial infarction, or
other cardiovascular disease or related complaint. The lack of
oxygen causes the death of the cells in the surrounding area,
leaving an infarct, which will eventually scar.
[0104] As used herein, "home" refers to the attraction and
mobilization of somatic stem cells towards damaged myocardium
and/or myocardial cells.
[0105] As used herein, "assemble" refers to the assembly of
differentiated somatic stem cells into functional structures i.e.,
myocardium and/or myocardial cells, coronary arteries, arterioles,
and capillaries etc. This assembly provides functionality to the
differentiated myocardium and/or myocardial cells, coronary
arteries, arterioles and capillaries.
[0106] Thus, the invention involves the use of somatic stem cells.
These are present in animals in small amounts, but methods of
collecting stem cells are known to those skilled in the art.
[0107] In another aspect of the invention, the stem cells are
selected to be lineage negative. The term "lineage negative" is
known to one skilled in the art as meaning the cell does not
express antigens characteristic of specific cell lineages.
[0108] Advantageously, the lineage negative stem cells are selected
to be c-kit positive. The term "c-kit" is known to one skilled in
the art as being a receptor which is known to be present on the
surface of stem cells, and which is routinely utilized in the
process of identifying and separating stem cells from other
surrounding cells.
[0109] The invention further involves a therapeutically effective
dose or amount of stem cells applied to the heart. An effective
dose is an amount sufficient to effect a beneficial or desired
clinical result. Said dose could be administered in one or more
administrations. In the examples that follow,
2.times.10.sup.4-1.times.10.sup.5 stem cells were administered in
the mouse model. While there would be an obvious size difference
between the hearts of a mouse and a human, it is possible that this
range of stem cells would be sufficient in a human as well.
However, the precise determination of what would be considered an
effective dose may be based on factors individual to each patient,
including their size, age, size of the infarct, and amount of time
since damage. One skilled in the art, specifically a physician or
cardiologist, would be able to determine the number of stem cells
that would constitute an effective dose without undue
experimentation.
[0110] In another aspect of the invention, the stem cells are
delivered to the heart, specifically to the border area of the
infarct. As one skilled in the art would be aware, the infarcted
area is visible grossly, allowing this specific placement of stem
cells to be possible.
[0111] The stem cells are advantageously administered by injection,
specifically an intramyocardial injection. As one skilled in the
art would be aware, this is the preferred method of delivery for
stem cells as the heart is a functioning muscle. Injection of the
stem cells into the heart ensures that they will not be lost due to
the contracting movements of the heart.
[0112] In a further aspect of the invention, the stem cells are
administered by injection transendocardially or trans-epicardially.
This preferred embodiment allows the stem cells to penetrate the
protective surrounding membrane, necessitated by the embodiment in
which the cells are injected intramyocardially.
[0113] A preferred embodiment of the invention includes use of a
catheter-based approach to deliver the trans-endocardial injection.
The use of a catheter precludes more invasive methods of delivery
wherein the opening of the chest cavity would be necessitated. As
one skilled in the art is aware, optimum time of recovery would be
allowed by the more minimally invasive procedure, which as outlined
here, includes a catheter approach.
[0114] Further embodiments of the invention require the stem cells
to migrate into the infarcted region and differentiate into
myocytes, smooth muscle cells, and endothelial cells. It is known
in the art that these types of cells must be present to restore
both structural and functional integrity. Other approaches to
repairing infarcted or ischemic tissue have involved the
implantation of these cells directly into the heart, or as cultured
grafts, such as in U.S. Pat. Nos. 6,110,459, and 6,099,832.
[0115] Another embodiment of the invention includes the
proliferation of the differentiated cells and the formation of the
cells into cardiac structures including coronary arteries,
arterioles, capillaries, and myocardium. As one skilled in the art
is aware, all of these structures are essential for proper function
in the heart. It has been shown in the literature that implantation
of cells including endothelial cells and smooth muscle cells will
allow for the implanted cells to live within the infarcted region,
however they do not form the necessary structures to enable the
heart to regain full functionality. The ability to restore both
functional and structural integrity is yet another aspect of this
invention.
[0116] The restoration or some restoration of both functional and
structural integrity of cardiac tissue--advantageously over that
which has occurred previously--is yet another aspect of this
invention.
[0117] The present invention is additionally described by way of
the following, non-limiting examples, that provide a better
understanding of the present invention and of its many
advantages.
[0118] All of the materials, reagents, chemicals, assays,
cytokines, antibodies, and miscellaneous items referred to in the
following examples are readily available to the research community
through commercial suppliers, including but not limited to,
Genzyme, Invitrogen, Gibco BRL, Clonetics, Fisher Scientific,
R& D Systems, MBL International Corporation, CN Biosciences
Corporate, Sigma Aldrich, and CedarLane Laboratories, Limited.
[0119] For example,
[0120] stem cell factor is available under the name SCF (multiple
forms of recombinant human, recombinant mouse, and antibodies to
each), from R & D Systems (614 McKinley Place N.E.,
Minneapolis, Minn. 55413);
[0121] granulocyte-colony stimulating factor is available under the
name G-CSF (multiple forms of recombinant human, recombinant mouse,
and antibodies to each), from R & D Systems;
[0122] stem cell antibody-1 is available under the name SCA-1 from
MBL International Corporation (200 Dexter Avenue, Suite D,
Watertown, Mass. 02472);
[0123] multidrug resistant antibody is available under the name
Anti-MDR from CN Biosciences Corporate;
[0124] c-kit antibody is available under the name c-kit (Ab-1)
Polyclonal Antibody from CN Biosciences Corporate (Affiliate of
Merck KgaA, Darmstadt, Germany. Corporate headquarters located at
10394 Pacific Center Court, San Diego, Calif. 92121).
EXAMPLES
Example 1
Hematopoietic Stem Cell (HSC) Repair of Infarcted Myocardium
[0125] A. Harvesting of Hematopoietic Stem Cells
[0126] Bone marrow was harvested from the femurs and tibias of male
transgenic mice expressing enhanced green fluorescent protein
(EGFP). After surgical removal of the femurs and tibias, the muscle
was dissected and the upper and lower surface of the bone was cut
on the surface to allow the collecting buffer to infiltrate the
bone marrow. The fluid containing buffer and cells was collected in
tubes such as 1.5 ml Epindorf tubes. Bone marrow cells were
suspended in PBS containing 5% fetal calf serum (FCS) and incubated
on ice with rat anti-mouse monoclonal antibodies specific for the
following hematopoietic lineages: CD4 and CD8 (T-lymphocytes),
B-220 (B-lymphocytes), Mac-1 (macrophages), GR-1 (granulocytes)
(Caltag Laboratories) and TER-119 (erythrocytes) (Pharmingen).
Cells were then rinsed in PBS and incubated for 30 minutes with
magnetic beads coated with goat anti-rat immunoglobulin
(Polysciences Inc.). Lineage positive cells (Lin.sup.+) were
removed by a biomagnet and lineage negative cells (Lin.sup.-) were
stained with ACK-4-biotin (anti-c-kit mAb). Cells were rinsed in
PBS, stained with streptavidin-conjugated phycoerythrin (SA-PE)
(Caltag Labs.) and sorted by fluorescence activated cell sorting
(FACS) using a FACSVantage instrument (Becton Dickinson).
Excitation of EGFP and ACK-4-biotin-SA-EP occurred at a wavelength
of 488 run. The Lin.sup.- cells were sorted as c-kit positive
(c-kit.sup.POS) and c-kit negative (c-kit.sup.NEG) with a 1-2 log
difference in staining intensity (FIG. 1). The c-kit.sup.POS cells
were suspended at 2.times.10.sup.4 to 1.times.10.sup.5 cells in 5
.mu.l of PBS and the c-kit.sup.NEG cells were suspended at a
concentration of 1.times.10.sup.5 in 5 .mu.l of PBS.
[0127] B. Induction of Myocardial Infarction in Mice
[0128] Myocardial infarction was induced in female C57BL/6 mice at
2 months of age as described by Li et al. (1997). Three to five
hours after infarction, the thorax of the mice was reopened and 2.5
.mu.l of PBS containing Lin.sup.-c-kit.sup.POS cells were injected
in the anterior and posterior aspects of the viable myocardium
bordering the infarct (FIG. 2). Infarcted mice, left uninjected or
injected with Lin.sup.-c-kit-.sup.NEG cells, and sham-operated mice
i.e., mice where the chest cavity was opened but no infarction was
induced, were used as controls. All animals were sacrificed 9.+-.2
days after surgery. Protocols were approved by institutional review
board. Results are presented as mean.+-.SD. Significance between
two measurements was determined by the Student's t test, and in
multiple comparisons was evaluated by the Bonferroni method
(Scholzen and Gerdes, 2000). P<0.05 was considered
significant.
[0129] Injection of male Lin.sup.-c-kit.sup.POS bone marrow cells
in the peri-infarcted left ventricle of female mice resulted in
myocardial regeneration. The peri-infarcted region is the region of
viable myocardium bordering the infarct. Repair was obtained in 12
of 30 mice (40%). Failure to reconstitute infarcts was attributed
to the difficulty of transplanting cells into tissue contracting at
600 beats per minute (bpm). However, an immunologic reaction to the
histocompatibility antigen on the Y chromosome of the donor bone
marrow cells could account for the lack of repair in some of the
female recipients. Closely packed myocytes occupied 68.+-.11% of
the infarcted region and extended from the anterior to the
posterior aspect of the ventricle (FIGS. 2A-2D). New myocytes were
not found in mice injected with Lin.sup.-c-kit.sup.NEG cells (FIG.
2E).
[0130] C. Determination of Ventricular Function
[0131] Mice were anesthetized with chloral hydrate (400 mg/kg body
weight, i.p.), and the right carotid artery was cannulated with a
microtip pressure transducer (model SPR-671, Millar) for the
measurements of left ventricular (LV) pressures and LV+ and -dP/dt
in the closed-chest preparation to determine whether developing
myocytes derived from the HSC transplant had an impact on function.
Infarcted mice non-injected or injected with Lin.sup.-c-kit.sup.NEG
cells were combined in the statistics. In comparison with
sham-operated groups, the infarcted groups exhibited indices of
cardiac failure (FIG. 3). In mice treated with Lin-c-kit.sup.POS
cells, LV end-diastolic pressure (LVEDP) was 36% lower, and
developed pressure (LVDP) and LV+ and -dP/dt were 32%, 40%, and 41%
higher, respectively (FIG. 4A).
[0132] D. Determination of Cell Proliferation and EGFP
Detection
[0133] The abdominal aorta was cannulated, the heart was arrested
in diastole by injection of cadmium chloride (CdCl.sub.2), and the
myocardium was perfused retrogradely with 10% buffered formalin.
Three tissue sections, from the base to the apex of the left
ventricle, were stained with hematoxylin and eosin. At 9.+-.2 days
after coronary occlusion, the infarcted portion of the ventricle
was easily identifiable grossly and histologically (see FIG. 2A).
The lengths of the endocardial and epicardial surfaces delimiting
the infarcted region, and the endocardium and epicardium of the
entire left ventricle were measured in each section. Subsequently,
their quotients were computed to yield the average infarct size in
each case. This was accomplished at 4.times. magnification
utilizing an image analyzer connected to a microscope. The fraction
of endocardial and epicardial circumference delimiting the
infarcted area (Pfeffer and Braunwald, 1990; Li et al., 1997) did
not differ in untreated mice, 78.+-.18% (n=8) and in mice treated
with Lin.sup.-c-kit.sup.POS cells (n=12), 75.+-.14% or
Lin.sup.-c-kit.sup.NEG cells (n=11), 75.+-.15%.
[0134] To establish whether Lin.sup.-c-kit.sup.POS cells resulted
in myocardial regeneration, BrdU (50 mg/kg body weight, i.p.) was
administered daily to the animals for 4-5 consecutive days before
sacrifice to determine cumulative cell division during active
growth. Sections were incubated with anti-BrdU antibody and BrdU
labeling of cardiac cell nuclei in the S phase was measured.
Moreover, expression of Ki67 in nuclei (Ki67 is expressed in
cycling cells in GI, S, G2, and early mitosis) was evaluated by
treating samples with a rabbit polyclonal anti-mouse Ki67 antibody
(Dako Corp.). FITC-conjugated goat anti-rabbit IgG was used as
secondary antibody. (FIGS. 5 and 6). EGFP was detected with a
rabbit polyclonal anti-GFP (Molecular Probes). Myocytes were
recognized with a mouse monoclonal anti-cardiac myosin heavy chain
(MAB 1548; Chemicon) or a mouse monoclonal anti-.alpha.-sarcomeric
actin (clone SC5; Sigma), endothelial cells with a rabbit
polyclonal anti-human factor VIII (Sigma) and smooth muscle cells
with a mouse monoclonal anti-.alpha.-smooth muscle actin (clone
1A4; Sigma). Nuclei were stained with propidium iodide (PI), 10
.mu.g/ml. The percentages of myocyte (M), endothelial cell (EC) and
smooth muscle cell (SMC) nuclei labeled by BrdU and Ki67 were
obtained by confocal microscopy. This was accomplished by dividing
the number of nuclei labeled by the total number of nuclei
examined. Number of nuclei sampled in each cell population was as
follows; BrdU labeling: M=2,908; EC=2,153; SMC=4,877. Ki67
labeling: M=3,771; EC=4,051; SMC=4,752. Number of cells counted for
EGFP labeling: M=3,278; EC=2,056; SMC=1,274. The percentage of
myocytes in the regenerating myocardium was determined by
delineating the area occupied by cardiac myosin stained cells
divided by the total area represented by the infarcted region in
each case. Myocyte proliferation was 93% (p<0.001) and 60%
(p<0.001) higher than in endothelial cells, and 225% (p<0.001
and 176% (p<0.001) higher than smooth muscle cells, when
measured by BrdU and Ki67, respectively.
[0135] The origin of the cells in the forming myocardium was
determined by the expression of EGFP (FIGS. 7 and 8). EGFP
expression was restricted to the cytoplasm and the Y chromosome to
nuclei of new cardiac cells. EGFP was combined with labeling of
proteins specific for myocytes, endothelial cells and smooth muscle
cells. This allowed the identification of each cardiac cell type
and the recognition of endothelial cells and smooth muscle cells
organized in coronary vessels (FIGS. 5, 7, and 8). The percentage
of new myocytes, endothelial cells and smooth muscle cells that
expressed EGFP was 53.+-.9% (n=7), 44.+-.6% (n=7) and 49.+-.7%
(n=7), respectively. These values were consistent with the fraction
of transplanted Lin.sup.-c-kit.sup.POS bone marrow cells that
expressed EGFP, 44.+-.10% (n=6). An average 54.+-.8% (n=6) of
myocytes, endothelial cells and smooth muscle cells expressed EGFP
in the heart of donor transgenic mice.
[0136] E. Detection of the Y-Chromosome
[0137] For the fluorescence in situ hybridization (FISH) assay,
sections were exposed to a denaturing solution containing 70%
formamide. After dehydration with ethanol, sections were hybridized
with the DNA probe CEP Y (satellite III) Spectrum Green (Vysis) for
3 hours. Nuclei were stained with PI.
[0138] Y-chromosomes were not detected in cells from the surviving
portion of the ventricle. However, the Y-chromosome was detected in
the newly formed myocytes, indicating their origin as from the
injected bone marrow cells (FIG. 9).
[0139] F. Detection of Transription Factors and Connexin 43
[0140] Sections were incubated with rabbit polyclonal anti-MEF2
(C-21; Santa Cruz), rabbit polyclonal anti-GATA-4 (H-112; Santa
Cruz), rabbit polyclonal anti-Csx/Nkx2.5 (obtained from Dr. Izumo)
and rabbit polyclonal anti-connexin 43 (Sigma). FITC-conjugated
goat anti-rabbit IgG (Sigma) was used as secondary antibody.
[0141] To confirm that newly formed myocytes represented maturing
cells aiming at functional competence, the expression of the
myocyte enhancer factor 2 (MEF2), the cardiac specific
transcription factor GATA-4 and the early marker of myocyte
development Csx/Nkx2.5 was examined. In the heart, MEF2 proteins
are recruited by GATA-4 to synergistically activate the promoters
of several cardiac genes such as myosin light chain, troponin T,
troponin I, .alpha.-myosin heavy chain, desmin, atrial natriuretic
factor and .alpha.-actin (Durocher et al., 1997; Morin et al.,
2000). Csx/Nkx2.5 is a transcription factor restricted to the
initial phases of myocyte differentiation (Durocher et al., 1997).
In the reconstituting heart, all nuclei of cardiac myosin labeled
cells expressed MEF2 (FIGS. 7D-7F) and GATA-4 (FIG. 10), but only
40.+-.19% expressed Csx/Nkx2.5 (FIGS. 7G-7I). To characterize
further the properties of these myocytes, the expression of
connexin 43 was determined. This protein is responsible for
intercellular connections and electrical coupling through the
generation of plasma membrane channels between myocytes (Beardsle
et al., 1998; Musil et al., 2000); connexin 43 was apparent in the
cell cytoplasm and at the surface of closely aligned
differentiating cells (FIGS. 11A-11D). These results were
consistent with the expected functional competence of the heart
muscle phenotype. Additionally, myocytes at various stages of
maturation were detected within the same and different bands (FIG.
12).
Example 2
Mobilization of Bone Marrow Cells to Repair Infarcted
Myocardium
[0142] A. Myocardial Infarction and Cytokines.
[0143] Fifteen C57BL/6 male mice at 2 months of age were
splenectomized and 2 weeks later were injected subcutaneously with
recombinant rat stem cell factor (SCF), 200 .mu.g/kg/day, and
recombinant human granulocyte colony stimulating factor (G-CSF), 50
.mu.g/kg/day (Amgen), once a day for 5 days (Bodine et al., 1994;
Orlic et al., 1993). Under ether anesthesia, the left ventricle
(LV) was exposed and the coronary artery was ligated (Orlic et al.,
2001; Li et al., 1997; Li et al., 1999). SCF and G-CSF were given
for 3 more days. Controls consisted of splenectomized infarcted and
sham-operated (SO) mice injected with saline. BrdU, 50 mg/kg body
weight, was given once a day, for 13 days, before sacrifice; mice
were killed at 27 days. Protocols were approved by New York Medical
College. Results are mean.+-.SD. Significance was determined by the
Student's t test and Bonferroni method (Li et al., 1999). Mortality
was computed with log-rank test. P<0.05 was significant.
[0144] Given the ability of bone marrow Lin.sup.-c-kit.sup.POS
cells to transdifferentiate into the cardiogenic lineage (Orlic et
al., 2001), a protocol was used to maximize their number in the
peripheral circulation in order to increase the probability of
their homing to the region of dead myocardium. In normal animals,
the frequency of Lin.sup.-c-kit.sup.POS cells in the blood is only
a small fraction of similar cells present in the bone marrow
(Bodine et al., 1994; Orlic et al., 1993). As documented
previously, the cytokine treatment used here promotes a marked
increase of Lin.sup.-c-kit.sup.POS cells in the bone marrow and a
redistribution of these cells from the bone marrow to the
peripheral blood. This protocol leads to a 250-fold increase in
Lin.sup.-c-kit.sup.POS cells in the circulation (Bodine et al.,
1994; Orlic et al., 1993).
[0145] In the current study, BMC mobilization by SCF and G-CSF
resulted in a dramatic increase in survival of infarcted mice; with
cytokine treatment, 73% of mice (11 of 15) survived 27 days, while
mortality was very high in untreated infarcted mice (FIG. 13A). A
large number of animals in this group died from 3 to 6 days after
myocardial infarction (MI) and only 17% (9 of 52) reached 27 days
(p<0.001). Mice that died within 48 hours post-MI were not
included in the mortality curve to minimize the influence of the
surgical trauma. Infarct size was similar in the cytokine-,
64.+-.11% (n=11), and saline-, 62.+-.9% (n=9), injected animals as
measured by the number of myocytes lost in the left ventricular
free wall (LVFW) at 27 days (FIG. 14).
[0146] Importantly, bone marrow cell mobilization promoted
myocardial regeneration in all 11 cytokine-treated infarcted mice,
sacrificed 27 days after surgery (FIG. 13B). Myocardial growth
within the infarct was also seen in the 4 mice that died
prematurely at day 6 (n=2) and at day 9 (n=2). Cardiac repair was
characterized by a band of newly formed myocardium occupying most
of the damaged area. The developing tissue extended from the border
zone to the inside of the injured region and from the endocardium
to the epicardium of the LVFW. In the absence of cytokines,
myocardial replacement was never observed and healing with scar
formation was apparent (FIG. 13C). Conversely, only small areas of
collagen accumulation were detected in treated mice.
[0147] B. Detection of BMC Mobilization by Echocardiography and
Hemodynamics.
[0148] Echocardiography was performed in conscious mice using a
Sequoia 256c (Acuson) equipped with a 13-MHz linear transducer
(15L8). The anterior chest area was shaved and two dimensional (2D)
images and M-mode tracings were recorded from the parasternal short
axis view at the level of papillary muscles. From M-mode tracings,
anatomical parameters in diastole and systole were obtained
(Pollick et al., 1995). Ejection fraction (EF) was derived from LV
cross sectional area in 2D short axis view (Pollick et al., 1995):
EF=[(LVDA-LVSA)/LVDA]*100 where LVDA and LVSA correspond to LV
areas in diastole and in systole. Mice were anesthetized with
chloral hydrate (400 mg/kg body weight, ip) and a microtip pressure
transducer (SPR-671, Millar) connected to a chart recorder was
advanced into the LV for the evaluation of pressures and + and
-dP/dt in the closed-chest preparation (Orlic et al., 2001; Li et
al., 1997; Li et al., 1999).
[0149] EF was 48%, 62% and 114% higher in treated than in
non-treated mice at 9, 16 and 26 days after coronary occlusion,
respectively (FIG. 15D). In mice exposed to cytokines, contractile
function developed with time in the infarcted region of the wall
(FIGS. 15E-M; FIGS. 16H-P, www.pnas.org). Conversely, LV
end-diastolic pressure (LVEDP) increased 76% more in non-treated
mice. The changes in LV systolic pressure (not shown), developed
pressure (LVDP), + and -dP/dt were also more severe in the absence
of cytokine treatment (FIGS. 17A-D). Additionally, the increase in
diastolic stress in the zone bordering and remote from infarction
was 69-73% lower in cytokine-treated mice (FIG. 15N). Therefore,
cytokine-mediated infarct repair restored a noticeable level of
contraction in the regenerating myocardium, decreasing diastolic
wall stress and increasing ventricular performance. Myocardial
regeneration attenuated cavitary dilation and mural thinning during
the evolution of the infarcted heart in vivo.
[0150] Echocardiographically, LV end-systolic (LVESD) and
end-diastolic (LVEDD) diameters increased more in non-treated than
in cytokine-treated mice, at 9, 16 and 26 days after infarction
(FIGS. 16A-B). Infarction prevented the evaluation of systolic
(AWST) and diastolic (AWDT) anterior wall thickness. When
measurable, the posterior wall thickness in systole (PWST) and
diastole (PWDT) was greater in treated mice (FIGS. 16C-D).
Anatomically, the wall bordering and remote from infarction was 26%
and 22% thicker in cytokine-injected mice (FIG. 16E). BMC-induced
repair resulted in a 42% higher wall thickness-to-chamber radius
ratio (FIG. 15A). Additionally, tissue regeneration decreased the
expansion in cavitary diameter, -14%, longitudinal axis, -5% (FIGS.
16F-G), and chamber volume, -26% (FIG. 15B). Importantly,
ventricular mass-to-chamber volume ratio was 36% higher in treated
animals (FIG. 15C). Therefore, BMC mobilization that led to
proliferation and differentiation of a new population of myocytes
and vascular structures attenuated the anatomical variables which
define cardiac decompensation.
[0151] C. Cardiac Anatomy and Determination of Infarct Size.
[0152] Following hemodynamic measurements, the abdominal aorta was
cannulated, the heart was arrested in diastole with CdCl.sub.2 and
the myocardium was perfused with 10% formalin. The LV chamber was
filled with fixative at a pressure equal to the in vivo measured
end-diastolic pressure (Li et al., 1997; Li et al., 1999). The LV
intracavitary axis was measured and three transverse slices from
the base, mid-region and apex were embedded in paraffin. The
mid-section was used to measure LV thickness, chamber diameter and
volume (Li et al., 1997; Li et al., 1999). Infarct size was
determined by the number of myocytes lost from the LVFW (Olivetti
et al., 1991; Beltrami et al., 1994).
[0153] To quantify the contribution of the developing band to the
ventricular mass, firstly the volume of the LVFW (weight divided by
1.06 g/ml) was determined in each group of mice. The data was
56.+-.2 mm.sup.3 in sham operated (SO), 62.+-.4 mm.sup.3 (viable
FW=41.+-.3; infarcted FW=21.+-.4) in infarcted non-treated animals,
and 56.+-.9 mm.sup.3 (viable FW=37.+-.8; infarcted FW=19.+-.5) in
infarcted cytokine-treated mice. These values were compared to the
expected values of spared [EXPLAIN] and lost myocardium at 27 days,
given the size of the infarct in the non-treated and
cytokine-treated animals. From the volume of the LVFW (56 mm.sup.3)
in SO and infarct size in non-treated, 62%, and treated, 64%, mice,
it was possible to calculate the volume of myocardium destined to
remain (non-treated=21 mm.sup.3; treated=20 mm.sup.3) and destined
to be lost (non-treated=35 mm.sup.3; treated=36 mm.sup.3) 27 days
after coronary occlusion (FIG. 18A). The volume of newly formed
myocardium was detected exclusively in cytokine-treated mice and
found to be 14 mm.sup.3 (FIG. 18A). Thus, the repair band reduced
infarct size from 64% (36 mm.sup.3/56 mm.sup.3=64%) to 39% [(36
mm.sup.3-14 mm.sup.3)/56 mm.sup.3=39%]. Since the spared portion of
the LVFW at 27 days was 41 and 37 mm.sup.3 in non-treated and
treated mice (see above), the remaining myocardium, shown in FIG.
18.alpha., underwent 95% (p<0.001) and 85% (p<0.001)
hypertrophy, respectively. Consistently, myocyte cell volume
increased 94% and 77% (FIG. 18B).
[0154] D. Determination the Total Volume of Formed Myocardium
[0155] The volume of regenerating myocardium was determined by
measuring in each of three sections the area occupied by the
restored tissue and section thickness. The product of these two
variables yielded the volume of tissue repair in each section.
Values in the three sections were added and the total volume of
formed myocardium was obtained. Additionally, the volume of 400
myocytes was measured in each heart. Sections were stained with
desmin and laminin antibodies and propidium iodide (PI). Only
longitudinally oriented cells with centrally located nuclei were
included. The length and diameter across the nucleus were collected
in each myocyte to compute cell volume, assuming a cylindrical
shape (Olivetti et al., 1991; Beltrami et al., 1994). Myocytes were
divided in classes and the number of myocytes in each class was
calculated from the quotient of total myocyte class volume and
average cell volume (Kajstura et al., 1995; Reiss et al., 1996).
Number of arteriole and capillary profiles per unit area of
myocardium was measured as previously done (Olivetti et al., 1991;
Beltrami et al., 1994).
[0156] Sections were incubated with BrdU or Ki67 antibody. Myocytes
(M) were recognized with a mouse monoclonal anti-cardiac myosin,
endothelial cells (EC) with a rabbit polyclonal anti-factor VIII
and smooth muscle cells (SMC) with a mouse monoclonal
anti-.alpha.-smooth muscle actin myosin. The fractions of M, EC and
SMC nuclei labeled by BrdU and Ki67 were obtained by confocal
microscopy (Orlic et al., 2001). Nuclei sampled in 11
cytokine-treated mice; BrdU: M=3,541; EC=2,604; SMC=1,824. Ki67:
M=3,096; EC=2,465; SMC=1,404.
[0157] BrdU was injected daily between days 14 to 26 to measure the
cumulative extent of cell proliferation while Ki67 was assayed to
determine the number of cycling cells at sacrifice. Ki67 identifies
cells in G1, S, G2, prophase and metaphase, decreasing in anaphase
and telophase (Orlic et al., 2001). The percentages of BrdU and
Ki67 positive myocytes were 1.6- and 1.4-fold higher than EC, and
2.8- and 2.2-fold higher than SMC, respectively (FIGS. 18C, 19).
The forming myocardium occupied 76.+-.11% of the infarct; myocytes
constituted 61.+-.12%, new vessels 12.+-.5% and other components
3.+-.2%. The band contained 15.times.10.sup.6 regenerating myocytes
that were in an active growing phase and had a wide size
distribution (FIGS. 18D-E). EC and SMC growth resulted in the
formation of 15.+-.5 arterioles and 348.+-.82 capillaries per
mm.sup.2 of new myocardium. Thick wall arterioles with several
layers of SMC and luminal diameters of 10-30 .mu.m represented
vessels in early differentiation. At times, incomplete perfusion of
the coronary branches within the repairing myocardium during the
fixation procedure led to arterioles and capillaries containing
erythrocytes (FIGS. 18F-H). These results provided evidence that
the new vessels were functionally competent and connected with the
coronary circulation. Therefore, tissue repair reduced infarct size
and myocyte growth exceeded angiogenesis; muscle mass replacement
was the prevailing feature of the infarcted heart.
[0158] E. Determination of Cell Differentiation
[0159] Cytoplasmic and nuclear markers were used. Myocyte nuclei:
rabbit polyclonal Csx/Nkx2.5, MEF2, and GATA4 antibodies (Orlic et
al., 2001; Lin et al., 1997; Kasahara et al., 1998); cytoplasm:
mouse monoclonal nestin (Kachinsky et al., 1995), rabbit polyclonal
desmin (Hermann and Aebi, 1998), cardiac myosin, mouse monoclonal
.alpha.-sarcomeric actin and rabbit polyclonal connexin 43
antibodies (Orlic et al., 2001). EC cytoplasm: mouse monoclonal
flk-1, VE-cadherin and factor VIII antibodies (Orlic et al., 2001;
Yamaguchi et al., 1993; Breier et al., 1996). SMC cytoplasm: flk-1
and .alpha.-smooth muscle actin antibodies (Orlic et al., 2001;
Couper et al., 1997). Scar was detected by a mixture of collagen
type I and type III antibodies.
[0160] Five cytoplasmic proteins were identified to establish the
state of differentiation of myocytes (Orlic et al., 2001; Kachinsky
et al., 1995; Hermann and Aebi, 1998): nestin, desmin,
.alpha.-sarcomeric actin, cardiac myosin and connexin 43. Nestin
was recognized in individual cells scattered across the forming
band (FIG. 20A). With this exception, all other myocytes expressed
desmin (FIG. 20B), .alpha.-sarcomeric actin, cardiac myosin and
connexin 43 (FIG. 20C). Three transcription factors implicated in
the activation of the promoter of several cardiac muscle structural
genes were examined (Orlic et al., 2001; Lin et al., 1997; Kasahara
et al., 1998): Csx/Nkx2.5, GATA-4 and MEF2 (FIGS. 21A-C). Single
cells positive for flk-1 and VE-cadherin (Yamaguchi et al., 1993;
Breier et al., 1996), two EC markers, were present in the repairing
tissue (FIGS. 20D,E); flk-1 was detected in SMC isolated or within
the arteriolar wall (FIG. 20F). This tyrosine kinase receptor
promotes migration of SMC during angiogenesis (Couper et al.,
1997). Therefore, repair of the infarcted heart involved growth and
differentiation of all cardiac cell populations resulting in de
novo myocardium.
Example 3
Migration of Primitive Cardiac Cells in the Adult Mouse Heart
[0161] To determine whether a population of primitive cells was
present in the adult ventricular myocardium and whether these cells
possessed the ability to migrate, three major growth factors were
utilized as chemoattractants: hepatocyte growth factor (HGF), stem
cell factor (SCF) and granulocyte monocyte colony stimulating
factor (GM-CSF). SCF and GMCSF were selected because they have been
shown to promote translocation of herriatopoietic stem cells.
Although HGF induces migration of hematopoietic stem cells, this
growth factor is largely implicated in mitosis, differentiation and
migration of cardiac cell precursors during early cardiogenesis. On
this basis, enzymatically dissociated cells from the mouse heart
were separated according to their size. Methods for dissociating
cardiac cells from heart tissue are well-known to those skilled in
the art and therefore would not involve undue experimentation (Cf
U.S. Pat. No. 6,255,292 which is herein incorporated by reference
in its entirety) A homogenous population of the dissociated cardiac
cells containing small undifferentiated cells, 5-7 .mu.m in
diameter, with a high nucleus to cytoplasm ratio were subjected to
migration assay in Boyden microchambers characterized by
gelatin-coated filters containing pores, 5 .mu.m (Boyden et al.,
1962, J. Exptl. Med. 115:453-456)
[0162] No major differences in the dose-response curve of migrated
cells in the presence of the three growth factors were detected.
However, HGF appeared to mobilize a larger number of cells at a
concentration of 100 ng/ml. In addition, the cells that showed a
chemotactic response to HGF consisted of 15% of c-kit positive
(c-kit.sup.POS) cells, 50% of multidrug resistance-1 (MDR-1)
labeled cells and 30% of stem cell antigen-1 (Sca-1) expressing
cells. When the mobilized cells were cultured in 15% fetal bovine
serum, they differentiated into myocytes, endothelial cells, smooth
muscle cells and fibroblasts. Cardiac myosin positive myocytes
constituted 50% of the preparation, while factor VIII labeled cells
included 15%, alpha-smooth muscle actin stained cells 4%, and
vimentin positive factor VIII negative fibroblasts 20%. The
remaining cells were small undifferentiated and did not stain with
these four antibodies. In conclusion, the mouse heart possesses
primitive cells which are mobilized by growth factors. HGF
translocates cells that in vitro differentiate into the four
cardiac cell lineages.
Example 4
Cardiac C-Kit Positive Cells Proliferate In Vitro and Generate New
Myocardium Vivo
[0163] To determine whether primitive c-kit.sup.POS cells were
present in senescent Fischer 344 rats, dissociated cardiac cells
were exposed to magnetic beads coated with c-kit receptor antibody
(ACK-4-biotin, anti-c-kit mAb). Following separation, these small
undifferentiated cells were cultured in 10% fetal calf serum. Cells
attached in a few days and began to proliferate at one week.
Confluence was reached at 7-10 days. Doubling time, established at
passage P2 and P4, required 30 and 40 hours, respectively. Cells
grew up to P18 (90th generation) without reaching senescence.
Replicative capacity was established by Ki67 labeling: at P2,
88.+-.14% of the cells contained Ki67 protein in nuclei. Additional
measurements were obtained between P1 and P4; 40% of cells
expressed alpha-sarcomeric actin or cardiac myosin, 13% desmin, 3%
alpha-smooth muscle actin, 15% factor VIIII or CD31, and 18%
nestin. Under these in vitro conditions, cells showed no clear
myofibrillar organization with properly aligned sarcomeres and
spontaneous contraction was never observed. Similarly, Ang II,
norepinephrine, isoprotererol, mechanical stretch and electrical
field stimulation failed to initiate contractile function. On this
basis, it was decided to evaluate whether these cells pertaining to
the myogenic, smooth muscle cell and endothelial cell lineages had
lost permanently their biological properties or their role could be
reestablished in vivo. Following BrdU labeling of cells at P2,
infarcted Fischer 344 rats were injected with these BrdU positive
cells in the damaged region, 3-5 hours after coronary artery
occlusion. Two weeks later, animals were sacrificed and the
characteristics of the infarcted area were examined. Myocytes
containing parallel arranged myofibrils along their longitudinal
axis were recognized, in combination with BrdU labeling of nuclei.
Moreover, vascular structures comprising arterioles and capillary
profiles were present and were also positive to BrdU. In
conclusion, primitive c-kit positive cells reside in the senescent
heart and maintain the ability to proliferate and differentiate
into parenchymal cells and coronary vessels when implanted into
injured functionally depressed myocardium.
Example 5
Cardiac Stem Cells Mediate Myocyte Replication in the Young and
Senescent Rat Heart
[0164] The heart is not a post-mitotic organ but contains a
subpopulation of myocytes that physiologically undergo cell
division to replace dying cells. Myocyte multiplication is enhanced
during pathologic overloads to expand the muscle mass and maintain
cardiac performance. However, the origin of these replicating
myocytes remains to be identified. Therefore, primitive cells with
characteristics of stem/progenitor cells were searched for in the
myocardium of of Fischer 344 rats. Young and old animals were
studied to determine whether aging had an impact on the size
population of stem cells and dividing myocytes. The numbers of
c-kit and MDR1 positive cells in rats at 4 months were 11.+-.3, and
18.+-.6/100 mm.sup.2 of tissue, respectively. Values in rats at 27
months were 35.+-.10, and 42.+-.13/100 mm.sup.2. A number of newly
generated small myocytes were identified that were still c-kit or
MDR1 positive. Ki67 protein, which is expressed in nuclei of
cycling cells was detected in 1.3.+-.0.3% and 4.1.+-.1.5% of
myocytes at 4 and 27 months, respectively. BrdU localization
following 6 or 56 injections included 1.0.+-.0.4% and 4.4.+-.1.2%
at 4 months, and 4.0.+-.1.5% and 16.+-.4% at 27 months. The mitotic
index measured in tissue sections showed that the fraction of
myocyte nuclei in mitosis comprised 82.+-.28/10.sup.6 and
485.+-.98/10.sup.6 at 4 and 27 months, respectively. These
determinations were confirmed in dissociated myocytes to obtain a
cellular mitotic index. By this approach, it was possible to
establish that all nuclei of multinucleated myocytes were in
mitosis simultaneously. This information could not be obtained in
tissue sections. The collected values showed that 95.+-.31/10.sup.6
myocytes were dividing at 4 months and 620.+-.98/10.sup.6 at 27
months. At both age intervals, the formation of the mitotic
spindle, contractile ring, disassembly of the nuclear envelope,
karyokinesis and cytokinesis were documented. In conclusion,
primitive undifferentiated cells reside in the adult heart and
their increase with age is paralleled by an increase in the number
of myocytes entering the cell cycle and undergoing karyokinesis and
cytokinesis. This relationship suggests that cardiac stem cells may
regulate the level and fate of myocyte growth in the aging
heart.
Example 6
Chimerism of the Human Heart and the Role of Stem Cells
[0165] The critical role played by resident primitive cells in the
remodeling of the injured heart is well appreciated when organ
chimerism, associated with transplantation of a female heart in a
male recipient, is considered. For this purpose, 8 female hearts
implanted in male hosts were analyzed. Translocation of male cells
to the grafted female heart was identified by FISH for Y chromosome
(see Example 1E). By this approach, the percentages of myocytes,
coronary arterioles and capillary profiles labeled by Y chromosome
were 9%, 14% and 7%, respectively. Concurrently, the numbers of
undifferentiated c-kit and multidrug resistance-1 (MDR1) positive
cells in the implanted female hearts were measured. Additionally,
the possibility that these cells contained the Y chromosome was
established. Cardiac transplantation involves the preservation of
portions of the atria of the recipient on which the donor heart
with part of its own atria is attached. This surgical procedure is
critical for understanding whether the atria from the host and
donor contained undifferentiated cells that may contribute to the
complex remodeling process of the implanted heart. Quantitatively,
the values of c-kit and, MDR1 labeled cells were very low in
control non-transplanted hearts: 3 c-kit and 5 MDR1/100 mm.sup.2 of
left ventricular myocardium. In contrast, the numbers of c-kit and
MDR1 cells in the atria of the recipient were 15 and 42/100
mm.sup.2. Corresponding values in the atria of the donor were 15
and 52/100 mm.sup.2 and in the ventricle 11 and 21/100 mm.sup.2.
Transplantation was characterized by a marked increase in primitive
undifferentiated cells in the heart. Stem cells in the atria of the
host contained Y chromosome, while an average of 55% and 63% of
c-kit and MDRI cells in the donor's atria and ventricle,
respectively, expressed the Y chromosome. All c-kit and MDRI
positive cells were negative for CD45. These observations suggest
that the translocation of male cells to the implanted heart has a
major impact on the restructuring of the donor myocardium. In
conclusion, stem cells are widely distributed in the adult heart
and because of their plasticity and migration capacity generate
myocytes, coronary arterioles and capillary structures with high
degree of differentiation.
Example 7
Identification and Localization of Stem Cells In The Adult Mouse
Heart
[0166] Turnover of myocytes occurs in the normal heart, and
myocardial damage leads to activation of myocyte proliferation and
vascular growth. These adaptations raise the possibility that
multipotent primitive cells are present in the heart and are
implicated in the physiological replacement of dying myocytes and
in the cellular growth response following injury. On this basis,
the presence of undifferentiated cells in the normal mouse heart
was determined utilizing surface markers including c-kit, which is
the receptor for stem cell factor, multidrug resistance-1 (MDR1),
which is a P-glycoprotein capable of extruding from the cell dyes,
toxic substances and drugs, and stem cell antigen-1 (Sca-1), which
is involved in cell signaling and cell adhesion. Four separate
regions consisting of the left and right atria, and the base,
mid-section and apical portion of the ventricle were analyzed. From
the higher to the lower value, the number of c-kit positive cells
was 26.+-.11, 15.+-.5, 10.+-.7 and 6.+-.3/100 mm.sup.2 in the
atria, and apex, base and mid-section of the ventricle,
respectively. In comparison with the base and mid-section, the
larger fraction of c-kit positive cells in the atria and apex was
statistically significant. The number of MDR1 positive cells was
higher than those expressing c-kit, but followed a similar
localization pattern; 43.+-.14, 29.+-.16, 14.+-.7 and 12.+-.10/100
mm.sup.2 in the atria, apex, base and mid-section. Again, the
values in the atria and apex were greater than in the other two
areas. Sca-1 labeled cells showed the highest value; 150.+-.136/100
mm.sup.2 positive cells were found in the atria. Cells positive for
c-kit, MDR1 and Sca-1 were negative for CD45, and for myocyte,
endothelial cell, smooth muscle cell and fibroblast cytoplasmic
proteins. Additionally, the number of cells positive to both c-kit
and MDR1 was measured to recognize cells that possessed two stem
cell markers. In the entire heart, 36% of c-kit labeled cells
expressed MDR1 and 19% of MDR1 cells had also c-kit. In conclusion,
stem cells are distributed throughout the mouse heart, but tend to
accumulate in the regions at low stress, such as the atria and the
apex.
[0167] Having thus described in detail preferred embodiments of the
present invention, it is to be understood that the invention
defined by the appended claims is not to be limited by particular
details set forth in the above description as many apparent
variations thereof are possible without departing from the spirit
or scope thereof.
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