U.S. patent application number 13/210955 was filed with the patent office on 2011-12-08 for methods for enhancing yield of stem cell cultures and enhancing stem cell therapy.
This patent application is currently assigned to CEDARS-SINAI MEDICAL CENTER. Invention is credited to Tao-Sheng Li, Eduardo Marban.
Application Number | 20110300112 13/210955 |
Document ID | / |
Family ID | 44858543 |
Filed Date | 2011-12-08 |
United States Patent
Application |
20110300112 |
Kind Code |
A1 |
Marban; Eduardo ; et
al. |
December 8, 2011 |
METHODS FOR ENHANCING YIELD OF STEM CELL CULTURES AND ENHANCING
STEM CELL THERAPY
Abstract
The present application relates to methods and compositions for
the generation of therapeutic cells having reduced incidence of
karyotypic abnormalities. In several embodiments cardiac stem cells
are cultured in an antioxidant-supplemented media that reduces
levels of reactive oxygen species, but does not down regulate DNA
repair mechanisms. In several embodiments, physiological oxygen
concentrations are used during culture in order to increase the
proliferation of stem cells, decrease the senescence of the cells,
decrease genomic instability, and/or augment the functionality of
such cells for cellular therapies.
Inventors: |
Marban; Eduardo; (Beverly
HIlls, CA) ; Li; Tao-Sheng; (Nanchang, CN) |
Assignee: |
CEDARS-SINAI MEDICAL CENTER
Los Angeles
CA
|
Family ID: |
44858543 |
Appl. No.: |
13/210955 |
Filed: |
August 16, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13096931 |
Apr 28, 2011 |
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13210955 |
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61330251 |
Apr 30, 2010 |
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Current U.S.
Class: |
424/93.7 ;
435/325; 435/375 |
Current CPC
Class: |
C12N 5/0662 20130101;
C12N 2500/38 20130101; A61P 9/00 20180101; C12N 5/0657 20130101;
C12N 2500/02 20130101; C12N 2501/998 20130101 |
Class at
Publication: |
424/93.7 ;
435/325; 435/375 |
International
Class: |
A61K 35/12 20060101
A61K035/12; A61P 9/00 20060101 A61P009/00; C12N 5/077 20100101
C12N005/077 |
Goverment Interests
STATEMENT REGARDING GOVERNMENT SPONSORED GRANT
[0002] This invention was made with Government support under the
Research Project Grant (R01HL083109) by the National Institutes of
Health. The United States Government has certain rights in this
invention.
Claims
1. A method of increasing the yield of cardiac stem cells in
culture, comprising: obtaining a population of cardiac stem cells
isolated from a source of cardiac tissue; restricting oxygen
concentrations in a culture environment to physiologic oxygen
concentrations, wherein said physiologic oxygen concentrations are
between about 4% and 7%; culturing said cardiac stem cells in said
restricted oxygen culture environment; wherein said physiologic
levels of oxygen increase the rate at which the cardiac stem cells
proliferate, thereby increasing the yield of cardiac stem cells as
compared to culture conditions that employ non-physiologic levels
of oxygen, wherein the yield of cardiac stem cells is increased per
unit weight of said source of cardiac tissue as compared to the
yield of cardiac stem cells cultured in conditions that employ
non-physiologic concentrations of oxygen, and wherein the per unit
weight yield is increased by at least about 5% for a given time
period of culturing.
2. The method of claim 1, wherein the per unit weight yield is
increased by at least about 20% for a given time period of
culturing.
3. The method of claim 1, wherein the increased yield reduces the
amount the amount of time that the cardiac stem cells are cultured
in order to reach a certain population as compared to the amount of
time cardiac stem cells are cultured in non-physiologic
concentrations of oxygen in order to reach said certain
population.
4. The method of claim 3, wherein the increased yield reduces the
amount the amount of time required for culturing by 20%.
5. The method of claim 3, wherein the increased yield reduces the
amount the amount of time required for culturing by 50%.
6. The method of claim 1, wherein said culturing in physiologic
oxygen concentrations reduces the incidence of karyotypic
abnormalities in the cultured cardiac stem cells as compared to
cardiac stem cells cultured in non-physiologic concentrations of
oxygen.
7. The method of claim 6, wherein said culturing in physiologic
oxygen concentrations reduces the incidence of aneuploidy in the
cultured cardiac stem cells as compared to cardiac stem cells
cultured in non-physiologic concentrations of oxygen.
8. The method of claim 1, wherein the cardiac stem cells comprise
cardiospheres, cardiosphere derived cells, or a subsequent
generation of cardiospheres.
9. The method of claim 1, wherein the cardiac stem cells are
suitable for administration of a subject having damaged or diseased
cardiac tissue.
10. The method of claim 9, wherein the tissue from which the
cultured cardiac stem cells were isolated is allogeneic with
respect to said subject.
11. The method of claim 9, wherein the tissue from which the
cultured cardiac stem cells were isolated is autologous with
respect to said subject.
12. The method of claim 9, wherein administration of said cultured
cardiac stem cells results in increased engraftment into the
cardiac tissue of said subject as compared to engraftment of
cardiac stem cells cultured in non-physiologic concentrations of
oxygen.
13. The method of claim 9, wherein administration of said cultured
cardiac stem cells results in one or more of increased myocardial
viability, increased wall thickness, and lower left ventricular
volume in the cardiac tissue of said subject as compared to that
resulting from administration of cardiac stem cells cultured in
non-physiologic concentrations of oxygen.
14. The method of claim 1, further comprising administering at
least a portion of said said population of cardiac stem cells to a
subject in need of cardiac stem cell therapy due to damaged or
diseased cardiac tissue, wherein said administered cardiac stem
cells engraft into the cardiac tissue of said subject to a greater
degree than cardiac stem cells expanded in non-physiologic
concentrations of oxygen, wherein said administered cardiac stem
cells survive in the cardiac tissue of said subject to a greater
degree than cardiac stem cells expanded in non-physiologic
concentrations of oxygen, and wherein said greater degree of
engraftment and survival lead to a greater increase in the cardiac
function of said subject as compared to the increase associated
with administration of cardiac stem cells expanded in
non-physiologic concentrations of oxygen.
15. The method of claim 14, wherein said population of cardiac stem
cells is allogeneic with respect to said subject.
16. The method of claim 14, wherein said cardiac stem cells are
cardiosphere-derived cells (CDCs).
17. The method of claim 14, wherein said cardiac function is left
ventricular ejection fraction and is increased by at least 5% as
compared to increased cardiac function due to administration of
cardiac stem cells expanded in non-physiologic concentrations of
oxygen.
18. A method of increasing the yield of cardiac stem cells in
culture, comprising: obtaining a population of cardiac stem cells
isolated from a source of cardiac tissue; restricting oxygen
concentrations in a culture environment to physiologic oxygen
concentrations, wherein said physiologic oxygen concentrations are
between about 1% and 8%; culturing said cardiac stem cells in said
restricted oxygen culture environment; wherein said physiologic
levels of oxygen increase the rate at which the cardiac stem cells
proliferate, thereby increasing the yield of cardiac stem cells as
compared to culture conditions that employ non-physiologic levels
of oxygen.
19. The method of claim 18, wherein the increased yield reduces the
amount the amount of time required for culturing by 20%.
20. The method of claim 18, wherein said culturing in physiologic
oxygen concentrations reduces the incidence of aneuploidy in the
cultured cardiac stem cells as compared to cardiac stem cells
cultured in non-physiologic concentrations of oxygen.
Description
RELATED CASES
[0001] This application is a continuation of U.S. application Ser.
No. 13/096,931, filed on Apr. 28, 2011, which claims the benefit of
U.S. Provisional Application No. 61/330,251 filed on Apr. 30, 2010,
the contents of which is expressly incorporated by reference
herein.
BACKGROUND
[0003] 1. Field of the Invention
[0004] The present application relates generally to methods and
compositions for generating genomically stable stem cells for the
repair or regeneration of damaged cells or tissue. For example, in
several embodiments the methods and compositions disclosed herein
may be used for the repair and/or regeneration of cardiac tissue.
In particular, isolated cardiac cells are cultured in oxygen
concentrations and/or in the presence of antioxidant compositions
that maintain an optimal balance between reduced oxidative-stress
induced DNA damage and functional DNA repair systems, thereby
reducing genomic instability (e.g., DNA damage or karyotypic
abnormalities) in the cultured cells.
[0005] 2. Description of the Related Art
[0006] The scope of human disease that involves loss of or damage
to cells is vast and includes, but is not limited to
neurodegenerative disease, endocrine diseases, cancers, and
cardiovascular disease. For example, coronary heart disease is
presently the leading cause of death in the United States, taking
more than 650,000 lives annually. According to the American Heart
Association, 1.2 million people suffer from a heart attack (or
myocardial infarction, MI) every year in America. Of those who
survive a first MI, many (25% of men and 38% of women survivors)
will still die within one year of the MI. Currently, 16 million
Americans are MI survivors or suffer from angina (chest pain due to
coronary heart disease). Coronary heart disease can deteriorate
into heart failure for many patients. 5 million Americans are
currently suffering from heart failure, with 550,000 new diagnoses
each year. Regardless of the etiology of their conditions, many of
those suffering from coronary heart disease or heart failure have
suffered permanent heart tissue damage, which often leads to a
reduced quality of life.
SUMMARY
[0007] Cell therapy, the introduction of new cells into a tissue in
order to treat a disease, represents a possible method for
repairing or replacing diseased tissue with healthy tissue.
However, cells generated for cell therapies have the potential to
develop genomic abnormalities when being processed for regenerative
therapies. Such abnormalities could lead to reduced efficacy of the
cell therapy or to neoplastic development at the target tissue.
Accordingly, it is highly desirable to provide methods and
compositions for generating cells for cellular therapy that have
enhanced genomic stability.
[0008] In several embodiments, there is provided a method for
reducing the incidence of karyotypic abnormalities in cardiac stem
cells for use in the repair or regeneration of cardiac tissue,
comprising isolating cardiac stem cells and culturing the isolated
stem cells in a culture media supplemented with an antioxidant
composition. In several embodiments, the cells are isolated from
healthy mammalian non-embryonic cardiac tissue, and then
cultured.
[0009] In several embodiments there is provided a composition for
reducing the incidence of karyotypic abnormalities in cultured
cardiac stem cells, the composition comprising at least one peptide
antioxidant, at least one non-peptide antioxidant; and a culture
media suitable for culturing cardiac stem cells, wherein the
culture media is supplemented with the at least one peptide
antioxidant and the at least one non-peptide antioxidant.
[0010] In several embodiments, there is provided a composition for
reducing the incidence of karyotypic abnormalities in cultured
cells, the composition comprising a culture media suitable for
culturing cells, and at least one antioxidant.
[0011] In several embodiments the at least one antioxidant is
present in a concentration ranging from about 0.1 to 200 .mu.M, and
functions to reduce reactive oxygen species (ROS) to a level which
decreases oxidative-stress induced DNA damage in the cultured
cells. In several embodiments, the antioxidant composition
comprises at least one peptide antioxidant and at least one
non-peptide antioxidant. Although the concentrations of the peptide
antioxidant(s) and non-peptide antioxidant(s) may vary according to
the embodiment (e.g.,. based on the cell type, the age of the
source tissue, or other factors), in several embodiments the at
least one peptide antioxidant and the at least one non-peptide
antioxidant are present in an individual or combined concentration
ranging from about 0.1 to 200 .mu.M. In several embodiments, the
antioxidant composition is suitable for reducing formation of ROS
such as peroxides and free radicals, among others. In some
embodiments, the reduced formation of ROS results in a level of ROS
which decreases oxidative-stress induced DNA damage, yet the
resulting ROS levels are not so low that markers of DNA repair
mechanisms are significantly reduced, and further the reduction
does not significantly induce markers of DNA damage in the cardiac
stem cells. Thus, in several embodiments, the balance of reduced
ROS generation, non-reduced DNA repair mechanisms and non-induction
of DNA damage reduces the overall incidence of karyotypic
abnormalities in the cardiac stem cells. In several embodiments,
this is particularly advantageous, as karyotypic abnormalities
reduce the percentage of usable cells in a pool of cells to be used
in cell therapy, but also present the risk of unwanted neoplastic
growth (teratoma formation). Thus, in several embodiments the
methods and compositions disclosed herein yield a greater number of
cells suitable for cell therapy (either in total number or based on
a percent of useable cells) and yield cells that are safer for use
in cell therapies. In several embodiments the markers of DNA repair
mechanisms comprise one or more DNA repair enzymes selected from
the group consisting of: ATM, ATR, Rad50, Rad51, Chk1, and Chk2 and
in several embodiments the markers of DNA damage comprise one or
more of .gamma.-H2AX foci in cultured cells, .gamma.-H2AX mRNA, or
.gamma.-H2AX protein. Other markers of DNA repair or DNA damage are
evaluated in other embodiments (e.g., phosphorylation of H2AX,
7-hydro-8-oxo-2'-deoxyguanosine concentrations). As a result of
culturing cells in the culture conditions and compositions provided
in several embodiments, ROS levels are reduced by at least 10%. In
other embodiments, greater reductions in ROS are achieved (e.g., at
least 15%, at least 20%, or more). In still additional embodiments,
a balance between reduced ROS generation, non-reduced DNA repair
mechanisms and non-induction of DNA damaged is not required to
realize reductions in karyotypic abnormalities. For example, in
some embodiments, alterations of one or more of the
above-referenced characteristics is sufficient to yield a reduction
in karyotypic abnormalities.
[0012] In several embodiments, the at least one peptide antioxidant
is present in a concentration ranging from about 0.1 to 200 .mu.M.
In several embodiments, the at least one peptide antioxidant
comprises glutathione. In some embodiments, glutathione can be
supplemented by including glutathione precursors in the culture
media (for example n-acetylcysteine, s-adenosylmethionine or whey
protein). As such, the cultured cells not only have the glutathione
in the media available, but have precursor compounds present to
increase the amount of glutathione production in response to
changing culture conditions.
[0013] Similarly, in several embodiments the at least one
non-peptide antioxidant is present in a concentration ranging from
about 0.1 to 200 .mu.M. In several embodiments the at least one
non-peptide antioxidant is selected from the group consisting of
thiols, vitamins and polyphenols. In some embodiments, these
non-peptide antioxidants function to terminate chain oxidation
reactions which are responsible, at least in part, for generation
of free radicals. In some embodiments, these antioxidants
"sacrifice" themselves by being oxidized, thereby sparing the
genetic material of the cell from damage. In several embodiments
vitamins are used as the non-peptide antioxidant, and may comprise
one or more vitamins selected from the group consisting of: vitamin
A, vitamin E, and vitamin C. In several embodiments the various
vitamins are present in an individual or total concentration
ranging from about 1 to 150 .mu.M. In still additional embodiments,
other antioxidants may be used to supplement culture media. For
example, enzymes such as catalase, superoxide dismutase and various
peroxidases are used in some embodiments.
[0014] Other concentrations are used in some embodiments, depending
on the cell type, the age of the cells, the time the cells have
been in culture, the passage number of the cell population. The
various components of the antioxidant composition may, in some
embodiments, be balanced to advantageously tailor the composition
to a particular set of characteristics possessed by a particular
cell population. For example, in some embodiments, an high passage
cell population may benefit from a greater concentration of a
peptide antioxidant as compared to a non-peptide antioxidant (or
vice versa).
[0015] In several embodiments, cells are cultured with the
antioxidant composition for about 24 hours. In some embodiments,
shorter culture times are used (e.g., about 4-6 hours, about 5-10
hours, about 8-16 hours, about 16 to 20 hours, and overlapping
ranges thereof). In some embodiments, longer culture times are used
(e.g., about 24-36 hours, about 36-48 hours, about 48-72 hours, or
longer). In some embodiments, time can be varied depending on a
variety of factors. For example, the age of the source tissue may
be a factor in determining how long a cell needs to be cultured for
the antioxidant composition to be effective. The overall metabolic
status of the cells may also be an important variable. Active cells
may more effectively metabolize the antioxidant compositions,
thereby benefiting from the antioxidant effects described herein.
However, in some cases, too great a metabolic rate may overwhelm
the antioxidant composition, thereby requiring additional time in
culture, increased concentrations of the compositions disclosed
herein, or combinations of both.
[0016] In one embodiment, cardiac cells are cultured with an
antioxidant composition comprising the peptide antioxidant
glutathione in a concentration ranging from about 0.1 to 20 .mu.M,
and a combination of non-peptide antioxidants comprising vitamin C
and vitamin E, in a concentration ranging from about 0.1 to 20
.mu.M. In some embodiments, antioxidant compositions are used in
conjunction with other methods to reduce the incidence of
karyotypic abnormalities in cells. For example, in some
embodiments, cells are cultured in an environment that more closely
mimics the natural in vivo conditions for that cell (e.g.,
physiologic oxygen concentrations), in conjunction with the use of
antioxidant compositions.
[0017] In several embodiments, there is provided a method of
increasing the yield of stem cells in culture, comprising obtaining
a population of stem cells isolated from a source of tissue,
restricting oxygen concentrations in a culture environment to
physiologic oxygen concentrations, and culturing the stem cells in
the restricted oxygen culture environment. In several embodiments,
physiologic levels of oxygen increase the rate at which the stem
cells proliferate, thereby increasing the yield of stem cells as
compared to culture conditions that employ non-physiologic levels
of oxygen. In several embodiments, the yield of stem cells is
increased per unit weight of the source tissue as compared to the
yield of stem cells cultured in conditions that employ
non-physiologic concentrations of oxygen. In some embodiments, the
per unit weight yield is increased by at least about 5% for a given
time period of culturing. In some embodiments, the per unit weight
yield is increased by at least about 20% for a given time period of
culturing. In several embodiments, greater increases in per unit
weight yield are achieved (e.g., at least 25%, 30%, 35%, 40%, 50%,
or greater). In several embodiments the source tissue is cardiac
tissue and the stem cells are cardiac stem cells.
[0018] Advantageously, in several embodiments, the increased yield
reduces the amount the amount of time that the stem cells are
cultured in order to reach a certain population as compared to the
amount of time stem cells are cultured in non-physiologic
concentrations of oxygen in order to reach the certain population.
For example, in some embodiments, the increased yield reduces the
amount the amount of time required for culturing by 20%. In
additional embodiments, the increased yield reduces the amount the
amount of time required for culturing by 50%. Such increased yield
(and reduced culture time) are particularly advantageous in some
embodiments, wherein the cells are to be used for therapy (reduced
time from collection to therapy), or in generating a cell bank
(reduced time to generate a sizeable bank for future therapy).
Moreover, in either in autologous or allogeneic transplant
scenarios, the reduced time to generate a given population size
reduces the time between tissue collection and subsequent therapy.
In some allogeneic contexts, this time is negligible, because a
cell bank can be generated prior to the need for any other subject
to receive therapy.
[0019] In several embodiments, there is provided a method for
increasing the function of the cardiac tissue of a subject having
damaged or diseased cardiac tissue, comprising obtaining a
population of cardiac stem cells for administration to the subject,
wherein the cardiac stem cells are harvested from donor cardiac
tissue and expanded in a culture environment comprising oxygen
concentrations restricted to physiologic oxygen concentrations (to
generate an expanded population of cardiac stem cells), and
administering at least a portion of the expanded population of
cardiac stem cells to the subject. In several embodiments the
administered cardiac stem cells engraft into the cardiac tissue of
the subject to a greater degree than cardiac stem cells expanded in
non-physiologic concentrations of oxygen. In several embodiments
the administered cardiac stem cells survive in the cardiac tissue
of the subject to a greater degree than cardiac stem cells expanded
in non-physiologic concentrations of oxygen. In still additional
embodiments, the greater degree of engraftment and/or survival lead
to increased cardiac function in the subject.
[0020] In several embodiments, there is provided a method for
enhancing the efficacy of cardiac stem cell therapy, comprising
obtaining a population of cardiac stem cells for administration to
a subject in need of cardiac stem cell therapy due to damaged or
diseased cardiac tissue wherein the cardiac stem cells are
harvested from donor cardiac tissue and expanded in a culture
environment comprising oxygen concentrations restricted to
physiological oxygen concentrations to generate an expanded
population of cardiac stem cells, and administering at least a
portion of the expanded population of cardiac stem cells to the
subject.
[0021] In several embodiments the physiologic oxygen concentrations
are between about 1% to about 8%, about 2% to about 7% about 3% to
about 6%, about 4% to about 5%, and overlapping ranges thereof. In
one embodiment, the oxygen concentration ranges from about 4% to
about 7%. In several embodiments, the oxygen concentrations are
tailored to a particular cell type. For example, depending on the
region of tissue from which a population of cells originated (e.g.,
a tissue having low oxygen concentrations in vivo versus a tissue
having high oxygen concentrations in vivo) oxygen concentrations
can be adjusted to be appropriately physiologic for that cell type.
Even within a particular organ, the degree of oxygenation may vary.
Tissue oxygen concentrations are readily discerned by one of
ordinary skill in the art and can thus be used to tailor the
methods disclosed herein to generate greater numbers of cells, more
genetically stable cells, and/or cells with increased
functionality.
[0022] In several embodiments, the methods provided comprise
culturing cells in physiologic oxygen concentrations to reduce the
incidence of karyotypic abnormalities in the cultured as compared
to cells cultured in non-physiologic concentrations of oxygen. In
some such embodiments, culturing in physiologic oxygen
concentrations reduces the incidence of aneuploidy in the cultured
cells as compared to cells cultured in non-physiologic
concentrations of oxygen. In some embodiments, DNA strand breaks
are reduced. In some embodiments, combinations of reduction in
karyotypic abnormalities, aneuploidy, and DNA strand breaks are
reduced. In several embodiments the cultured cells are cardiac stem
cells. In some embodiments, the cells are for use in allogeneic
cardiac cell therapy. In other embodiments, the cells are for use
in autologous therapy. In several embodiments, the cardiac stem
cells comprise cardiospheres, cardiosphere derived cells, or a
subsequent generation of cardiospheres. In some embodiments, the
cardiac stem cells are suitable for administration of a subject
having damaged or diseased cardiac tissue.
[0023] In several embodiments, the administration of the cardiac
stem cells cultured in physiologic oxygen to a subject results in
increased engraftment into the cardiac tissue of the subject as
compared to engraftment of cardiac stem cells cultured in
non-physiologic concentrations of oxygen. In several embodiments,
the administration of the cardiac stem cells cultured in
physiologic oxygen to a subject results in one or more of increased
myocardial viability, increased wall thickness, and lower left
ventricular volume in the cardiac tissue of the subject as compared
to that resulting from administration of cardiac stem cells
cultured in non-physiologic concentrations of oxygen. In several
embodiments, increased function due to administration of cells
cultured in physiologic oxygen concentrations is realized as an
improved left ventricular ejection fraction (by at least 5% as
compared to increased function due to administration of cardiac
stem cells expanded in non-physiologic concentrations of oxygen).
In several embodiments, the administration of the cardiac stem
cells cultured in physiologic oxygen to a subject results in
greater survival of the cells in the cardiac tissue of the subject
to a greater degree than cardiac stem cells expanded in
non-physiologic concentrations of oxygen
[0024] In several embodiments, a method for reducing the incidence
of karyotypic abnormalities in cardiac stem cells for use in the
repair or regeneration of cardiac tissue is provided. In one
embodiment, the method comprises isolating cardiac stem cells and
culturing the isolated stem cells in a culture media supplemented
with an antioxidant composition. In one embodiment, the method
comprises combining cells susceptible to chromosomal damage with an
antioxidant composition at a concentration suitable to reduce the
incidence of chromosomal damage in the cells, when, for example,
the cells are administered to a mammal. In one embodiment, the
antioxidant composition is provided at a concentration that reduces
free radical damage while still permitting (or without
substantially impairing) the function of one or more of the cell's
endogenous repair mechanisms.
[0025] In several embodiments, a composition for reducing the
incidence of karyotypic abnormalities in cultured cardiac stem
cells is provided. In one embodiment, the composition comprises at
least one peptide antioxidant, at least one non-peptide
antioxidant, and a culture media suitable for culturing cardiac
stem cells that is supplemented (or suitable to supplementation)
with the at least one peptide antioxidant and the at least one
non-peptide antioxidant. In one embodiment, the antioxidant
composition comprises, consists or consists essentially of one or
more non-peptide antioxidants. In another embodiment, the
antioxidant composition comprises, consists or consists essentially
of one or more peptide antioxidants.
[0026] In several embodiments, a composition for reducing the
incidence of karyotypic abnormalities in cultured cells is
provided. In one embodiment, the composition comprises a culture
media suitable for culturing cells and at least one antioxidant
present in a concentration ranging from about 0.1 to 200 .mu.M. In
some embodiments, the cells are stem cells. In some embodiments,
the cells are cardiac stem cells.
[0027] In several embodiments, a method for reducing cellular
and/or genetic (e.g., karyotypic) abnormalities in cells cultured
in a medium is provided. In one embodiment, the method comprises
contacting the cells with one or more of the compositions disclosed
herein. In another embodiment, the method comprises culturing the
cells in a hypoxic environment and, optionally, with an antioxidant
composition according to several embodiments disclosed herein.
[0028] In several embodiments, the cells are isolated from healthy
mammalian non-embryonic cardiac tissue. In some embodiments, the
cells are isolated from healthy mammalian non-embryonic non-cardiac
tissue. In some embodiments, the cells are isolated from embryonic
tissue.
[0029] In several embodiments, the antioxidant composition reduces
reactive oxygen species to a level which decreases oxidative-stress
induced DNA damage, but does not significantly impair DNA (and/or
other cellular) repair mechanisms. In one embodiment, antioxidant
compositions according to several embodiments disclosed herein
reduce oxidative-stress induced damage without adversely affecting
the cell's own repair mechanisms by more than 1%, 5%, 10%, 25%, or
50%.
[0030] In several embodiments, the reduction in reactive oxygen
species does not significantly induce DNA damage (as evidenced by
markers of DNA damage) in stem cells, such as cardiac stem cells.
In several embodiments, the balance of reduced reactive oxygen
species, non-reduced DNA repair mechanisms and non-induction of DNA
damage reduces the incidence of karyotypic abnormalities in cells,
including stem cells (such as cardiac stem cells).
[0031] In some embodiments, at least one peptide antioxidant is
selected from the group consisting of enzymes, proteins, peptides.
In some embodiments, at least one peptide antioxidant comprises
glutathione. In some embodiments, glutathione is present in a
concentration ranging from about 1 to 150 .mu.M. In some
embodiments, glutathione is present in a concentration ranging from
about 1 to 50 .mu.M. In some embodiments, glutathione is present in
a concentration ranging from about 0.1 to 20 .mu.M.
[0032] In some embodiments, at least one non-peptide antioxidant is
selected from the group consisting of thiols, vitamins and
polyphenols. In some embodiments, at least one non-peptide
antioxidant comprises one or more vitamins. In some embodiments,
the vitamins comprise one or more of vitamin A, vitamin E, and
vitamin C. In some embodiments, the vitamins are present in an
individual or total concentration ranging from about 1 to 150
.mu.M. In some embodiments, the vitamins are present in an
individual or total concentration ranging from about 1 to 50 .mu.M.
In some embodiments, the vitamins comprise vitamin C and vitamin E.
In some embodiments, vitamin C and vitamin E are present in an
individual or total concentration ranging from about 1 to 50 .mu.M.
In some embodiments, vitamin C and vitamin E are present in a
concentration ranging from about 0.1 to 20 .mu.M.
[0033] In some embodiments, the antioxidant composition comprises,
consists of, or consists essentially of glutathione at a
concentration ranging from about 0.1 to 20 .mu.M and vitamin C and
vitamin E, which are each present in a concentration ranging from
about 0.1 to 20 .mu.M.
[0034] In several embodiments, cells are exposed to antioxidant
compositions and/or hypoxic conditions to cause a reduction in
reactive oxygen species of at least 10% as compared to cells
cultured in 20% oxygen. In some embodiments, the reactive oxygen
species are reduced by at least 50% as compared to cells cultured
in 20% oxygen. In some embodiments, the reactive oxygen species are
reduced by about 60%-70% (e.g., 65%) as compared to cells cultured
in 20% oxygen. In some embodiments, a reduction in reactive oxygen
species of at least 10%, 25%, 50% or 75% is achieved as compared to
cells cultured in hyperoxic conditions. In one embodiment, the use
of antioxidant compositions disclosed herein reduces karyotypic
damage when cells are cultured in about 20% oxygen, or higher. In
one embodiment, the combined use of antioxidant compositions and
hypoxic conditions result in a synergistic effect (e.g., enhanced
reduction of cellular/DNA damage).
[0035] In several embodiments, the markers of DNA repair mechanisms
comprise one or more DNA repair enzymes. In some embodiments, DNA
repair enzymes are selected from the group consisting of ATM, ATR,
Rad50, Rad51, Chk1, Chk2, or combinations thereof. In several
embodiments of the invention, compositions or hypoxic conditions
disclosed herein, do not impair DNA repair mechanism at by more
than 0% (e.g., no effect), 2%, 5%, 10%, 20%, 30%, and 50%. DNA
repair mechanisms include, but are not limited to, base excision,
nucleotide excision and mismatch repair. In one embodiment,
compositions or hypoxic conditions disclosed herein maintain or
up-regulate at least one repair mechanism while down-regulating
another repair mechanism, wherein the overall impairment of DNA
repair is no more than 0%, 2%, 5%, 10%, 20%, 30%, or 50%. In one
embodiment, compositions or hypoxic conditions disclosed herein do
not impair base excision, nucleotide excision or mismatch repair.
In another embodiment, compositions or hypoxic conditions disclosed
herein do not impair at least two of base excision, nucleotide
excision or mismatch repair. In one embodiment, compositions or
hypoxic conditions disclosed herein reduce the incidence of
karyotypic abnormalities in cells by more than 10%, 25%, 50%, 75%,
or 95% as compared to control cells.
[0036] In several embodiments, the markers of DNA damage comprise
one or more markers of DNA double-strand breaks. In some
embodiments, the markers of DNA double-strand breaks comprise
.gamma.-H.sub.2AX foci in cultured cells, .gamma.-H.sub.2AX mRNA,
or .gamma.-H.sub.2AX protein, or combinations thereof.
[0037] In several embodiments provided herein, the isolated cells
(e.g., stem cells) are cultured for at least about 24 hours, 3 days
or 7 days. In some embodiments, the isolated cells (e.g., stem
cells) are cultured for a period of one to 6 months (e.g., 1, 2, 3,
4, 5, or 6 months).
[0038] In several embodiments, a method for assessing the risk of
neoplastic disease in a subject is provided. In one embodiment, the
method comprises obtaining a blood or tissue sample from the
subject and measuring the concentration of one or more reactive
oxygen species in the sample. In some embodiments, the risk for
neoplastic disease is greater if the level of reactive oxygen
species is sufficiently high to induce oxidative DNA damage in the
cells of the subject and/or if the level of reactive oxygen species
is sufficiently low to promote down-regulation of DNA repair
mechanisms in the cells of the subject. Reduction in the risk of
neoplastic disease is achieved in several embodiments of the
invention by, for example, treating subjects with cells cultured in
compositions or conditions disclosed herein.
[0039] In several embodiments, a method for assessing the risk of
neoplastic disease in a subject is provided. In one embodiment, the
method comprises analyzing the subject's tissue for evidence of
karyotypic abnormalities (e.g., DNA damage) and correlating the
damage with the subject's antioxidant levels. Optionally, the
subject is then treated to reduce or increase antioxidant levels as
needed. In one embodiment, evidence of damage is determined by
analyzing markers of DNA damage, DNA damage-associated mRNA
expression levels, and/or DNA damage-associated protein expression
levels. In one embodiment, the risk for neoplastic disease
increases as the incidence of DNA damage increases, as the
expression of DNA damage-associated mRNA increases, and/or as the
expression of DNA damage-associated protein increases. In some
embodiments, DNA damage is measured by identifying
.gamma.-H.sub.2AX foci, by measuring .gamma.-H.sub.2AX mRNA
expression levels, and/or by measuring .gamma.-H.sub.2AX protein
expression levels.
[0040] In several embodiments, a method for optimizing the amount
of an antioxidant-containing supplement administered to a subject
is provided. In one embodiment, the method comprises measuring the
concentration of one or more reactive oxygen species in the
subject's sample (e.g., tissue samples, skin scrapings, blood,
isolated cells, saliva, urine, other bodily fluids, etc.) and the
incidence of DNA damage in cells isolated from the sample. In some
embodiments, in addition to, or instead of, ascertaining DNA
damage, the expression of DNA repair enzymes in cells isolated from
the sample is measured. In one embodiment, the method further
comprises treating the patient with an antioxidant composition, and
optionally adjusting the composition to balance the concentration
of reactive oxygen species in the subject with the subject's
endogenous DNA repair mechanism(s), as determined by, for example,
expression of DNA repair enzymes or other markers of DNA repair. In
several embodiments, compositions (e.g., supplements) comprising an
optimal amount of antioxidant(s) are provided that are formulated
to decrease oxidative-stress induced DNA damage without negatively
impacting (wholly or partially) DNA repair mechanisms in the
subject. In one embodiment, therapeutic antioxidant compositions
that balance oxidative damage with endogenous DNA repair mechanisms
are provided based on a subject's individual antioxidant and/or
karyotypic profile. In some embodiments, the invention comprises
determining .gamma.-H.sub.2AX foci to gauge risk of chromosomal
damage (and neoplasm) and/or to optimize antioxidant
supplements.
BRIEF DESCRIPTION OF THE FIGURES
[0041] FIG. 1 depicts histograms summarizing karyotyping data for
the various culture conditions utilized in the Examples.
[0042] FIGS. 2A-2C depict representative karyotypes made from CDCs
cultured in various conditions.
[0043] FIGS. 3A-3D depict CDC proliferation data. FIGS. 3A, 3B, and
3C depict representative microscopic images of CDCs cultured for 36
days in various conditions (A=20% O.sub.2; B=20% O.sub.2 plus
antioxidant supplement at 1:1000 dilution; C=20% O.sub.2 plus
custom antioxidant cocktail at 100 .mu.M). FIG. 3D depicts that no
significant differences in CDC proliferation were detected among
the various culture conditions.
[0044] FIGS. 4A and 4B depict ATP and mitochondrial membrane
potential data. FIG. 4A depicts intracellular ATP concentrations
and FIG. 4B depicts mitochondrial membrane potential of CDCs
cultured in various conditions.
[0045] FIGS. 5A-5D depict intracellular ROS concentrations in human
cardiosphere-derived cells after 24 hours culture under 20% O.sub.2
with different concentrations of antioxidants (5A and 5B), catalase
(5C), and hydrogen peroxide (H.sub.2O.sub.2; 5D).
[0046] FIGS. 6A-6D depict flow cytometry assessments of
intracellular ROS levels in human CDCs cultured for 24 hours with
different concentrations of antioxidants (6A and 6B), catalase
(6C), or hydrogen peroxide (H.sub.2O.sub.2; 6D).
[0047] FIGS. 7A-7D depict ROS and DNA damage data. FIG. 7A depicts
intracellular ROS concentration in human cardio sphere-derived
cells after long-term culture under 20% O.sub.2, 20% O.sub.2, or
under 20% O.sub.2 with added antioxidants. FIG. 7B depicts
intracellular ROS concentrations after culturing in hypoxic
conditions or with the addition of antioxidants. FIG. 7C depicts
.gamma.-H.sub.2AX foci in CDCs cultured in the indicated
conditions. FIG. 7D depicts quantitation of .gamma.-H.sub.2AX
foci.
[0048] FIGS. 8A-8D depict representative microscopic images
demonstrating .gamma.-H.sub.2AX foci in CDCs cultured with
antioxidants (8A and 8B); catalase (8C) and hydrogen peroxide
(H.sub.2O.sub.2; 8D).
[0049] FIGS. 9A-9D depict the bi-phasic DNA damage dose-response in
human CDCs and embryonic stem (ES) cells cultured with varying
amounts of anti-oxidants (9A and 9B), catalase (9C) and hydrogen
peroxide (H.sub.2O.sub.2; 9D).
[0050] FIGS. 10A-10D depict ATM protein levels in human
cardiosphere derived cells after 24 hours of culture under 20%
O.sub.2 with antioxidants (10A and 10B), catalase (10C), and
hydrogen peroxide (H.sub.2O.sub.2; 10D).
[0051] FIGS. 11A-11D depict representative western blot analysis of
the expression of various DNA repair-related factors after
culturing under 20% O.sub.2 with antioxidants (11A and 11B),
catalase (11C), and hydrogen peroxide (H.sub.2O.sub.2; 11D).
[0052] FIGS. 12A-12E depict semi-quantitative histograms of western
blot data for the expression of ATR (FIG. 12A), Rad50 (FIG. 12B),
Rad51 (FIG. 12C) Chk1 (FIG. 12D), and Chk2 (FIG. 12E).
[0053] FIGS. 13A and 13B depict DNA repair data. FIG. 13A depicts
protein expression and quantitation of ATM from human
cardiosphere-derived cells after 1-2 months long-term culture under
the indicated conditions. FIG. 13B depicts protein expression of
various DNA repair-related factors in human cardiosphere-derived
cells after 1-2 months long-term culture under different
conditions.
[0054] FIG. 14 depicts a schematic of the possible interactions
between intracellular ROS levels and genomic stability.
[0055] FIG. 15 depicts the difference in growth between culturing
CDCs in 20% versus 5% O.sub.2.
[0056] FIG. 16 depicts the number of euploid cells, aneuploid
cells, and cells with DNA breaks or translocations for 13 CDC lines
divided and cultured in 20% or 5% oxygen.
[0057] FIG. 17 depicts a significant improvement in the efficacy of
CDCs cultured in physiologic oxygen concentrations as compared to
those cultured in room oxygen.
[0058] FIGS. 18A-18D depict the growth and proliferation of cardiac
stem cells in 5% O.sub.2 and 20% O.sub.2. Panel A shows
representative images showing that cell outgrowth from `explants`
(red arrow) was much faster in 5% O.sub.2 (right) than in 20%
O.sub.2 (left). The number of cells harvested (Panel C) was more
than two-fold higher in 5% O.sub.2 than in 20% O.sub.2, although
the amount of starting material was equivalent. Panel B depicts
CDCs at earlier passages(shown in passage #2)showing no differences
in either morphology or proliferative activity (Panel D) under 5%
O.sub.2 and 20% O.sub.2, although greater proliferation was
observed in cells expanded under 5% O.sub.2 at later passages.
[0059] FIGS. 19A-19B depicts analysis of chromosomal abnormalities.
Panel A indicates fewer aneuploid cells in CDCs cultures expanded
in 5% O.sub.2 as compared to 20% O.sub.2. Panel B shows that the
percentages of aneuploid cells were also decreased in 5% O.sub.2
culture.
[0060] FIGS. 20A-20F depict analysis of cell senescence. Compared
with 20% O2 culture, cell senescence of CDCs was improved under 5%
O.sub.2 culture, by flow cytometry for p16.sup.INK4A (panels A and
D), immunostaining for telomerase activity (panels B and E), and
senescence-associated b-galactosidase staining (panels C and
F).
[0061] FIGS. 21A-21F depict analysis of intracellular ROS, DNA
damage, and resistance to oxidative stress. Panel A shows that the
levels of intracellular ROS are lower in CDCs expanded in 5%
O.sub.2 when compared with those in 20% O.sub.2. Panel C depicts
DNA damage as evidenced by the formation of .gamma.-H.sub.2AX foci.
DNA damage was lower in CDCs expanded in 5% O.sub.2 than in 20%
O.sub.2 (Panel D). Panel E shows representative images of
TUNEL-positive (red) CDCs after 24 h exposure to 100 mM
H.sub.2O.sub.2. The number of apoptotic cells (panel F) was lower
in CDCs expanded in 5% O.sub.2 than 20% O.sub.2.
[0062] FIGS. 22A-22E depicts analysis of cell engraftment, cardiac
functional recovery, and their relationships. Panel A depicts
quantitative data on the survival rate of human CDCs 24 h and 7
days after implantation into mice-infarcted heart. Panel B shows
cells positively stained by HNA are more frequently observed in
mice 3 weeks after implantation with CDCs expanded in 5% O.sub.2
than in 20% O.sub.2. Panel C shows quantitative data for cell
engraftment (% nuclei) in the infarcted heart. Panel D shows that
left ventricular ejection fraction (LVEF) at baseline does not
differ among groups, indicating a similar infarct size to begin
with. After 3 weeks, the LVEF was higher in mice implanted with
CDCs expanded in 5% O.sub.2 than in 20% O.sub.2, although the LVEF
was also higher in mice implanted with CDCs expanded in 20% O.sub.2
than in controls with PBS injection only. Panel D indicates that
the engraftment of human CDCs (% nuclei) within the infarcted
hearts of mice is strongly correlated with the absolute values of
LVEF at 3 weeks.
[0063] FIGS. 23A-23G depict histological assessments of infarct
size and ventricular morphology. Representative images of Masson's
staining show that, compared with an infarcted heart receiving PBS
injection only (panel A), the infarct size was much smaller in a
heart that had received CDCs expanded in both 5% O.sub.2 (panel C)
and 20% O.sub.2 (panel B). Quantitative analyses of viable
myocardium (panel D), LV wall thickness (panel E), LV chamber area
(panel F), and LV total area (panel G) show that better therapeutic
efficiency was achieved by the implantation of CDCs expanded in 5%
O.sub.2 than in 20% O.sub.2, although a significant improvement was
also observed by the implantation of CDCs expanded under 20%
O.sub.2 when compared with the control treatment with PBS injection
only.
[0064] FIG. 24 depicts a representative karyotype showing
aneuploidy (in this case, trisomy 8) in twice-passaged
cardiac-derived cells expanded under 20% O.sub.2.
[0065] FIGS. 25A-25D depict cell phenotype and in vitro myogenic
differentiation. Panel A shows flow cytometry data shows no
significant difference in the proportion of c-kit.sup.+ stem cells
in cardiac-derived cells expanded in 5% O.sub.2 versus 20% O.sub.2
(quantification in panel C). Panel B shows similar expression of
troponin T was also observed in the two groups (quantification in
panel D).
[0066] FIGS. 26A-26D depict analysis of the expression of adhesion
molecules and c-Myc (panel A shows Western blot protein expression
data). There is no significant difference in the expression levels
of integrin-.alpha..sub.2 (panel B), laminin-.beta..sub.1 (panel
C), or c-Myc (panel D) in cardiac-derived cells expanded in 5%
O.sub.2 versus 20% O.sub.2.
[0067] FIGS. 27A-27F depict a comparison of in vitro production of
growth factors between cardiac-derived cells expanded in 5% O.sub.2
and 20% O.sub.2. The concentrations of angiopoietin-2 (panel A),
bFGF (panel B), HGF (panel C), IGF-1 (panel D), SDF-1 (panel E),
and VEGF (panel F) measured by ELISA showed no significant
difference in conditioned medium culture with hypoxic
stimulation.
[0068] FIGS. 28A-28B depict an in vitro angiogenesis assay. Panel A
shows the formation of capillary-like tubes in extracellular matrix
from cardiac-derived cells expanded in 5% O.sub.2 and 20% O.sub.2
did not differ due to hypoxic stimulation. Panel B shows the
quantitative summary data for tube length.
[0069] FIGS. 29A-29D depict immunostaining analysis showing that
some human CDCs (identified by expression of human nuclear antigen,
HNA) expanded in 5% O.sub.2 (panel A) and 20% O.sub.2 (panel
B)expressed .alpha.-sarcomeric actin (.alpha.-SA), which is
indicative of myogenic differentiation 3 weeks after implantation
into infarcted hearts of mice. Image insets (panels C and D) show
that the human nuclei are actually within cardiomyocytes.
[0070] FIGS. 30A-30B depict immunostaining analysis shows some
human cardiac-derived cells (CDCs, identified by human nuclear
antigen, HNA) expanded in 5% O.sub.2 (panel A) and 20% O.sub.2
(panel B) expressed smooth muscle actin (SMA) 3 weeks after
implantation into infarcted hearts of mice.
[0071] FIGS. 31A-31B depict immunostaining analysis shows some
human cardiac-derived cells (CDCs, identified by human nuclear
antigen, HNA) expanded in 5% O.sub.2 (panel A) and 20% O.sub.2
(panel B) expressed the endothelial marker von Willebrand factor
(vWF) 3 weeks after implantation into infarcted hearts of mice.
DETAILED DESCRIPTION
[0072] In several embodiments described herein, methods of reducing
the incidence of karyotypic abnormalities in cultured stem cells
for use in the repair or regeneration of cardiac tissue are
provided. In several embodiments, compositions that reduce the
incidence of karyotypic abnormalities in the culturing of stem
cells are provided. Such embodiments are advantageous because
chromosomal abnormalities have been found in an unexpectedly large
percentage of stem cells isolated, cultured, and expanded for use
in cellular therapies. For example, chromosomal abnormalities have
been found in up to 50% of long-term cultured human embryonic stem
(ES) cells. Normal embryonic stem cells, which are typically
derived from an early stage embryo, have the potential to develop
into any type of cell in the body. In some instances, unplanned
growth of one cell type in a distinct type of tissue may result in
the formation of teratomas. Undesirable neoplastic growth may be
potentiated if ES cells with chromosomal abnormalities are used in
therapy.
[0073] In contrast to ES cells, adult stem cells generally develop
into cell types related to the tissue from which the stem cells
were isolated. However, the potential for undesirable neoplastic
growth still exists, particularly if adult stem cells with
chromosomal abnormalities are used in therapy. In fact, G-banding
karyotype analysis of primary cardiosphere-derived cells (CDCs, a
heart-derived mixed-cell population rich in cardiac stem cells)
revealed that .about.30% of preliminary CDC production runs
resulted in cells with chromosomal abnormalities (See Table 1). In
addition to potential neoplastic growth, genomic alterations of
stem cells may impair therapeutic potency of administered cells.
Thus, several embodiments of the present invention are particularly
beneficial because they enhance the therapeutic potency (e.g.,
efficacy and/or viability, etc.) of the administered cells.
TABLE-US-00001 TABLE 1 Summary of Chromosomal Abnormalities Samples
Karyotype 2424/p3 46, XX[20] 2377/p3 46, XX[20] 2404/p3 46, XY[20]
2482/p4 46, XY[20] R071215/p3 47, XY +8[3]/47, XY +18[3]/46, XY[44]
R071215/p6 47, XY +8[2]/46, XY[48] R071212/p6 46, XY[50] R071214/p4
47, XY +2[4]/46, XY[46] R071214/p5 47, XY +2[6]/46, XY[44]
R071214/p6 47, XY +2[13]/46, XY[7] 6-1-1/p5 46, XY[20] 6-1-3/p5 46,
XY[20] 10-1/p2 46, XX[20] CSB9/p4 46, XY, inv(9)(p11; q13) [20]
CSB11/p3 46, XY[20] CSB3/p3 45, X - Y[6]/46, XY[14] BX13/p4 46,
XY[20] BX14/p3 46, XX[20] BX15/p3 47, XY +8[20] BX16/p4 45, X -
Y[7]/47, XY +i(8)(q10)[6]/47, XY +8[1]/46, XY[6] BX17/p2 45, X -
Y[3]/46, XY[17] BX18/p3 46, XY[20] BX19/p3 46, XY[20] BX20/p2 46,
XY[20] BX21/p2 46, XY[20] BX22/p1 46, XY[20] BX23/p1 46, XY[20]
BX23/p2 46, XY[20] BX24/p1 45, X - Y[10]/47, XY +18[2]/46, XY[8]
BX27/p1 47, XY +8[6]/45, X - Y[3]/47, XY + Y[2]/46, XY[9] BX27/p2
47, XY +8[14]/46, XY[6] BX43/p0 46, XY[20] BX45/p0 46, XY[20]
BX46/p0 46, XY[20] BXJ4/p0 45, -Y, t(X; 11)(p10; p10)[11]/45, X,
-Y[3]/46, XY[6] BX45/p1 46, XY[20] BX46/p1 47, XY + 8[4]/46, XY[16]
BXJ4/p1 45, -Y, t(X; 11)(p10; p10)[19]/46, XY[1] BX34/p1 46, XY[20]
BX40/p2 46, XY[20] BXJ2/p1 46, XX[20]
[0074] Reactive oxygen species (ROS) are reactive molecules that
contain the oxygen atom. The superoxide ion (O.sub.2-) and
peroxides (e.g., hydrogen peroxide, H.sub.2O.sub.2) are well known
ROS. The superoxide ion leaks from active mitochrondria and is
converted to H.sub.2O.sub.2. Cellular enzymes such as catalase and
superoxide dismutase act on H.sub.2O.sub.2 to form hydrogen and
water; however these reactions are not efficient enough to remove
all the H.sub.2O.sub.2. ROS molecules are typically highly reactive
due to the presence of unpaired valence shell electrons. While ROS
are formed under normal circumstances where oxygen is metabolized,
and may play important roles in cell signaling, during times of
environmental or metabolic stress (e.g., UV or heat exposure,
ionizing radiation, hypoxia, ischemia, etc.) ROS levels may
increase, possibly resulting in damage to intracellular structures
or induction of programmed cell death mechanisms. The various
pathways that generate ROS are known as oxidative stress. Increases
in oxidative stress have the potential to damage RNA, DNA, or
protein that, if uncorrected, may lead to chromosomal
abnormalities. Chromosomal abnormalities, in turn, can lead to
poorly functioning or malfunctioning cells, uncontrolled
proliferation of cells, or apoptosis, among other outcomes.
[0075] Because cells will fail to function or function improperly
if oxidative stress corrupts the integrity or accessibility of a
cells genome, mammalian cells have developed a variety of innate
DNA repair mechanisms. In some instances, cells will use the
unmodified complementary DNA strand to recover any genetic
information that is lost due to oxidative stress. If both strands
of DNA are damaged, the cell may allow polymerases to replicate DNA
through the site of the lesion in order to replicate essential DNA
sequences. However, none of these repair mechanisms is completely
free of errors. Thus, the proper function and longevity of a cell
is a balance of the oxidative stress a cell experiences and the
ability of one or more DNA repair mechanism to maintain the cell's
genome in normal working order.
[0076] Often, cells to be used in research or clinical applications
are cultured in media equilibrated with 95% air and 5% CO.sub.2
(.about.20% O.sub.2). Certain cells thrive in such an environment,
as the oxygen concentration mimics what those cells would be
exposed to in vivo. However, in the case of many stem cell
varieties, depending on the tissue, cellular oxygen concentrations
may be as low as .about.1-5% in the in vivo physiological
microenvironment. Exposure of stem cells to a non-physiological
hyperoxic state in culture may lead to oxidative stress, which, as
discussed above, may induce ROS formation, DNA damage, and/or
genomic instability. Genomic instability may be manifest in several
ways, including karyotypic abnormalities, low viability cells,
cells prone to neoplastic formation and the like. As used herein,
the terms "physiologic oxygen concentrations" and "physiological
oxygen concentrations" shall be given its ordinary meaning and
shall also refer to oxygen concentrations ranging from about 1% to
about 8% oxygen. In some embodiments oxygen concentrations in which
the CDCs (or other cardiac stem cells) are cultured range from
about 1% to about 2%, about 2% to about 3%, about 3% to about 4%,
about 4% to about 5%, about 5% to about 6%, about 6% to about 7%,
and overlapping ranges thereof. In several embodiments physiologic
oxygen conditions are maintained throughout the entire culture
process, while in other embodiments, physiologic oxygen conditions
are used in only a portion of the culture process.
[0077] Therefore, in several embodiments, methods and compositions
for reducing the incidence of karyotypic abnormalities in cultured
stem cells for use in the repair or regeneration of tissue are
provided. In several embodiments, use of the compositions and
methods disclosed herein permit the use of hyperoxic cell culture
conditions. In several embodiments, cardiac tissue is repaired or
regenerated. Other types of tissue (e.g., kidney, lung, liver
pancreas, spleen, bone, bone marrow, muscle tissue, vascular
tissue, nervous tissue, skin, etc.) are repaired or regenerated in
other embodiments. In several embodiments the method comprises
providing a culture media supplemented with an antioxidant cocktail
(discussed in more detail below) that is capable of inducing a
balance between the formation of ROS and the ensuing DNA damage and
the innate DNA repair mechanisms of the cultured cells, thereby
reducing in the incidence of karyotypic abnormalities. In some
embodiments, the method optionally includes culturing the cells in
a "hypoxic" environment relative to standard 20% O.sub.2 cell
culture conditions (e.g., oxygen concentrations ranging from about
1% to about 8%). In several embodiments, such culture conditions
improve the viability of the cultured stem cells. In some
embodiments, short term viability is improved, while in some
embodiments, long-term viability is improved. In some embodiments,
both short and long-term viability is improved. As such, in several
embodiments, the proliferation rate of the cells in culture is
increased. However, due to the normoxic (vis-a-vis the normal
cellular environment) conditions, limited genetic alterations
occur. In some embodiments, this is particularly advantageous
because the rate of cell expansions reduces the amount of time
needed to reach a certain population of cells. In some embodiments,
wherein the cells are to be administered for therapy in a certain
dosage, the amount of time between inception of culture of the
cells and administration of a certain dose of cells is reduced
(e.g., the time to reach a certain population is reduced as
compared to culture methods employing non-physiologic
concentrations of oxygen.
[0078] In several embodiments, culturing stem cells in physiologic
oxygen concentrations (and/or in antioxidant compositions)
positively affects the cells even after the period of culturing is
complete (e.g., the cells have been administered to a subject). For
example, in some embodiments, the use of physiologic oxygen
concentrations imparts to stem cells a greater viability in vivo.
As such, cells cultured according to the methods and/or with the
use of compositions as disclosed herein, remain viable for a longer
period of time post-administration, thereby increasing the
potential effectiveness of these cells in cellular therapies. In
several embodiments, cells cultured according to the methods and/or
with the use of compositions as disclosed herein engraft into host
tissue to a greater degree than those cultured in non-physiologic
concentrations of oxygen. In certain embodiments, engraftment is a
threshold step to efficacious cell therapy. For example, in some
therapies, direct tissue regeneration plays a significant role in
the therapy. However, advantageously, in some embodiments, cells
cultured according to the methods and/or with the use of
compositions as disclosed herein generate a more effective amount
of certain cellular signaling factors (e.g., autocrine, paracrine,
intracrine or endocrine factors such as growth factors, hormones,
cytokines and the like) that invoke an indirect mechanism of
therapy. For example, in some embodiments, an indirect mechanism,
such as recruitment of other endogenous cells is induced by a
signaling factor. As such, in some embodiments, a greater efficacy
of therapy is achieved by virtue of the production (or reduced
production) of certain such factors. In some embodiments, these
signaling factors act in concert with direct mechanisms to achieve
an effective therapy, while in some embodiments, the signaling
factors function alone. In several embodiments, these direct and/or
indirect mechanisms yield a more effective therapy, either by
improving anatomical aspects of the target tissue, functional
aspects of the target tissue, or combinations thereof. For example,
in the context of cardiac stem cell therapy, culture of cardiac
stem cells in physiologic oxygen concentrations improves the
viability and engraftment of administered cells, as discussed
below. Moreover, these improved parameters, in the context of a
cell therapy for treating an adverse cardiac event, also yield
improved cardiac anatomy (e.g., reduced infarct size, lower left
ventricular area) and improved function (increased left ventricular
ejection fraction, increased cardiac output, etc.).
[0079] In some embodiments, the stem cells are embryonic stem
cells. In some embodiments, the stem cells are adult stem cells. In
some embodiments, the stem cells are hematopoietic stem cells,
neuronal stem cells, mesenchymal stem cells, insulin producing stem
cells, hepatocyte stem cells, or epithelial stem cells, or
combinations thereof. In some embodiments, the stem cells comprise
cardiac stem cells. In some such embodiments, the cardiac stem
cells are cardiospheres or cardiosphere-derived cells (CDCs). In
some embodiments, the stem cells are processed and prepared for
administration to a subject in order to repair damaged tissue. In
some embodiments, the subject has damaged cardiac tissue in need of
repair. In some embodiments, the subject is a mammal. In some
embodiments, the subject is human, while in some embodiments the
subject is a non-human mammal or other organism. Damaged cardiac
tissue in a subject may be caused by a variety of events,
including, but not limited to myocardial infarction, ischemic
cardiac tissue damage, congestive heart failure, aneurysm,
atherosclerosis-induced events, cerebrovascular accident (stroke),
and coronary artery disease.
[0080] In one embodiment of the invention, cardiospheres and CDCs
are isolated as according to the following general protocol.
Briefly, cardiac tissue samples are weighed, cut into small
fragments and cleaned of gross connective tissue, and washed in a
sterile solution, such as phosphate-buffered saline. In some
embodiments, the tissue fragments are at least partially digested
with protease enzymes such as collagenase, trypsin, and the like.
In certain embodiments, the digested pieces are placed in primary
culture as explants on sterile tissue culture dishes with a
suitable culture media. The digested pieces of tissue range in size
from about 0.1 mm to about 2.5 mm (e.g., about 0.1-0.5 mm, 0.5-1
mm, 1-2 mm, 2-2.5 mm, and overlapping ranges thereof). In several
embodiments, the digested pieces of tissue range 0.25 mm to about
1.5 mm. Smaller or larger pieces of tissue can be used in other
embodiments. The tissue culture dish and culture media are selected
so that the tissue fragments adhere to the tissue culture plates.
In some embodiments, the tissue culture plates are coated with
fibronectin or other extracellular matrix (ECM) proteins, such as
collagen, elastin, gelatin and laminin, for example. In other
embodiments, the tissue culture plates are treated with plasma. In
certain embodiments, the dishes are coated with fibronectin at a
final concentration of from about 10 to about 50 .mu.g/mL. In still
other embodiments, the fibronectin dishes are coated with
fibronectin at a final concentration of from about 20 to 40
.mu.g/mL, with still other embodiments employing a final
fibronectin concentration of about 25 .mu.g/mL.
[0081] In certain embodiments, the base component of the complete
explant medium comprises Iscove's Modified Dulbecco's Medium
(IMDM). In some embodiments, the culture media is supplemented with
fetal calf serum (FCS) or fetal bovine serum (FBS). In certain
embodiments, the media is supplemented with serum ranging from 5 to
30% v/v. In other embodiments, the culture media is serum-free and
is instead supplemented with specific growth factors or hydrolyzed
plant extracts. In other embodiments, the media is further
supplemented with antibiotics, essential amino acids, reducing
agents, or combinations thereof. In one embodiment, the complete
explant medium comprises IMDM supplemented with about 20% fetal
bovine serum, about 50 .mu.g/mL gentamicin, about 2 mM L-glutamine,
and about 0.1 mM 2-mercaptoethanol. In some embodiments, the
explant media is changed every 2-4 days while the explants
culture.
[0082] The tissue explants are cultured until a layer of
stromal-like cells arise from adherent explants. This phase of
culturing is further identifiable by small, round, phase-bright
cells that migrate over the stromal-cells. In certain embodiments,
the explants are cultured until the stromal-like cells grow to
confluence. At or before that stage, the phase-bright cells are
harvested. In certain embodiments, phase-bright cells are harvested
by manual methods, while in others, enzymatic digestion, for
example trypsin, is used. The phase-bright cells may be termed
cardiosphere-forming cells, and the two phrases are used
interchangeably herein.
[0083] Cardiosphere-forming cells may then be seeded on sterile
dishes and cultured in cardiosphere media. In certain embodiments,
the dishes are coated with poly-D-lysine, or another suitable
natural or synthetic molecule to deter cell attachment to the dish
surface. In other embodiments, for example, laminin, fibronectin,
poly-L-orinthine, or combinations thereof may be used.
[0084] In certain embodiments, the base component of the
cardiosphere medium comprises Iscove's Modified Dulbecco's Medium
(IMDM). In some embodiments, the culture media is supplemented with
fetal calf serum (FCS) or fetal bovine serum (FBS). In certain
embodiments, the media is supplemented with serum ranging from 5 to
30% v/v, including from about 5% to about 10%, about 10% to about
15%, about 15% to about 20%, about 20% to about 25%, about 25% to
about 30%, and overlapping ranges thereof. In other embodiments,
the culture media is serum-free and is instead supplemented with
specific growth factors or hydrolyzed plant extracts. In certain
other embodiments, the media is further supplemented with
antibiotics, essential amino acids, reducing agents, or
combinations thereof. In one embodiment the cardiosphere medium
comprises IMDM supplemented with about 10% fetal bovine serum,
about 50 .mu.g/mL gentamicin, about 2 mM L-glutamine, and about 0.1
mM 2-mercaptoethanol.
[0085] According to one embodiment, cardiospheres will form
spontaneously during the culturing of the cardiosphere forming
cells. Cardiospheres are recognizable as spherical multicellular
clusters in the culture medium. Cells that remain adherent to the
poly-D-lysine-coated dishes are discarded. In certain embodiments,
the cardiospheres are collected and used to seed a biomaterial or
synthetic graft. In other embodiments, the cardiospheres are
further cultured on coated cell culture flasks in
cardiosphere-derived stem cell (CDC) medium.
[0086] In some embodiments used to culture cardiospheres into CDCs,
the culturing flasks are fibronectin coated, though in other
embodiments other cellular attachment promoting coatings are
employed. The cultured cardiospheres attach to the surface of the
flask and are expanded as a monolayer of CDCs. CDC medium comprises
IMDM, and in certain embodiments is supplemented with fetal calf
serum (FCS) or fetal bovine serum (FBS). In some embodiments, the
media is supplemented with serum ranging from 5 to 30% v/v. In
other embodiments, the culture media is serum-free and is instead
supplemented with specific growth factors or hydrolyzed plant
extracts. In certain other embodiments, the media is further
supplemented with antibiotics, essential amino acids, reducing
agents, or combinations thereof. In one embodiment, the CDC medium
comprises IMDM supplemented with about 10% fetal bovine serum,
about 2 mM L-glutamine, and about 0.1 mM 2-mercaptoethanol. CDCs
may be repeatedly passaged by standard cell culture techniques and
in several embodiments are harvested and used to seed a biomaterial
or synthetic graft.
[0087] In some embodiments the cells to be administered to the
subject are obtained from healthy tissue of the subject, e.g., an
autologous transplant. In some embodiments the cells to be
administered to the subject are obtained from healthy tissue of an
individual other than the subject, e.g., an allogeneic transplant.
In some embodiments the cells to be administered to the subject are
obtained from healthy tissue an individual who is highly
genetically similar or identical to the subject, e.g., a syngeneic
transplant. In still further embodiments, the cells to be
administered to the subject are obtained from healthy tissue an
individual of a species distinct from the subject, e.g., a
xenogeneic transplant.
[0088] In some embodiments, the culture methods reduce the
concentration of ROS during the cell culturing process. As used
herein, the terms reactive oxygen species or ROS shall be given
their ordinary meaning and shall include, but not be limited to,
superoxide anions (O.sub.2--), hydrogen peroxide (H.sub.2O.sub.2),
hydroxyl radicals (OH--), organic hydroperoxides (ROOH), alkoxy
(RO) and peroxy (ROO) radicals, hypochlorous acid (HOCl), and
peroxynitrite (ONOO--), and combinations thereof.
[0089] In several embodiments, antioxidant compositions are
provided in a range of about 0.1 to about 1000 .mu.M. In some
embodiments, about 0.3 to about 50 .mu.M of a custom antioxidant
cocktail (e.g., a formulation comprising vitamin C, vitamin E, and
glutathione) reduces the formation of ROS. In some embodiments, ROS
production is reduced by about 10-30%. In some embodiments ROS
production is reduced by about 20-50%, or at least about 25, 28,
31, 34, 37, 40, 43, 46, and 49%. In some embodiments, ROS
production is reduced by about 40-80%. In some embodiments, ROS
production is reduced by up to about 90%. As discussed below, in
some embodiments, cells with a large reduction in ROS still exhibit
DNA damage. In some embodiments, use of the antioxidants described
herein reduce the formation, viability, and/or activity of ROS,
and/or enhance the degradation of ROS. In some embodiments, an
antioxidant cocktail (or composition) comprises one or more of the
following antioxidants: vitamin A, vitamin C, vitamin E,
glutathione, mixed carotenoids (e.g., beta carotene, alpha
carotene, gamma carotene, lutein, lycopene, phytopene, phytofluene,
and astaxanthin), selenium, Coenzyme Q10, indole-3-carbinol,
proanthocyanidins, resveratrol, quercetin, catechins, salicylic
acid, curcumin, bilirubin. oxalic acid, phytic acid, lipoic acid,
vanilic acid, polyphenols, flavanoids, ferulic acid, theaflavins,
derivatives thereof, and other antioxidants.
[0090] In some embodiments, in addition to or in lieu of the
decreased production of ROS, the scavenging (e.g., processing) of
ROS is increased. In some embodiments, the activity of ROS
scavenging enzymes is increased. In some embodiments, superoxide
dismutase expression and/or function is increased. In some
embodiments, glutathione peroxidase expression and/or function is
increased. In still further embodiments, one or more of
lactoperoxidase, catalase, and peroxiredoxins expression and/or
function is increased.
[0091] In some embodiments, the culture methods reduce the
mitochondrial metabolism and therefore reduce the formation of ROS
and/or the leakage of ROS from the mitochondrial membrane. In some
embodiments intracellular concentration of adenosine triphosphate
(ATP) is reduced, either alone or in combination with a decrease in
mitochondrial membrane potential. However, in other embodiments,
the incidence of karyotypic abnormalities is reduced in the absence
of significant changes in ATP concentrations or mitochondrial
membrane potential.
[0092] In several embodiments, the culture methods described herein
decrease the DNA damage caused by ROS formation. In some
embodiments, one or more markers of DNA damage is reduced, as
compared to stem cells cultured under standard conditions and/or
with standard media. In some embodiments, .gamma.-H.sub.2AX mRNA
and/or protein expression is decreased when a certain concentration
range of antioxidant cocktail is employed, as discussed in more
detail below. Numerous other markers for various types of DNA or
histone damage that are known in the art may also be used to
evaluate the efficacy of the methods and compositions disclosed
herein.
[0093] As discussed above, some tissues have lower in vivo oxygen
concentrations. Thus, in some embodiments, the culturing method
comprises reducing the oxygen concentration in which the cells are
cultured. In some embodiments, in vitro O.sub.2 concentrations that
are substantially equivalent to in vivo O.sub.2 concentrations are
used. In some embodiments, O.sub.2 concentrations less than 20% are
used. In some embodiments, O.sub.2 concentrations of about 10-15%
are used, while in some embodiments, O.sub.2 concentrations of
about 13-19% are used. In other embodiments, O.sub.2 concentrations
of less about 10% are used, including 9%, 8%, 7%, and 6%. In still
other embodiments, O.sub.2 concentrations of about 5% or less are
used, including 4%, 3%, 2%, and 1%. In one embodiment, the closer
the culture conditions in which a stem cell is culture are to its
native oxygen conditions, the fewer karyotypic abnormalities will
manifest, as the cells is being cultured in what is effectively a
more natural environment. In contrast, non-physiological hyperoxic
conditions in culture may lead to oxidative stress, which, as
discussed above, may induce ROS formation. In other embodiments,
karyotypic abnormalities are reduced when O.sub.2 concentrations
are about 5-75% lower in vitro, as compared to the in vivo
environment.
[0094] In several embodiments, the culture method comprises
supplementation of culture media with an antioxidant cocktail.
Several embodiments of the composition of the cocktail are
discussed in more detail below. In some embodiments, the addition
of antioxidants increases the consumption (e.g., scavenging) of
ROS. In turn, the reduction in ROS reduces the amount of DNA
damage. In several embodiments, the addition of antioxidants
increases the innate DNA repair mechanisms already present in the
cell. In some embodiments, the function of the existing DNA repair
enzymes is upregulated, while in some embodiments, the expression
of one or more components of one or more DNA repair mechanisms is
increased.
[0095] Despite the negative effects of excessive ROS formation,
excessive reduction in ROS levels can also be damaging to cultured
cells. In one embodiment, significant reductions in ROS levels may
cause a downregulation of the innate DNA repair mechanisms (either
expression, function, or both). Though unexpected, excessive
reduction of ROS levels, as could be achieved by certain levels of
antioxidant supplementation, could induce DNA damage due to the
lack of sufficient DNA repair mechanisms or activity.
[0096] Advantageously, several embodiments of the compositions and
culture methods disclosed herein strike an optimal balance between
ROS reduction and maintenance of adequate levels of innate DNA
repair. In other words, in some embodiments, the culture methods
and compositions used herein decrease ROS levels sufficient to
reduce DNA damage due to oxidative stresses, but at the same time
do not reduce ROS levels to the degree that innate DNA repair
function is compromised. In several embodiments, antioxidant
compositions are added in a range of about 0.1 to about 1000 .mu.M.
While both high and low concentrations of antioxidants appear to
result in high levels of DNA damage, intermediate concentrations
unexpectedly result in reduced levels of DNA damage. In some
embodiments, DNA damage is reduced when an antioxidant cocktail is
added in a concentration of about 0.1 to about 200 .mu.M (e.g.,
about 0.1-20 .mu.M, 1-20 .mu.M, 5-20 .mu.M, 10-20 .mu.M, 10-30
.mu.M, 0.1-50 .mu.M, 1-50 .mu.M, 10-50 .mu.M, 25-50 .mu.M, and
overlapping ranges thereof). In some embodiments, the antioxidant
cocktail is added in a concentration of about 5 .mu.M to about 30
.mu.M. In some embodiments, about 10 .mu.M is used. In some
embodiments, about 20 .mu.M is used.
[0097] In several embodiments, compositions comprising various
antioxidant compounds are provided in order to decrease the
incidence of karyotypic abnormalities in the cultured cells, e.g.,
as disclosed above. Compositions disclosed herein may be used to
affect any of the methods disclosed herein, unless otherwise
specified. In some embodiments, the compositions are used to
supplement a culture media.
[0098] In several embodiments, the composition comprises one or
more non-peptide antioxidants. As used herein, the term
"non-peptide antioxidant" shall be given its ordinary meaning and
shall also be understood to include vitamins and other chemical
compounds capable of reducing ROS molecules, such as thiols (e.g.,
mercaptans) or polyphenols. In some embodiments, the composition
comprises one or more antioxidant vitamins, including, but not
limited to, vitamin A, vitamin C (ascorbic acid), and/or vitamin E.
In some embodiments, one or more of vitamin A, C or E are provided
at a concentration ranging from about 0.1 to about 1000 .mu.M. In
some embodiments, one or more of Vitamin A, C, or E are added in a
concentration ranging from about 1 to about 500 .mu.M. In some
embodiments, one or more of Vitamin A, C, or E are added in a
concentration ranging from about 0.1 to about 200 .mu.M (e.g.,
about 0.1-20 .mu.M, 1-20 .mu.M, 5-20 .mu.M, 10-20 .mu.M, 10-30
.mu.M, 0.1-50 .mu.M, 1-50 .mu.M, 10-50 .mu.M, 25-50 .mu.M, 50-100
.mu.M, 100-200 .mu.M, and overlapping ranges thereof). In some
embodiments, one or more of Vitamin A, C, or E are added in a
concentration ranging from about 75 to about 150 .mu.M, including
about 80, 90, 100, 110, 120, 130, and 140 .mu.M. In some
embodiments, one or more of Vitamin A, C, or E are added in a
concentration ranging from about 1 to about 50 .mu.M, including
about 5, 10, 20, 30, and 40 .mu.M. Other non-peptide antioxidants
include, but are not limited to: mixed carotenoids (e.g., beta
carotene, alpha carotene, gamma carotene, lutein, lycopene,
phytopene, phytofluene, and astaxanthin), selenium, Coenzyme Q10,
indole-3-carbinol, proanthocyanidins, resveratrol, quercetin,
catechins, theaflavins, salicylic acid, curcumin, bilirubin. oxalic
acid, phytic acid, lipoic acid, vanilic acid, polyphenols,
penicillamine, flavanoids, ferulic acid, theaflavins, derivatives
thereof, and other antioxidants. In several embodiments, such
non-peptide antioxidants are used in the concentrations described
above.
[0099] In several embodiments, the composition comprises one or
more peptide antioxidants. As used herein, the term "peptide
antioxidant" shall be given its ordinary meaning and shall also be
understood to include full-length proteins, protein fragments,
enzymes or polypeptides, amino acids, or poylpeptide precursor
molecules. In some embodiments, the peptide antioxidant is added in
addition to the non-peptide antioxidant described above. In other
embodiments, the peptide antioxidant (or the non-peptide
antioxidant) is used alone to supplement the culture media. In some
embodiments polypeptides such as glutathione are incorporated into
the composition. In some embodiments, glutathione is incorporated
into the composition at a concentration ranging from about 0.1 to
about 1000 .mu.M. In some embodiments, glutathione is added in a
concentration ranging from about 1 to about 500 .mu.M. In some
embodiments, glutathione is added in a concentration ranging from
about 0.1 to about 200 .mu.M (e.g., about 0.1-20 .mu.M, 1-20 .mu.M,
5-20 .mu.M, 10-20 .mu.M, 10-30 .mu.M, 0.1-50 .mu.M, 1-50 .mu.M,
10-50 .mu.M, 25-50 .mu.M, 50-100 .mu.M, 100-200 .mu.M, and
overlapping ranges thereof). In some embodiments, glutathione is
added in a concentration ranging from about 75 to about 150 .mu.M,
including about 80, 90, 100, 110, 120, 130, and 140 .mu.M. In some
embodiments, glutathione is added in a concentration ranging from
about 1 to about 50 .mu.M, including about 5, 10, 20, 30, and 40
.mu.M. Other peptide antioxidants include, but are not limited to:
superoxide dismutases, peroxiredoxins, thioredoxin, ceruloplasmin,
transferrins, hydroperoxide reductases, n-acetylcysteine and, in
several embodiments, are used in the concentrations described
above.
[0100] In several embodiments, methods for assessing a subject's
risk of developing a neoplastic disease are provided. In one
embodiment, the method comprises obtaining and evaluating a tissue
sample from the subject for evidence of karyotypic abnormalities
(e.g., DNA damage) and correlating any detected abnormalities with
the subject's antioxidant levels. Thereafter, the subject is
optionally treated to reduce or increase antioxidant levels as
needed. In several embodiments, DNA damage is detected by analysis
of markers of DNA damage, quantifying DNA damage-associated mRNA
expression levels, and/or quantifying DNA damage-associated protein
expression levels. In one embodiment, the risk of developing
neoplastic disease increases as the incidence of DNA damage
increases, as the expression of DNA damage-associated mRNA
increases, and/or as the expression of DNA damage-associated
protein increases. In some embodiments, identification of
.gamma.-H.sub.2AX foci, quantifying .gamma.-H.sub.2AX mRNA
expression levels, and/or by quantifying .gamma.-H.sub.2AX protein
expression levels, or combinations thereof, are used to detect
and/or quantify DNA damage.
[0101] In several embodiments, a method for optimizing a subject's
intake of an antioxidant-containing supplement is provided. In one
embodiment, the method comprises measuring the concentration of one
or more reactive oxygen species in a sample obtained from the
subject (e.g., a tissue or blood sample) and measuring the
incidence of DNA damage in cells isolated from said sample. In some
embodiments, the expression of DNA repair enzymes in cells isolated
from said sample is measured. In some embodiments, both DNA damage
and expression of DNA repair enzymes are measured.
[0102] In one embodiment, the method further comprises
administration of an antioxidant composition to the subject (and
optionally adjusting said composition in subsequent
administrations) in order to achieve an optimal degree of
homeostasis between the concentration of reactive oxygen species in
the subject and the subject's endogenous DNA repair mechanism(s).
Such a level of reactive oxygen species can be assessed by, for
example, expression of DNA repair enzymes or other markers of DNA
repair. In several embodiments, antioxidant compositions (e.g.,
supplements) comprising an optimal amount of antioxidant-containing
supplement are provided. Such compositions are formulated to reduce
DNA damage caused by oxidative-stress but to minimize negatively
impacting (wholly or partially) the subject's endogenous DNA repair
mechanisms. In several embodiments, the methods comprise serial
administrations of antioxidant compositions having different
concentrations of antioxidant compounds to a subject. In some
embodiments, the invention comprises optimizing antioxidant therapy
for an individual. In one embodiment, a first sample (e.g., tissue
or blood sample) is obtained from the subject prior to
administration, in order to establish a baseline. Thereafter, a
first antioxidant composition having a first concentration of an
antioxidant composition is administered. An additional sample is
obtained from the subject and evaluated for concentrations of
reactive oxygen species, markers of DNA damage, changes in
expression of DNA repair enzymes, or combinations thereof. In some
embodiments, a plurality of additional administrations of an
antioxidant composition (optionally having varying concentrations
of antioxidant compounds) may be made in conjunctions with
obtaining and evaluating a plurality of additional samples from the
subject. As such, an optimal concentration of antioxidant compounds
may be reached over time by the serial evaluation of the subject's
response to the antioxidant compositions. Thus, in several
embodiments, therapeutic antioxidant compositions that balance
oxidative damage with endogenous DNA repair mechanisms are provided
based on a subject's individual antioxidant and/or karyotypic
profile. Antioxidant compositions according to several embodiments
herein comprise, consist, or consist essentially of one, two,
three, five, ten, or more antioxidants.
EXAMPLES
[0103] Examples provided below are intended to be non-limiting
embodiments of the invention.
Materials And Methods
[0104] The following materials, methods, and protocols may be used
to perform the examples disclosed herein as well as practice the
various embodiments of the invention disclosed herein.
Long-Term Culture Conditions For Human CDCs
[0105] Adult human cardiac stem cells were isolated from
percutaneous septal endomyocardial heart tissue biopsies (about
10-20 .mu.g), which were obtained from patients during
clinically-indicated procedures (to monitor heart transplant
recipients for rejection) after informed consent. Biopsies were
minced into small fragments and digested with 0.2 mg/ml collagenase
for 30 minutes. The digested tissue fragments were then equally
moved to each of four 6-cm diameter culture dishes coated with 20
.mu.g/ml fibronectin (BD Biosciences), and randomly selected to
culture as "explants" in the following four conditions: [0106] 1)
in a typical 20% O.sub.2 incubator (95% air/5% CO.sub.2); [0107] 2)
in a 5% O.sub.2 "hypoxia incubator"; [0108] 3) in a typical 20%
O.sub.2 incubator with the addition of 1000-fold diluted
proprietary antioxidant supplement (Sigma-Aldrich, Catalogue
Number: Sigma A1345, Antioxidant A); [0109] or [0110] 4) a custom
antioxidant cocktail consisting of 100 .mu.M L-ascorbate,
L-glutathione, and alpha-tocopherol acetate (Sigma-Aldrich,
Antioxidant B).
[0111] The cardiosphere and CDC amplification steps were performed
under the same conditions in each group. IMDM basic medium (Gibco)
supplemented with 10% FBS (Hyclone) and 20 mg/ml gentamycin was
used for all cultures. As used herein, the term "short-term
culture" shall be given its ordinary meaning and also be read to
include culture periods ranging from about 1 to about 48 hours,
including, 8, 16, 20, 24, 32, 36, and 40 hours. As used herein, the
term "long-term culture" shall be given its ordinary meaning and
also be read to include culture periods ranging from 48 hours to 6
months. In some embodiments, long-term culture lasts for several
days to several months, including 2, 3, 4, and 5 months. In some
embodiments, long-term culture lasts for several days to several
weeks, include 1-3 weeks, 2-5, weeks, 3-7 weeks, and 5-10 weeks. In
some embodiments long-term culture lasts for about 6 to about 10
months, and in some embodiments from about 10 months to several
years.
Karyotype Analysis
[0112] Twice-passaged CDCs (with long-term culture for 1-2 months
from the date of tissue biopsy) were seeded onto fibronectin-coated
25-cm.sup.2 tissue culture flask (10.sup.4 cells/cm.sup.2). After
.about.24 hours of incubation, cells were treated with 0.1 .mu.g/ml
colcemid (Invitrogen) for 4 hours, then trypsinized, treated with
hypotonic solution, and fixed. Metaphases were spread on microscope
slides, and karyotype analysis was done by using standard G banding
technique. The chromosomes were classified according to the
International System for Human Cytogenetic Nomenclature. At least
20 metaphases were analyzed per cell sample.
Determination of ROS Levels
[0113] To assay intracellular ROS levels (ROS levels),
twice-passaged CDCs were seeded in 6- or 96-well plates coated with
20 .mu.g/ml fibronectin, and continuously cultured under the
abovementioned four different conditions. At about 90% confluence,
cells were incubated with 10 .mu.M 2',7'-dichlorodihydrofluorescein
diacetate (DCFH-DA) (Invitrogen) for 60 min to allow DCFH-DA to
diffuse into cells. The DCF fluorescence intensity in cells
cultured in 96-well plates is directly determined using
SpectraMax.RTM. M5 (Molecular Devices Corp.) with an excitation
wavelength of 495 nm and an emission wavelength of 520 nm. Cells
cultured in 6-well plates were trypsin-treated and fixed. The DCF
fluorescence intensity in cells was analyzed using a FACS Calibur
flow cytometer with CellQuest software (BD Biosciences).
[0114] To observe the changes of ROS levels during short-term
exposure to different concentrations of antioxidants, catalase, and
H.sub.2O.sub.2, CDCs were expanded by traditional conditions under
20% O.sub.2 as described above. Twice-passaged cells were seeded in
6- or 96-well plates coated with 20 .mu.g/ml fibronectin. When
about 70% confluent, cell cultures were supplemented with
antioxidant A (100-1,000,000-fold dilution), custom antioxidant
cocktail "B" (0.1-1000 .mu.M), catalase (0.1-1000 units/ml), or
H.sub.2O.sub.2 (0.1-1000 .mu.M), as indicated. After 24 hours of
culture, the DCF fluorescence intensity in cells was measured using
the same methods as disclosed above.
Analysis of DNA Damage In CDCs And ES Cells
[0115] DNA damage in human CDCs and ES cells was evaluated by
immunostaining for phosphorylation of histone H.sub.2AX on serine
139 (.gamma.-H.sub.2AX), a marker of DNA double-strand breaks,
after short-term culture with different concentrations of
antioxidants, catalase, and H.sub.2O.sub.2. CDCs were expanded in
conventional conditions under 20% O.sub.2 as described above.
Twice-passaged CDCs and ES cells were used in these Examples. Cells
were seeded in 96-well plates coated with 20 .mu.g/ml fibronectin.
When about 70% confluent, cell cultures were supplemented with
antioxidant A (100-1,000,000-fold dilution), custom antioxidant
cocktail "B" (0.1-1000 .mu.M), catalase (0.1-1000 units/ml), or
H.sub.2O.sub.2 (0.1-1000 .mu.M), as indicated. After 24 hours of
culture, cells were fixed, permeabilized and stained with rabbit
polyclonal antibody against .gamma.-H.sub.2AX (phosphor S139, Abcam
Inc.). After being washed, the cells were stained with a
PE-conjugated secondary antibody and 4,6-diamidino-2-phenylindole
(DAPI). Quantification of cells positive for .gamma.-H.sub.2AX foci
was performed by fluorescence microscopy (.times.40 magnification).
Briefly, at least 5 images were captured from each culture
condition from randomly-selected fields using Q-imaging (RETIGA EXi
FAST, Canada) with the same exposure time. Cells with
.gamma.-H.sub.2AX foci in the nuclei were counted by a single
observer blinded to treatment regimen, and the percentage of cells
with .gamma.-H.sub.2AX foci in each culture condition was used for
statistical analysis.
[0116] To quantify DNA damage in long-term cultured CDCs under the
abovementioned four different conditions, twice-passaged CDCs were
seeded in 6- or 96-well plates coated with 20 .mu.g/ml fibronectin,
and continuously cultured for 24 hours. The analysis of
.gamma.-H.sub.2AX foci was done as described above.
Western Blotting
[0117] To examine the protein levels of ATM and other DNA
repair-related factors, total protein was purified from
twice-passaged CDCs cultured under the abovementioned four
different conditions, using well-established laboratory techniques.
Briefly, harvested cells were homogenized in a lysis buffer
containing a protease inhibitor mixture (Roche Applied Science) on
ice. After centrifugation at 15,000 rpm for 10 min, the supernatant
was collected for experiments. The equivalent of 30 .mu.g of total
protein was loaded onto 5% or 10% SDS-PAGE gels, and then
transferred to PVDF membranes. After overnight blocking in 3% milk
TBS-T, membranes were incubated with the following primary
antibodies: 1:5000 dilution of rabbit anti-ATM polyclonal antibody,
1:1000 dilution of rabbit anti-ATR polyclonal antibody, 1:500
dilution of rabbit anti-Chk1 (phosphor S317) polyclonal antibody,
1:200 dilution of rabbit anti-Chk2 (phosphor T26) polyclonal
antibody, 1:1000 dilution of mouse anti-Rad50 monoclonal antibody,
1:1000 dilution of mouse anti-Rad51 monoclonal antibody (all from
Abcam Inc.) and 1:3000 dilution of rabbit anti-B-actin monoclonal
antibody. The appropriate horseradish peroxidase-conjugated
secondary antibodies were used, and then the blots were visualized
by using SuperSignal West Femto maximum sensitivity substrate
(Thermo Scientific)) and exposed to Gel Doc XR System (Bio-Rad
Lab., Inc.). Quantitation for blots was done by Quantity One
software, and expressions were normalized by .beta.-actin.
[0118] To observe the expression of ATM and other DNA
repair-related factors during short-term exposure to different
concentrations of antioxidants, catalase, and H.sub.2O.sub.2, CDCs
were expanded by traditional culture in 20% O.sub.2 as described
above. Twice-passaged cells were seeded in 6-well plates coated
with 20 .mu.g/ml fibronectin. When about 70% confluent, cells were
cultured in 20% O.sub.2 with the supplement of antioxidant A
(100-1,000,000-fold dilution), custom antioxidant cocktail "B"
(0.1-1000 .mu.M), catalase (0.1-1000 units/ml), or H.sub.2O.sub.2
(0.1-1000 .mu.M), as indicated. After 24 hours of culture, cells
were harvested and total protein was purified. The expression of
ATM and other DNA repair-related factors was assessed by Western
blotting as described above.
Measurement of the Intracellular ATP Level And Mitochondrial
Transmembrane Potential
[0119] The intracellular ATP level was measured by the
luciferin-luciferase method using an ATP-determination kit
(Invitrogen). Briefly, twice-passaged CDCs (2.times.10.sup.5
cells/well) were seeded in 6-well plates coated with 20 .mu.g/ml
fibronectin, and continuously cultured under the abovementioned
four different conditions for 24 hours. The cells were washed twice
with ice-cold PBS and lysed in 200 .mu.l lysis buffer with protease
inhibitors. The lysates (20 .mu.l normalized by protein content)
were added to the reaction solution (200 .mu.l) containing 0.5
.mu.M luciferin, 1.25 .mu.g/ml luciferase, and 1 .mu.M DTT, and the
bioluminescence was measured using a Monolight.TM. 3010
(Pharmingen).
[0120] To measure mitochondrial transmembrane potential,
twice-passaged CDCs (2.times.10.sup.5 cells/well) were seeded in
6-well plates coated with 20 .mu.g/ml fibronectin, and continuously
cultured under the abovementioned four different conditions for 24
hours. Cells were loaded with TMRE at 37.degree. C. for 30 minutes,
and harvested by trypsinization. The fluorescence intensity in
cells was analyzed using a FACS Calibur flow cytometer with
CellQuest software (BD Biosciences).
Statistical Analysis
[0121] All results are presented as mean.+-.SD. Statistical
significance was determined using the 2-tailed chi-square test for
karyotype data and ANOVA followed by Bonferroni post hoc test for
other data (Dr. SPSS II). Differences were considered statistically
significant when p<0.05.
Example 1
Genomic Alterations Decrease In Physiological Oxygen But
Unexpectedly Increase With Antioxidant Supplements
[0122] Source human heart biopsies (n=16) were divided and
processed in parallel in the various culture conditions,
facilitating direct comparisons. CDCs grown under conventional
conditions not infrequently included cells with genomic alterations
(6 of 16 samples; FIG. 1, Table 1 and Table 2). In reference to
FIG. 1, each bar represents a histogram of one sample of stem
cells; blue denotes cells with a normal karyotype. Compared with
culture in traditional 20% O.sub.2 incubator (95% room air/5%
CO.sub.2), the number of cells with DNA breaks or translocations
(colored green) and losses or gains of chromosomes (red) was
decreased when cells were cultured in 5% O.sub.2 (p=0.007). When
CDCs were cultured in 5% O.sub.2, genomic alterations were detected
in only 3 of 16 samples (FIG. 1, Table 2). The genomic changes were
relatively innocuous: one sample contained one cell with a balanced
translocation, another had 8 cells with a derivative chromosome,
and the third included one cell with loss of the Y chromosome (FIG.
1, Table 2). The reduction of the frequency of chromosomal
abnormalities in CDCs cultured in 5% O.sub.2 indicates that
physiological oxygen concentrations during culture enhance the
genomic stability of stem cells.
TABLE-US-00002 TABLE 2 Karyotyping Date of Twice-Passaged Human
CDCs Samples 20% O.sub.2 Antioxidant A Antioxidant B 5% O.sub.2
CSB59m 46, XY[20] 46, XY[20] 46, XY[20] 46, XY[20] CSB59s 46,
XY[20] 45, X - Y[10]/46, XY[10] 46, XY[20] 46, XY[20] CSB61m 46,
XY[20] 47, XY, +7[3]/46, XY[17] 47, XY, +7[3]/46, XY[18] 46, XY[20]
CSB61s 46, XY[20] 47, XY, +7[1]/46, XY[19] 46, XY[20] 46, XY[20]
CSB64m 46, XY[20] 48, XY, +2, +20[4]/46, XY, 46, XY[20] 46, XY[20]
der(13)t(9; 13)(q12; p11, 2)[1]/46, XY[15] CSB64s 46, XY[20] 46,
XY[20] 46, XY[20] 46, XY[20] CSB65m 47, XY, +8[9]/48, s1, +2, -9,
46, XY[20] 46, X, +8[7]/47, XY, +12 46, XY[20] der(13)t(9; 13)(q12;
p11.1), [10]/46, XY[3] +14[1]/46, XY[11] CSB65s 45, X, -Y[2]/47,
XY, 47, XYY[4]/46, XY[16] 47, XYY[11]/47, XY, +2 46, XY[20]
+8[1]/46, XY[20] [1]/47, XY, +8[5]/46, XY[3] CSB66m 47, XX, +2[1]/
46, XX[20] 46, XX 46, XX[20] 47, XX, m[1]/46, XX[18] CSB66s 46,
XX[20] 47, XX, +8[1]/46, XX[19] 46, XX, add(1)(p36.1)[3]/ 46,
XX[20] 46, XX[18] CSB69m 47, XY, +8[2]/45, X, -Y 47, XY, +8[5]/46,
XY[15] 47, XY, +8[4]/ 46, XY, der(18)t(14; 18) Y[1]/46, XY[18] 46,
XY[16] (q22; q23)[3]/46, XY[17] CSB69s 46, XY[20] 47, XY,
+8[17]/46, XY[3] 47, XY, +8[5]/ 46, XY, der(18)t(14; 18) 46, XY[15]
(q22; q23)[5]/45, X, -Y [1]/46, XY[15] CSB73m 46, XY[20] ND ND 46,
XY, t(6; 19)[1]/ 46, XY[19] CSB73s 46, XY[20] ND ND 46, XY[20]
CSB76m 45, X, -Y[6]/46, XY[14] ND ND 46, XY[20] CSB76s 45, X,
-Y[1]/47, XY, ND ND 46, XY[20] +8[1]/46, XY[18]
[0123] In contrast, karyotypic abnormalities were dramatically
increased in frequency and severity when CDCs were cultured in 20%
O.sub.2 with either of two antioxidant cocktails: a proprietary
antioxidant supplement for cell culture (product A1345,
Sigma-Aldrich, 1000-fold dilution; Antioxidant A), or a custom
antioxidant mixture (L-ascorbate, L-glutathione, and
.alpha.-Tocopherol acetate, each 100 .mu.M; Antioxidant B) (FIG. 1,
Table 2). Among the 12 samples of CDCs cultured with Antioxidant A
or B, 8 and 6 samples included 46 and 49 cells with genomic
alterations, respectively (p<0.001 vs. 20% O.sub.2 culture by
chi-square test, FIG. 1).
[0124] Unlike conventional 20% O.sub.2 culture, where trisomy 8 and
loss of Y predominated, the karyotypic abnormalities seen with
antioxidants were numerous and varied (trisomy 2, 7, 8, 12, 18, and
20). FIG. 2 depicts (A) CDCs cultured in a traditional 20% O.sub.2
incubator (95% air/5% C O.sub.2), (B) gain of chromosome 7 in the
presence of Antioxidant A, and (C) gains of chromosome 2 and 20 in
the presence of Antioxidant B. To the Applicant's knowledge, some
of these, namely trisomy 7 and trisomies 2 and 20 have not been
previously reported (FIG. 2).
[0125] The effects of antioxidants on genomic stability appear not
to reflect generalized toxicity, as CDCs proliferated normally
without obvious morphologic abnormalities (FIG. 3). Panel A depicts
twice-passaged CDCs grown for 36 days in 20% O.sub.2 without
addition of antioxidants. Panel B depicts CDCs grown in the same
O.sub.2 concentration in media supplemented with an antioxidant
supplement at 1:1000 dilution. Panel C depicts CDCs grown in the
same O.sub.2 concentration in media supplemented with a custom
antioxidant cocktail at 100 .mu.M. Panel D indicates that that no
significant differences in CDC proliferation were detected among
the various culture conditions.
[0126] Furthermore, neither the intracellular ATP level nor
mitochondrial transmembrane potential showed any obvious
differences among the four various culture conditions (FIGS. 4A and
B, respectively). Indeed, the similarities in cell proliferative
activity and intracellular ATP levels indicate that energy
metabolism is not severely undermined in any of the long-term
culture conditions.
Example 2
Antioxidants Decrease Intracellular ROS Monotonically But DNA
Damage Shows A Biphasic Response In Stem Cells
[0127] CDCs were initially maintained in traditional 5%
CO.sub.2/20% O.sub.2 culture condition and then seeded into 96-well
plates and cultured for 24 hours under experimental conditions.
Intracellular ROS levels in CDCs exposed for 24 hours to a wide
range of antioxidant concentrations (A-B), with catalase (a pure
ROS scavenger, C) and hydrogen peroxide (H.sub.2O.sub.2, a powerful
oxidant, D) as controls were measured. The results shown in FIG. 5
are means.+-.Std. Dev. for six separate experiments using different
twice-passaged CDCs. (a.u.: arbitrary units. *p<0.01,
.dagger.p<0.05 vs. the baseline levels., represented by "0" on
the x-axis). The results shown in FIG. 6 are representative
histograms of the intracellular ROS data obtained by flow
cytometry
[0128] Catalase decreased, and H.sub.2O.sub.2 increased, ROS levels
in a progressive dose-dependent manner (FIG. 5C-D, FIG. 6C-D). Like
catalase, antioxidants A and B both decreased ROS levels
monotonically (FIG. 5A-B, FIG. 6A-B). At the concentrations used to
culture CDCs for karyotyping analysis (1000-fold dilution of
antioxidant A and 100 .mu.M antioxidant B, respectively), ROS
levels was very low.
[0129] Likewise, ROS levels was depressed in CDCs sent for
karyotyping analysis after 1-2 months in 5% oxygen, and even more
so in antioxidants, relative to routine culture conditions (FIG.
7). As shown in Panel A, the intracellular ROS was lower in cells
cultured long-term under 5% O.sub.2 than under 20% O.sub.2.
Intracellular ROS was decreased to very low levels by the addition
of 1000-fold diluted antioxidant supplement (Antioxidant A) or 100
.mu.M custom antioxidant cocktail (Antioxidant B), as shown in
Panel B. Shown in Panel C are representative images of
.gamma.-H.sub.2AX foci in CDCs (arrows) cultured for long-term
under different conditions. As depicted in Panel D, compared to
traditional 20% O.sub.2 culture, quantitative data showed that
.gamma.-H.sub.2AX foci in CDCs was significantly decreased in 5%
O.sub.2 culture, but increased by the supplement with antioxidants.
*p<0.01 vs. other groups, .dagger. p<0.01 vs. 5% O.sub.2 and
20% O.sub.2. Thus, assuming that ROS levels in CDCs cultured under
5% O.sub.2 approximates the physiological intracellular
concentration, long-term culture with antioxidants suppresses ROS
levels to sub-physiological levels (FIGS. 5, 6, and 7).
[0130] The concept of "reductive stress" (an extreme suppression of
ROS) may underlie a form of cardiomyopathy due to protein
aggregation. To identify whether an excessive decrease of ROS
levels likewise induces DNA damage and genomic instability,
.gamma.-H.sub.2AX foci was quantified in CDCs and human ES cells
(FIG. 8). .gamma.-H.sub.2AX is a marker of DNA double-strand breaks
and was detected in several CDCs whether cultured with 1:1000
diluted antioxidant supplement (Panel A), with 100 .mu.M custom
antioxidant cocktail (Panel B), with 100 units/mL catalase (Panel
C), or with 100 .mu.M H.sub.2O.sub.2 (Panel D).
[0131] It was also demonstrated that oxidative stress induced by
H.sub.2O.sub.2 increased DNA damage dose-dependently (FIG. 9D).
Here, however, the effects were unexpectedly biphasic: the
percentage of CDCs with .gamma.-H.sub.2AX foci was decreased at low
antioxidant concentrations, but increased at higher doses (FIG.
9A-B). A similar result was observed with increasing concentrations
of catalase (FIG. 9C). The biphasic response is not limited to
CDCs: human ES cells exhibited a similar pattern (red crosses with
dotted lines, FIG. 9A-C), although the overall percentages of ES
cells with .gamma.-H.sub.2AX foci were lower than in adult CDCs.
Interestingly, the number of .gamma.-H.sub.2AX foci was minimal at
modest concentrations of antioxidants (10,000-fold dilution of
antioxidant A, 10 .mu.M of antioxidant B, and 10 units/ml of
catalase; FIG. 9A-C), the same concentrations that drive ROS levels
to "physiological" levels. These results motivate the concept of an
"oxidative optimum", a narrow range of ROS levels within which stem
cells maintain optimal genomic stability.
[0132] This dose-response data correlated with the results of FIGS.
7C and 7D discussed above, where .gamma.-H.sub.2AX foci were
significantly decreased in 5% O.sub.2 culture, but increased by
supplementation with 1000-fold diluted antioxidant supplement
(Antioxidant A) or 100 .mu.M custom antioxidant cocktail
(Antioxidant B), when compared to traditional 20% O.sub.2 culture
(FIG. 7C, D).
Example 3
Extreme Suppression of Intracellular ROS Down-Regulates ATM And
Other DNA Repair Factors
[0133] The protein kinase ATM (ataxia-telangiectasia mutated), is
believed to play a role in DNA repair. Intracellular ROS enhances
the expression of ATM, which phosphorylates a host of downstream
targets in response to DNA double-strand breaks, inducing cell
cycle arrest and inhibiting apoptosis. It is possible that
excessive suppression of ROS levels might down-regulate ATM,
thereby promoting genomic instability. ATM protein levels were
indeed decreased at high concentrations of antioxidants A and B
(.gtoreq.1000-fold dilution, (>100 .mu.M, respectively), or
catalase (.gtoreq.100 units/ml) (FIG. 10A-C). As shown in Panel A,
the protein levels of ATM in CDCs were decreased at high doses
(.ltoreq.1000-fold dilution), but not at low doses
(.gtoreq.10,000-fold dilution) of antioxidant supplements
(Antioxidant A). Panel B shows that ATM protein in CDCs decreases
at .gtoreq.100 .mu.M, but not at .ltoreq.10 .mu.M, of custom
antioxidant cocktail (Antioxidant B). Panel C shows that catalase
also decreases ATM protein level in CDCs at .gtoreq.100 units/ml,
but not at .ltoreq.10 units/mL. Finally, Panel D shows that the
expression of ATM in CDCs was slightly increased at a high dose
(.gtoreq.100 .mu.M) of H.sub.2O.sub.2. Quantitative data are
means.+-.standard deviation for four separate experiments using
different twice-passaged CDCs.
[0134] Various other DNA repair factors, including ATR and
downstream factors Rad50, Rad51, Chk1, and Chk2, were also
decreased by antioxidants (FIGS. 11 and 12), at the same
concentrations that suppressed ROS levels significantly. As shown
in FIG. 11, Panel A (antioxidant supplement), Panel B (custom
antioxidant cocktail), Panel C (catalase), the expression levels of
ATR, Rad50, Rad51, Chk-1, and Chk-2 were slightly or obviously
decreased by high concentrations of catalase or antioxidants. In
contrast, as shown in Panel D, expression levels were increased by
high-dose H.sub.2O.sub.2. FIG. 12 shows representative quantitative
histograms of the expression data from FIG. 11.
[0135] According to one embodiment of the invention, the expression
levels of ATM and other DNA repair-related factors were
down-regulated when ROS levels were decreased to
"sub-physiological" levels at very high concentrations of
antioxidants. In one embodiment, a low concentration of
antioxidants drives ROS levels to an optimal "physiological" range,
which reduces oxidative stress-induced DNA damage without impairing
the DNA repair system. In one embodiment, given that ATM protein
levels fell within 24 hours of exposure to antioxidants,
physiological ROS levels concentrations may stabilize ATM and other
DNA repair-related protein kinases, consistent with the notion that
reductive stress induces intracellular protein aggregation.
According to one embodiment, transcriptional downregulation plays
an additional or supplemental role. In one embodiment, while ATM
and related DNA repair factors may underlie the genomic instability
seen with high antioxidants, the levels of these proteins do not
change when CDCs are grown in certain conditions (FIG. 13).
According to one embodiment, as shown in FIG. 13A, as compared to
cells cultured under traditional 20% O.sub.2, ATM expression did
not significantly change in cells cultured under 5% O.sub.2, but
was significantly decreased by the addition of 1000-fold diluted
antioxidant supplement (Antioxidant A) or 100 .mu.M custom
antioxidant cocktail (Antioxidant B). According to one embodiment,
as shown in FIG. 13 B, expression of ATR, Rad50, Rad51, Chk1, and
Chk2 did not significantly change in cells cultured under 5%
O.sub.2. However, according to some embodiments, many of these
factors were decreased in cells cultured under traditional 20%
O.sub.2 with the addition of 1000-fold diluted antioxidant
supplement (Antioxidant A) or 100 .mu.M custom antioxidant cocktail
(Antioxidant B).
[0136] According to several embodiments, the invention comprises
compositions and methods to balance DNA injury during oxidative
stress and faulty DNA repair in reductive stress.
[0137] According to several embodiments, a biphasic relationship
between intracellular ROS levels and genomic stability in human
cardiac and ES cells is provided. In one embodiment, a modest ROS
suppression by culture in physiological oxygen (5%) decreases
karyotypic abnormalities, but profound ROS suppression by
antioxidant supplements paradoxically enhances genomic alterations.
FIG. 14 depicts schematically, one possible model for such results.
In one embodiment, oxidative stress induces DNA damage, accounting
for the high frequency of karyotypic abnormalities in 20% O.sub.2
culture (FIG. 14, right panels). On the other hand, excessive
suppression of ROS to subphysiological levels down-regulates DNA
repair pathways, thereby contributing to genomic instability (FIG.
14, left panels). Thus, according to one embodiment, optimal
"physiological" levels of ROS are provided for activation of DNA
repair pathways to maintain genomic stability in stem cells (FIG.
14, center panels). In one embodiment, excessive inhibition of ROS
production leads to defective DNA repair and, thereby, increased
frequency of karyotypic abnormalities seen with high antioxidants.
In one embodiment, maintenance or enhancement of DNA repair
pathways using the compositions disclosed herein contribute to the
reduction in random (rather then systematic) chromosomal changes.
In other embodiments, reduction of systematic chromosomal changes
is achieved. In yet other embodiments, reduction in both random and
systematic chromosomal changes is achieved.
[0138] According to some embodiments of the invention, CDCs derived
from post-transplant patients are used. In one embodiment, such
CDCs exhibit chromosomal abnormalities at a higher frequency than
non-immunosuppressed patients. Thus, in several embodiments, the
invention is particularly beneficial for reducing chromosomal
damage in cells obtained from subjects with suppressed immune
systems or other conditions that enhance karyotypic
abnormalities.
[0139] Several embodiments of the invention are unexpected because
free radicals are popularly viewed as harmful by-products of cell
metabolism, and antioxidant dietary supplements, even at high
doses, are touted to be beneficial in the prevention of cancer and
a host of other diseases. In one embodiment, a balanced formulation
comprising vitamin C, vitamin E, and glutathione prevents the
extreme suppression of ROS by high-dose antioxidants which would
otherwise increase DNA damage and genomic instability associated
with down-regulation of DNA repair-related protein kinases. Several
embodiments of the invention facilitate the dual role of ROS, in
which a physiological level of ROS is aids in effective DNA repair,
but high ROS induces DNA damage. In one embodiment, compositions or
hypoxic conditions disclosed herein reduce the incidence of
karyotypic abnormalities in cells by more than 10%, 25%, 50%, 75%,
or 95% as compared to control cells.
Example 4
Effect of Physiologic Oxygen Concentrations On Stem Cell
Proliferation
[0140] In order to determine other effects of that culturing in
physiologic oxygen has, CDCs were generated in duplicate from 13
clinical biopsy samples. One portion of the tissue was used to
generate cells which were cultured in room oxygen, while another
portion was cultured in physiologic oxygen. The mean CDC yield for
room oxygen cultures was 77.1.+-.80.3 million from a mean starting
mass of 263.+-.185 mg of tissue. This yield was achieved in a mean
time of 35.1.+-.6.4 days. During the same average manufacturing
time, mean CDC yield increased 3.2-fold for specimens cultured in
physiologic oxygen. Moreover, the amount of starting material
decreased 6.1-fold. The per mg yield was therefore 19.6-fold
higher. To confirm these findings, parallel runs with the same
amount of human starting tissue (.about.60 mg) were performed.
Culture in physiologic oxygen conditions produced over six times
more CDCs than room oxygen conditions (see FIG. 15).
[0141] In several embodiments, physiological oxygen concentrations
also reduce the amount of time needed in culture to generate a
given number of cells. Thus, in several embodiments, use of
physiological oxygen concentrations is particularly advantageous
because such conditions increase the overall yield of CDCs. In some
embodiments, reduced quantities of starting cardiac tissue are
required. In some embodiments, this is advantageous because a given
sample has a greater capacity to produce CDCs, which increases the
number of CDCs that are available for therapy and/or banking.
[0142] As discussed above, CDCs have been shown to occasionally
acquire cytogenetic abnormalities, most frequently aneuploidy,
during culture. In still additional embodiments, physiological
oxygen concentrations also reduce decrease the incidence of one or
more of aneuploidy, DNA breaks, base-pair mismatches, or
translocations (see, e.g., FIGS. 1 and 16). Therefore, in several
embodiments CDCs are cultured in physiologic oxygen to reduce
cytogenetic abnormalities.
Example 5
In Vivo Efficacy of CDCs Grown In Physiologic Oxygen
[0143] As discussed above, culture conditions comprising
physiological oxygen levels are used in some embodiments. The in
vivo efficacy of CDCs cultured in physiologic oxygen was tested in
comparison to CDCs cultured in room oxygen (20%). A mouse
myocardial infarction model was used. In brief, myocardial
infarction (MI) was created by ligation of the mid-left anterior
descending coronary artery. Cells were delivered intramyocardially
by direct injection at two peri-infarct sites immediately following
ligation. CDCs (105) were injected in calcium-free PBS (5-7 .mu.L
at each site), with 10.sup.5 normal human dermal fibroblasts
(NHDFs) or PBS as control. Cardiac function measured 3 weeks
post-MI, indicated that CDCs grown in physiologic oxygen were
yielded improved left ventricular ejection fraction (LVEF) as
compared to CDCs grown in room oxygen (see FIG. 17). Both
CDC-treated groups outperformed the PBS group. These data indicate
that CDC potency is enhanced when culture is performed under
physiologic oxygen conditions, as is done in several embodiments.
In several embodiments, the potency of CDCs cultured in physiologic
oxygen is increased in a statistically significant manner. In some
embodiments, the potency of CDCs cultured in physiologic oxygen is
increased by at least about 5%, about 10%, about 15%, about 20%,
about 25% or greater.
Example 6
Expansion of Human Cardiac Stem Cells In Physiologic Oxygen
Improves Cell Production, Efficiency, And Potency For Myocardial
Repair
[0144] Further to the discoveries detailed above, additional
studies were conducted to obtain additional details regarding the
impact of culturing human cardiac stem cells in physiologic
concentrations of oxygen.
[0145] Expansion of cells in culture is the foundation for a wide
variety of applications of adult stem cells and embryonic stem (ES)
cells, including regenerative medicine and drug development.
Chromosomal abnormalities are frequently found in stem cells after
long-term culture and such chromosomal abnormalities may enhance
carcinogenesis and impair functional potency, thereby complicating
the therapeutic application of stem cells. Therefore, it is
important to expand stem cells with fewer chromosomal abnormalities
for clinical applications. Resident cardiac stem cells are
considered to be particularly promising for myocardial
regeneration, as they mediate cardiogenesis and angiogenesis. In
several embodiments, the methods and compositions disclosed herein
allow the expansion of a relatively small number of cells into a
larger population (e.g., tens of millions of cells in some
embodiments) of cardiac stem cells and supporting cells
(collectively termed CDCs) using ex vivo culture. As discussed
above, in some embodiments, the initial sample of cardiac tissue is
from a minimally invasive human heart biopsy. In other embodiments,
other tissue sources are used (e.g., surgically obtained cardiac
tissue, donor cardiac tissue, etc.). While clinical application of
CDCs is presently under way in the CADUCEUS (CArdiosphere-Derived
aUtologous Stem CElls to Reverse ventricUlar dySfunction,
ClinicalTrials.gov. Identifier NCT00893360) trial, karyotyping
revealed that approximately one-third of preliminary CDC production
runs included cytogenetically abnormal cells, most often due to
changes in chromosome number (aneuploidy; see e.g., FIG. 1).
[0146] As discussed above, ex vivo expansion of stem cells,
including CDCs, is generally performed by incubating cells in
incubators equilibrated with 95% air and 5% CO.sub.2 (.about.20%
O.sub.2). However, the oxygen concentration of the in vivo
microenvironment of stem cells in biological tissues is often much
lower, ranging from about 1% to about 8%, (depending on the
tissue). As a result, stem cells for use in a tissue having a low
oxygen microenvironment in vivo may suffer from oxidative stress
under such conventional culture conditions. Given that oxidative
stress may play a role in DNA damage and genomic instability, the
present study investigated the effects of ex vivo expansion of
human cardiac stem cells in `hypoxic` conditions (mimicking the low
oxygen tensions operative in vivo)
Ex Vivo Expansion of Human Cardiac Stem Cells Under Low Oxygen
[0147] Adult human cardiac stem cells were expanded using similar
methods as described above. Briefly, endomyocardial heart tissue
biopsies (.about.10 mg) were obtained from patients during
clinically indicated procedures after informed consent, in an
Institutional Review Board-approved protocol. As discussed above,
in some embodiments, non-biopsy tissue sources are used. All
investigations conform to the Declaration of Helsinki. Biopsies
were minced into small fragments and digested with 0.2 mg/mL of
collagenase for 30 min. The digested tissue fragments were then
equally divided and moved to two 6 cm diameter culture dishes
coated with 20 mg/mL of fibronectin (BD Biosciences) and randomly
selected to culture as `explants` in a typical CO.sub.2 incubator
(20% O.sub.2) or a incubator with physiological low oxygen (5% 02).
Following cardiosphere formation, CDCs were grown and expanded by
passaging under 20% O.sub.2 (20% O.sub.2 CDCs) or 5% O.sub.2 (5%
O.sub.2 CDCs), respectively. IMDM basic medium (Gibco) supplemented
with 10% FBS (Hyclone) and 20 mg/mL gentamycin was used for all
cultures. Twice-passaged CDCs (1-2 months culture from the date of
tissue biopsy) were used for experiments unless otherwise
indicated.
Karyotype Analysis
[0148] CDCs were seeded onto fibronectin-coated 25 cm.sup.2 tissue
culture flasks (10.sup.4 cells/cm.sup.2) and continuously cultured
under 5% O.sub.2 or 20% O.sub.2, respectively. The karyotyping data
in this example are extended from those discussed above. After
.about.24 h of incubation, cells were treated with 0.1 mg/mL of
colcemid (Invitrogen) for 4 h. Then the cells were trypsinized,
treated with hypotonic solution, and fixed. Metaphases were spread
on microscope slides, and karyotype analysis was done using a
well-established G banding technique. The chromosomes were
classified according to the International System for Human
Cytogenetic Nomenclature. At least 20 metaphases were analyzed per
cell sample. Notably, all but one of the patients whose samples
underwent karyotypic analysis were immunosuppressed heart
transplant recipients. Cytogenetic abnormalities may have been more
frequent in the immunosuppressed heart transplant patient
population which predominated in the samples studied, as
immunosuppressed post-transplant patients may be predisposed to
lymphomas with clonal chromosomal abnormalities. Notably, in the
immunocompetent population studied in CADUCEUS, only 1 of 13
clinical-production samples to date has been abnormal by
cytogenetic screening (unpublished results). However, based on the
reduction of chromosomal abnormalities in 5% O.sub.2 culture
according to several embodiments, disclosed herein, cytogenetic
screening may no longer be required as a clinical-grade product
release criterion. In some embodiments, reduction in chromosomal
abnormalities due to culturing in physiological oxygen
concentrations results, regardless of the immune status of the
subject from whom the original tissue samples were collected.
Advantageously, this broadens the scope of individuals who can
serve as tissue donors for generation of cardiac stem cells. In
some embodiments, an immunocompromised transplant patient can serve
as a donor for cells for use in autologous therapy (e.g., expanded
cells to be administered to that patient) or even for allogeneic
therapy.
Flow Cytometry
[0149] To investigate how physiological low-oxygen culture affects
the subpopulation of c-kit.sup.+ stem cells and the expression of
p16INK4A, a marker for cell senescence, CDCs expanded in 5% O2 and
20% O2 were harvested as single-cell suspensions using trypsin
digestion. Cells were then incubated with PE-conjugated mouse
anti-human c-kit antibody (eBioscience) or mouse anti-human
p16INK4A antibody (BD Biosciences) for 60 min. The expressions of
c-kit and p16INK4A were quantitatively measured using a FACS
Calibur flow cytometer with CellQuest software (BD
Biosciences).
Immunostaining
[0150] To determine the myogenic differentiation in vitro, CDCs
were seeded on fibronectin-coated four-chamber culture slides and
continuously cultured in 5% O.sub.2 or 20% O.sub.2. After 3 days of
culture, cells were fixed, blocked with goat serum for 30 min, and
then incubated with mouse anti-human troponin T antibody (R&D
Systems Inc.) for 1 h at room temperature. Culture slides were
washed and then incubated with a PE-conjugated secondary antibody.
Cell nuclei were stained with DAPI. Myogenic differentiation was
quantified by calculating the positive-stained area using the
Image-Pro Plus software (version 5.1.2, Media Cybernetics Inc.,
Carlsbad, Calif., USA).
[0151] The telomerase activity and DNA damage in CDCs were also
estimated by immunostaining with mouse anti-human telomerase
catalytic subunit (TERT) antibody (Lifespan Bioscience) or rabbit
polyclonal antibody against .gamma.-H.sub.2AX (phosphor 5139, Abcam
Inc.), as described above. Positively stained cells were counted by
fluorescence microscopy.
Senescence-Associated Beta-Galactosidase Staining
[0152] Third-passage CDCs were seeded on fibronectin-coated
four-chamber culture slides and continuously cultured in 5% O.sub.2
or 20% O.sub.2. After 3 days of culture, senescence-associated
beta-galactosidase (SA-.beta.-Gal) was performed according to
established techniques. The SA-.beta.-Gal-positive cells were
counted under a microscope.
TUNEL Assay
[0153] To quantify the resistance to oxidative stress in vitro,
CDCs were seeded on fibronectin-coated four-chamber culture slides
and continuously cultured in 5% O.sub.2 or 20% O.sub.2. After 2
days of culture, cells were moved into a general incubator (20%
O.sub.2), and cultured with or without the addition of 100 mM
H.sub.2O.sub.2 in the medium for another 24 h. Cells were fixed,
and apoptotic cells were detected by TUNEL assay (Roche
Diagnostics, Mannheim, Germany), according to the manufacturer's
instructions. Cell nuclei were stained with DAPI, and
TUNEL-positive cells were counted under fluorescence
microscopy.
Measurement of Intracellular Reactive Oxygen Species
[0154] To measure intracellular reactive oxygen species (ROS), CDCs
were seeded in 6- or 96-well plates coated with 20 mg/mL of
fibronectin and continuously cultured in 5% O.sub.2 or 20% O.sub.2.
At .about.90% confluence, cells were incubated with 10 mM
2',7'-dichlorodihydrofluorescein diacetate (DCF; Invitrogen) for 60
min. The fluorescence of 2',7'-dichlorodihydrofluorescein in cells
cultured in 96-well plates is directly determined using SpectraMax
M5 (Molecular Devices Corp.) with an excitation wavelength of 495
nm and an emission wavelength of 520 nm. Cells cultured in six-well
plates were trypsin-treated and fixed. The DCF fluorescence
intensity in cells was analysed by flow cytometry as described
above.
Western Blotting
[0155] Western blot analysis was performed to compare the
expressions of integrin-.alpha..sub.2, laminin-.beta..sub.1, and
c-Myc in CDCs cultured under 5% O.sub.2 and 20% O.sub.2. The
equivalent of 30 mg of total protein was loaded onto SDS-PAGE gels,
and then transferred to PVDF membranes. After overnight blocking,
membranes were incubated with mouse anti-human
integrin-.alpha..sub.2 antibody, mouse anti-human
laminin-.beta..sub.1 antibody, rabbit anti-.beta.-actin monoclonal
antibody (Lifespan Bioscience), or mouse anti-human c-Myc antibody
(BD Biosciences). The appropriate horseradish peroxidase-conjugated
secondary antibodies were used, and then the blots were visualized
by using SuperSignal West Femto maximum sensitivity substrate
(Thermo Scientific) and exposed to Gel Doc.TM. XR System (Bio-Rad
Lab. Inc.). Quantitative analysis for blots was done by Quantity
One software, and expressions were normalized by b-actin.
ELISA
[0156] To compare the potency of the productions of growth factors,
CDCs expanded under 5% O.sub.2 or 20% O.sub.2 were seeded in
24-well culture plates at a density of 1.times.10.sup.5 mL and
incubated for 3 days with hypoxic stimulation (under 1% O.sub.2 to
mimic the microenvironment of the ischemic heart). The supernatants
were collected and the concentrations of Angiopoietin-2, bFGF, HGF,
IGF-1, SDF-1, and VEGF were measured with human ELISA kits (R&D
Systems Inc.), according to the manufacturer's instructions.
In Vitro Angiogenesis Assay
[0157] Angiogenic potency was estimated by tube formation using an
in vitro angiogenesis assay kit (Chemicon Int.), according to the
manufacturer's instructions. Briefly, CDCs expanded under 5%
O.sub.2 or 20% O.sub.2 were seeded on ECMatrix.TM.-coated 96-well
plates at a density of 2.times.10.sup.4 cells per well. After 6 h
incubation with hypoxic stimulation (under 1% O.sub.2 to mimic the
microenvironment of ischemic heart), tube formation were imaged.
The total tube length was then measured with Image-Pro Plus
software.
Myocardial Infarction Model And Cell Implantation
[0158] An acute myocardial infarction was created in adult male
SCID-beige mice (10-12 weeks old), as described above. The study
was approved by the Institutional Animal Care and Use Committee of
Cedars-Sinai Medical Center and conformed to the Guide for the Care
and Use of Laboratory Animals published by the US National
Institutes of Health (NIH Publication No. 85-23, revised 1996).
Briefly, after general anaesthesia and tracheal intubation, mice
were artificially ventilated with room air. A left thoracotomy was
performed through the fourth intercostal space and the left
anterior descending artery was ligated with 9-0 prolene under
direct vision. The mice were then randomized and subjected to
intramyocardial injections with a 30 G needle at four points in the
infarct border zone with 40 mL of PBS (PBS group, n=10),
1.times.10.sup.5 CDCs expanded under 20% O.sub.2 (20% O.sub.2 CDCs,
n=10), or 1.times.10.sup.5 CDCs expanded under 5% O.sub.2 (5%
O.sub.2 CDCs, n=12).
Quantification of Engraftment By Real-Time PCR
[0159] Quantitative PCR was performed 24 h and 7 days after cell
injection in six to seven animals from each cell-injected group to
quantify cell retention/engraftment. Male human CDCs were injected
to enable detection of the SRY gene located on the Y chromosome.
The whole mouse heart was harvested, weighed, and homogenized. The
TaqMan assay was used to quantify the number of transplanted cells
with the human SRY gene as template (Applied Biosystems, CA, USA).
A standard curve was generated with multiple dilutions of genomic
DNA isolated from the injected CDCs to quantify the absolute gene
copy numbers. All samples were spiked with equal amounts of genomic
DNA from non-injected mouse hearts as control. For each reaction,
50 ng of genomic DNA was used. Real-time PCR was performed with an
Applied Biosystems 7900 HT Fast real-time PCR machine. Experiments
were performed in triplicate. The number of engrafted cells per
heart was quantified by calculating the copy number of SRY gene in
the total amount of DNA based on the standard curve.
Echocardiography
[0160] Mice underwent echocardiography at 3 h (baseline) and 3
weeks after surgery using Vevo 770.TM. Imaging System
(VISUALSONICS.TM., Toronto, Canada). After the induction of light
general anaesthesia, the hearts were imaged two-dimensionally (2D)
in long-axis views at the level of the greatest LV diameter. LV end
diastolic volume, LV end systolic volume, and LV ejection fraction
(LVEF) were measured with VisualSonics V1.3.8 software from 2D
long-axis views taken through the infarcted area.
Histology
[0161] Mice were sacrificed 3 weeks after treatment. Hearts were
sectioned in 5 mm slices and fixed with 4% paraformaldehyde. The
engraftment of implanted human CDCs was identified by
immunostaining with antihuman nuclear antigen (HNA) antibody
(Chemicon). To measure cell engraftment, 10 images of the
infarction and border zones were selected randomly from each animal
(three sections/animal; 1 mm separation between sections; 20.times.
magnification, Eclipse TE2000-U). The percentages of human nuclei
per total nuclei were quantified using Image-Pro Plus software
(version 5.1.2, Media Cybernetics Inc.), and the average value from
each heart was used for statistical analysis. 24 The
differentiation of cardiac stem cells into myocytes, smooth muscle
cells, and endothelial cells was identified by immunostaining with
monoclonal antibodies against human-specific a-sarcomeric actin,
smooth muscle actin, and von Willebrand factor (vWF) (Sigma),
respectively, as described above. Masson's trichrome staining was
also performed to examine the infarction size, LV wall thickness,
LV chamber area, and viable myocardium, as described
previously.
Statistical Analysis
[0162] All results are presented as mean.+-.SD. Statistical
significance between two groups was determined using the two-tailed
paired t-test and among groups by ANOVA followed by Bonferroni post
hoc test (SPSS II, Chicago, Ill., USA), unless otherwise indicated.
Differences were considered statistically significant when
P<0.05.
Results
Cell Production
[0163] Compared with culture in 20% O.sub.2, the outgrowth of cells
from the `explants` was faster in 5% O.sub.2, resulting in more
than two-fold higher yield of outgrowth cells (P, 0.001, FIG. 18A).
The proliferation of CDCs was similar at subsequent early passages,
although proliferation at later passages was higher in CDCs
expanded in 5% O.sub.2 (P<0.05, FIG. 18B). In several
embodiments, the increased proliferation is due to induction of
HIF-1.alpha. (or other hypoxia-mediated factors) and subsequent
adaptation of the cells to the reduced oxygen. In some embodiments,
5% O.sub.2 culture conditions increases cell migration out from the
`explants` through the induction of HIF-1.alpha. (or other
hypoxia-mediated factors). Interestingly, the degree of hypoxia in
5% O.sub.2 culture was insufficient to increase the concentrations
of certain chemokines and cytokines in the media, including VEGF
and SDF-1, (data not shown). Thus in some embodiments, increased
proliferation of cardiac stem cells occurs in a
migratory-independent fashion (e.g., in the absence of HIF-1.alpha.
(or other hypoxia-mediated factors such as VEGF or SDF-1). In
several embodiments, increased proliferation is due to hypoxia
induced increases in cell cycle entry, or reduced apoptosis, or a
combination of these mechanisms (and/or in combination with other
mechanisms disclosed herein).
[0164] Other studies have suggested that culturing cells in
physiological low-oxygen culture increases the `sternness` of
cells, for example embryonic stem cells. However, according to the
presently disclosed methods and compositions, the c-kit+
subpopulation and differentiation potency were not augmented in
these twice-passaged CDCs expanded in 5% O.sub.2 relative to 20%
O.sub.2. Furthermore, the expression levels of
integrin-.alpha..sub.2, laminin-.beta..sub.1, and c-Myc were also
comparable in the two conditions. Thus, in some embodiments,
cardiac stem cells respond in a different way to physiologic oxygen
concentrations as compared to other types of stem cells. These
differences likely reflect differences in the responses of specific
cell types to hypoxia. In some embodiments, such differences are
advantageous, the differential responses of different cell types
are used to optimize the reproduction (e.g., expansion), viability,
engraftment, and/or functional effect of a population of cells to
be administered to a certain patient. For example, different
degrees of hypoxia (e.g., a sliding scale of oxygen concentrations)
impact cardiac stem cells differently, in some embodiments. In some
embodiments, cardiac stem cells isolated from highly oxygenated
regions of the cardiac tissue respond differently to oxygen
concentrations that differ from their in vivo micro-environment. As
a non-limiting example, a cardiac stem cell that originated from
cardiac tissue exposed to a 5% concentration of oxygen in vivo was
exposed to a 3% concentration in culture, the resultant expanded
population may proliferate slightly less, but provide more
efficient engraftment as compared to the same cells cultured in 5%
oxygen.
Chromosomal Abnormalities
[0165] Consistent with the preliminary karyotyping data discussed
above, 6 of 16 (37.5%) CDC samples expanded in 20% O.sub.2 included
aneuploid cells (26 of 323 examined cells, FIG. 19A). The most
common changes were trisomy 8 (FIG. 24) and Y chromosome loss. In
contrast, in 16 CDC samples expanded under 5% O.sub.2, there was
only one aneuploid cell (from a total of 321 examined cells;
P<0.01 by .chi..sup.2 test, FIG. 19A). The aneuploidy here
reflected loss of the Y chromosome, an innocuous cytogenetic
abnormality. These differences in genomic stability cannot be due
to differences in the source tissue, as the same biopsies were
subdivided and cultured in parallel to facilitate direct
comparison. The percentages of aneuploid cells were also lower in
CDCs expanded under 5% O.sub.2 than in 20% O.sub.2 (P=0.028 by
unpaired t-test, FIG. 19B). Thus, in several embodiments, ex vivo
expansion of human cardiac stem cells under physiological low
oxygen conditions (e.g., about 5% O.sub.2) reduces the incidence of
chromosomal abnormalities. In several embodiments, oxygen
concentrations are tailored specifically to the region of tissue
from which the original cardiac cells were collected and
subsequently expanded. For example, if cells were collected from
the interior wall of the heart, which is frequently exposed to
oxygenated blood, an oxygen concentration that mimics this in vivo
environment may be used to culture cardiac stem cells collected
from this region. In other embodiments, cells are collected from a
surgical sample, in particular from within the myocardial wall, a
lower oxygen concentration may be used during several embodiments,
of the expansion protocol. In several embodiments, oxygen
concentrations are tailored specifically to the region of tissue to
be treated. For example, if a subject has damaged cardiac tissue on
the interior wall of the heart, which is frequently exposed to
oxygenated blood, a first oxygen concentration may be used to
culture cardiac stem cells to treat this region. In other
embodiments, if damaged tissue is within the myocardial wall, a
lower oxygen concentration may be used.
Phenotype And In Vitro Myogenic Differentiation
[0166] The subpopulation of c-kit+ stem cells was comparable in
CDCs expanded in 5% O.sub.2 and in 20% O.sub.2 (P=0.106 FIGS. 25A
and 25C). Likewise, the propensity for cardiomyogenic
differentiation, by immunostaining for human-specific cardiac
troponin T, was equivalent in the two conditions (FIGS. 25B and
25D). Thus, in some embodiments, the use of physiological oxygen
concentrations does not alter the "stemness" profile of the
cultured cells, at least with regard to expression of certain
markers (e.g., c-kit). In other embodiments, however, alterations
in stem cell marker profiles result from alterations in oxygen
concentration. As such, in some embodiments, the profile of the
resultant cells may be controlled at least in part by the oxygen
concentration used in the culturing process.
Cell Senescence
[0167] Cell senescence was evaluated by the expression of
p16.sup.INK4A, telomerase activity, and SA-.beta.-Gal staining. The
expression of p16.sup.INK4A was lower (FIGS. 20A and 20D,
P<0.001), and the telomerase activity (evaluated by the
expression of TERT) was higher (FIGS. 20B and 20E, P<0.01) in
CDCs expanded in 5% O.sub.2. The fraction of SA-.beta.-Gal-positive
cells was also lower in third-passage CDCs expanded in 5% O.sub.2
than in 20% O.sub.2 (FIGS. 20C and 20F, P<0.05). Thus, in some
embodiments, the culture conditions and methods disclosed herein
reduce the "aging" rate of cultured cells. In some embodiments,
this is realized as a decrease in expression in one or more genes
(or proteins) related to the aging of a cell, cell cycle entry (or
activity), tumor suppressor genes, and the like. Moreover, in some
embodiments, karyotypic abnormalities and/or aging of cells is
reduced by virtue of increase telomerase activity (e.g., reduced
telomere shortening).
Intracellular ROS, DNA Damage, And Resistance To Oxidative
Stress
[0168] Intracellular ROS concentrations were measured by DCF
staining. Intracellular ROS level was lower in 5% O.sub.2-cultured
CDCs than in 20% O.sub.2-cultured CDCs (FIGS. 21A and 21B;
P<0.01). The percentage of cells with .gamma.-H.sub.2AX foci, a
marker of DNA damage was likewise lower in 5% O.sub.2-cultured CDCs
(FIGS. 21C and 21D; P<0.01). This correlates with the
karyotyping data discussed above, which showed a dramatic reduction
of aneuploidy in CDCs expanded under 5% O.sub.2 (see e.g., FIG.
19). Thus, in several embodiments, the culturing methods and
compositions disclosed herein reduce the level of intracellular
ROS, which in turn, reduces the incidence of karyotypic
abnormalities. In some embodiments, the decrease in ROS also
increases cellular viability. In some embodiments, increased
viability is manifest by increased proliferation in culture,
increased viability after in vivo administration, and/or improved
engraftment after in vivo administration.
[0169] In order to determine whether cells cultured in
physiological O.sub.2 were more resistant (or more sensitive), to
oxidative stress, CDCs were therefore exposed to H.sub.2O.sub.2, a
powerful oxidant. After 24 h of exposure to 100 mM H.sub.2O.sub.2,
the number of apoptotic cells was lower in 5% O.sub.2 CDCs than in
those grown in 20% O.sub.2 (FIGS. 21E and 21F, P<0.01). The
basal frequency of apoptosis was also lower in the 5% O.sub.2 CDCs
(P<0.05). Thus, in some embodiments, not only do the disclosed
culture methods and compositions reduce formation of ROS that
naturally appear, but they reduce the sensitivity of cells to
sources of oxidative stress. In some embodiments, as a result,
cells are more robust in a wider variety of environments in vivo,
and thus, have greater viability.
Expression of Adhesion Molecules And c-Myc
[0170] The expression levels of integrin-.alpha..sub.2,
laminin-.beta..sub.1, and c-Myc were comparable in CDCs expanded in
5% O.sub.2 and in 20% O.sub.2 (see FIGS. 26A-26D).
In Vitro Production of Growth Factors And Tube Formation
[0171] While CDCs are known to secrete a variety of growth factors
the present study analysed conditioned media to determine whether
hypoxic culture influences the paracrine secretion of selected
growth factors by CDCs. Cells cultured either in 5% O.sub.2 or 20%
O.sub.2 were plated in fresh media and grown for 3 days with
hypoxic stimulation (1% O.sub.2) to mimic the microenvironment of
ischemic heart. The production of the majority of growth factors
under hypoxic stimulation, including angiopoietin-2, bFGF, HGF,
IGF-1, SDF-1, and VEGF, was comparable in CDCs expanded under 5%
O.sub.2 or 20% O.sub.2 (see FIGS. 27A-27F), although some factors
tended to be higher in CDCs expanded under 5% O.sub.2.
[0172] By in vitro angiogenesis assay, CDCs expanded under both 5%
O.sub.2 and 20% O.sub.2 could form capillary-like networks (tube
formation) on matrigel in 6 h with hypoxic stimulation, and there
was no significant difference between groups (see FIGS.
28A-28B).
[0173] In several embodiments, in vivo multilineage differentiation
from human CDCs expanded under either 5% O.sub.2 or 20% O.sub.2, is
achieved as well as the robust production of various angiogenic and
anti-apoptotic growth factors in vitro. However, several
embodiments are particularly advantageous in that the above results
are achieved without the increased incidence of genomic
instability. As such, cells cultured under physiologic oxygen
concentrations are particularly advantageous for use in cellular
therapies (e.g., cardiac stem cell therapy).
In Vivo Cell Engraftment And Differentiation
[0174] The ultimate test of cell quality for cells to be used in
cardiac regeneration and repair is the efficacy of the cells in
vivo. Cell retention (24 h) and engraftment (7 days) in hearts
injected with CDCs cultured in 5% O.sub.2 or 20% O.sub.2 was
quantified. Quantitative PCR analysis showed that CDCs expanded in
5% O.sub.2 survived better than CDCs expanded in 20% O.sub.2, both
at 24 h and at 7 days after implantation into the infarcted hearts
of SCID mice (FIG. 22A). Furthermore, in the scar and marginal
regions of the infarcted mouse heart 3 weeks after treatment,
greater survival of CDCs expanded in 5% O.sub.2 than in 20% O.sub.2
was detected (FIG. 22A). Quantitative analysis of percentages of
human nuclei revealed better cell engraftment after implantation of
CDCs expanded under 5% O.sub.2 than under 20% O.sub.2 (3.40+0.94
vs. 1.77+0.52%, P<0.01, FIGS. 22B and 22C). The improvements in
retention and engraftment suggest greater transplanted cell
resilience, consistent with the observed increase in resistance to
oxidative stress (FIGS. 21A and 21D) and the decrease in senescence
(FIG. 20) in 5% O.sub.2-cultured CDCs. One distinctive feature of
CDCs is the ability to detect consistent cardiomyogenic and
angiogenic differentiation of injected cells (e.g., direct
regenerative effects), even though the mechanism of functional
benefit appears, in some embodiments, to be largely
paracrine-mediated. The present study also confirmed that CDCs can
differentiate into cardiomyocytes, endothelial and vascular smooth
muscle cells, irrespective of the level of oxygen in which they
were cultured. Histology of mouse hearts that had been injected
with CDCs 3 weeks earlier revealed expression of .alpha.-sarcomeric
actin (see FIGS. 29A-29D), smooth muscle actin (see FIGS. 30A-30B),
and vWF (see FIGS. 31A-31B) in some of the surviving progeny of
human CDCs expanded in either 5% O.sub.2 or 20% O.sub.2, indicative
of multilineage differentiation. Although CDCs expanded in 5%
O.sub.2 exhibited greater survival, their differentiation potential
was not noticeably different than that of 20% O.sub.2-cultured
CDCs.
[0175] When implanted into infarcted hearts of SCID mice, 5%
O.sub.2-cultured human CDCs engrafted to a greater degree and
improved function to a greater degree as compared to 20%
O.sub.2-cultured CDCs. As discussed above, the decrease in cell
senescence and increased resistance to oxidative stress in vitro
are, in some embodiments, at least partially responsible for the
superior effects observed in vivo. Also as discussed above, it is
believed that implantation of CDCs into infarcted heart leads to
functional improvement via a direct regeneration, paracrine
effects, or a combination thereof. Thus, in several embodiments,
expansion of cardiac stem cells in physiologic oxygen
concentrations augments the ability of the cells to effect cardiac
repair and/or regeneration. In some embodiments, the cells cultured
in physiologic oxygen concentrations engraft more readily than
cells cultured in 20% O.sub.2. In some embodiments, engraftment is
more efficient (e.g., a greater proportion of administered cells
engraft). In some embodiments, engraftment is for a longer term
(e.g., engrafted cells cultured in physiologic oxygen
concentrations survive longer than those cultured in 20% O.sub.2.
In several embodiments, increased engraftment or longer term
engraftment is related to the decreased senescence of cells
cultured in physiologic oxygen concentrations (e.g., the cells are
more metabolically active and more readily survive in a new host
environment).
[0176] While the present study did not attempt to quantify the
relative roles of direct regeneration vs. indirect paracrine
effects, in some embodiments, both direct and indirect effects are
operative. As discussed above, direct effects (e.g., the
differentiation of the administered cells into functional cardiac,
vascular, or endothelial tissue are responsible for repair and/or
regeneration of cardiac tissue, in some embodiments. In some
embodiments, paracrine effects (e.g., growth factors, cytokines, or
other signals/factors) generated by the administered cells serve to
mediate cardiac tissue repair or regeneration. In some embodiments,
combinations of direct and indirect mechanisms are involved.
Cardiac Function And Infarct Size
[0177] To assess efficacy in myocardial repair, left ventricular
function in hearts injected with vehicle or with CDCs cultured in
5% O.sub.2 or 20% O.sub.2 was compared. Although LVEF at baseline
did not differ between groups, LEVF measured 3 weeks after
treatment was higher in mice implanted with 5% O.sub.2-cultured
CDCs as compared to those expanded in 20% O.sub.2 (41.5+3.2 vs.
36.5+4.1%, P=0.043, FIG. 22D). Both cell groups outperformed the
vehicle-treated group. In addition, the absolute values of LVEF
correlate strongly with the degree of cell engraftment
(r.sup.2=0.80, P=0.002, FIG. 22E). Compared with the control
treatment with PBS injection only, the implantation of CDCs
expanded under either 5% O.sub.2 or 20% O.sub.2 results in
obviously smaller infarct size, more viable myocardium, increased
infarct wall thickness, and lower LV chamber area 3 weeks after
treatment (P<0.05 vs. PBS, FIG. 23A-23G). These parameters
showed greater improvements with administration of 5%
O.sub.2-cultured CDCs as compared to those expanded in 20% O.sub.2
(P, 0.05). Thus, as discussed above, in several embodiments,
culture of cells in physiologic oxygen concentrations augments the
ability of the cells to effect cardiac repair. In some embodiments,
use of physiologic oxygen concentrations during culture of cells to
be administered improves the functional or anatomical endpoints (as
compared to cells cultured in 20% oxygen) by at least about 5%. In
some embodiments, cells cultured in physiologic oxygen
concentrations improve functional or anatomic endpoints by about 5%
to about 10%, about 10% to about 15%, about 15% to about 20%, and
overlapping ranges thereof. In some embodiments, depending on the
endpoint, use of physiologic oxygen concentrations during culture
of cells to be administered improves the effects the therapy (e.g.,
the repair or regeneration of cardiac tissue) by about 2-fold,
about 4-fold, about 6-fold, about 10-fold, about 20-fold, and
overlapping ranges lying between these values.
[0178] In several embodiments, ex vivo expansion of human CDCs
under physiological oxygen (5% O.sub.2) results in one or more of
the following: increases in cell yield, decreases in aneuploidy and
cell senescence, and increases in resistance to oxidative stress.
Furthermore, in some embodiments, CDCs expanded in 5% O.sub.2
results in improved engraftment and functional benefit after
implantation into infarcted hearts (or other damaged or diseased
heart tissue).
[0179] Various modifications and applications of embodiments of the
invention may be performed without departing from the true spirit
or scope of the invention. Method steps disclosed herein need not
be performed in the order set forth. It should be understood that
the invention is not limited to the embodiments set forth herein
for purposes of exemplification, but is to be defined only by a
reading of the appended claims, including the full range of
equivalency to which each element thereof is entitled.
* * * * *