U.S. patent application number 16/358443 was filed with the patent office on 2020-01-16 for methods of reducing teratoma formation during allogeneic stem cell therapy.
The applicant listed for this patent is The Johns Hopkins University. Invention is credited to Eduardo Marban.
Application Number | 20200016210 16/358443 |
Document ID | / |
Family ID | 47293374 |
Filed Date | 2020-01-16 |
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United States Patent
Application |
20200016210 |
Kind Code |
A1 |
Marban; Eduardo |
January 16, 2020 |
Methods of Reducing Teratoma Formation During Allogeneic Stem Cell
Therapy
Abstract
The present application relates to methods and compositions for
treating diseased or damaged cardiac tissue comprising regenerative
cells harvested from donor cardiac tissue. In one embodiment,
regenerative cells are harvested from an allogeneic source and
after administration result in increased viability and/or
functional improvement of damaged or diseased cardiac tissue.
Inventors: |
Marban; Eduardo; (Baltimore,
MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Johns Hopkins University |
Baltimore |
MD |
US |
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|
Family ID: |
47293374 |
Appl. No.: |
16/358443 |
Filed: |
March 19, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13412051 |
Mar 5, 2012 |
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16358443 |
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11666685 |
Apr 21, 2008 |
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PCT/US2005/040359 |
Nov 8, 2005 |
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13412051 |
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60625695 |
Nov 8, 2004 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 5/0668 20130101;
C12N 5/0657 20130101; A61K 35/28 20130101; A61P 35/00 20180101 |
International
Class: |
A61K 35/28 20060101
A61K035/28; C12N 5/077 20060101 C12N005/077; C12N 5/0775 20060101
C12N005/0775 |
Goverment Interests
STATEMENT REGARDING GOVERNMENT SPONSORED GRANT
[0002] The studies disclosed herein were made with Government
support under one or more of the National Institutes of Health
Research Project Grants HL095203 HL103356, HL081028, and HL083109.
The United States Government has certain rights in this invention.
Claims
1. A method for the reduction of teratoma formation following the
delivery of nonself cells to a first subject, comprising:
delivering to a first subject a population of regenerative cells,
wherein said regenerative cells are isolated from a tissue source
harvested from a second subject, wherein said regenerative cells
express one or more factors that reduce teratoma formation, wherein
said at least a portion of said regenerative cells engraft into a
target tissue of said first subject after delivery to said subject,
wherein said engraftment persists for a time period ranging from
about 1 week to about 6 weeks, wherein during said period of
engraftment at least a portion of said regenerative cells are
destroyed by the immune system of said first subject, and wherein
said engraftment of a portion of said regenerative cells, said
period of engraftment, and said destruction of said regenerative
cells reduces teratoma formation.
Description
RELATED CASES
[0001] This application is a continuation-in-part application of
U.S. patent application Ser. No. 11/666,685, filed Apr. 21, 2008,
which is the U.S. National Phase application under 35 U.S.C. .sctn.
371 International Application No. PCT/US2005/040359 filed on Nov.
8, 2005, which claims the benefit of U.S. Provisional Application
No. 60/625,695 filed Nov. 8, 2004, the disclosures of each of which
are expressly incorporated herein.
BACKGROUND
Field of the Invention
[0003] Several embodiments of the present application relate
generally to regenerative cells, methods of preparing regenerative
cells, and compositions comprising regenerative cells for use in
transplant for repair of damaged tissue. In one embodiment,
regenerative cells isolated from donor heart tissue may be
cultured, expanded, and administered to a recipient in order to
repair damaged cardiac tissue of the recipient.
Description of the Related Art
[0004] Stem cells are characterized by the ability to renew
themselves through mitotic cell division and the ability to
differentiate into a diverse range of specialized cell types. The
two primary types of mammalian stem cells are embryonic stem cells
and adult stem cells (i.e., non-embryonic stem cells). Embryonic
stem cells are isolated from the inner cell mass of blastocysts and
are pluripotent, meaning that the cells have the capacity to
differentiate into all of the specialized embryonic tissues. Adult
stem cells are isolated from adult tissues and function as an
ongoing repair system for adult organs.
[0005] 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 long lasting and
severe heart tissue damage, which often leads to a reduced quality
of life.
SUMMARY
[0006] Given the vast potential of stem cell therapy to
revolutionize medical treatment, there exists a need in for
effective and efficient administration of stem cells, compositions
comprising stem cells, or derivatives of stem cells to a recipient
in order to elicit therapeutic effects, including, among others,
tissue regeneration. In particular, in the context of coronary
heart disease, there is a need for improved methods to isolate,
prepare and administer cell-based compositions to recipients in
order to ameliorate and/or treat the cardiac tissue damage that
results from adverse cardiac events associated with coronary heart
disease.
[0007] In several embodiments of the invention, a method of
treating an adverse cardiac event (such as myocardial infarction)
is provided, wherein the method comprises obtaining a cardiac
biopsy sample from a first patient, culturing the sample to obtain
regenerative cells, and implanting the regenerative cells into a
second patient. This type of allogeneic transplant, according to
several embodiments, is particularly advantageous because the
regenerative cells do not evoke a significant chronic immune
response that is adverse to the patient. Instead, the regenerative
cells trigger a cascade of therapeutic signaling effects (e.g., a
paracrine effect) prior to destruction via an acute immune response
that destroys the regenerative cells. In this manner, according to
several embodiments, "off-the-shelf" regenerative cells can be
produced to treat patients suffering from cardiac diseases. Thus
the patient need not have healthy tissue from which to harvest his
or her own cells (as is the case for an autologous transplant).
Moreover, even when a patient has healthy heart tissue for biopsy,
the patient need not have to wait for the culturing process.
Instead, the "off-the-shelf" allogeneic cells may be available with
little or no time delay.
[0008] According to several embodiments, methods for increasing
function of a damaged or diseased heart of a mammal are provided.
In one embodiment, a population of cells is administered to the
mammal, wherein the population of cells increases cardiac function
in the mammal. The population of cells, in one embodiment, is
obtained by the process of culturing cells obtained from
cardiospheres on a surface as a monolayer. In several embodiments,
a population of in vitro-expanded cells is administered to the
mammal. In some embodiments, the cells have the capacity to form
cardiospheres in suspension culture. According to one embodiment,
the cells are not, however, in the form of cardiospheres when
administered.
[0009] Methods for treating a mammal with a damaged or diseased
heart are provided which comprise, in some embodiments, obtaining
heart tissue from the damaged or diseased heart of the mammal. In
several embodiments, heart tissue is obtained from a heart of a
donor. In some embodiments, the heart tissue is obtained by way of
a percutaneous endomyocardial biopsy. In some embodiments, the
heart tissue is treated to obtain and expand a population of
cardiac stem cells and the cardiac stem cells and/or their progeny
are subsequently introduced into the damaged or diseased heart.
[0010] In some embodiments, methods are provided for treating a
mammal with a damaged or diseased organ (e.g., not necessarily the
heart). In one embodiment, tissue is obtained from the damaged or
diseased organ of the mammal or from a healthy organ of a donor by,
for example, a percutaneous biopsy. In several embodiments, the
tissue is treated to obtain and expand a population of stem cells.
The stem cells and/or their progeny are introduced into the damaged
or diseased organ of the mammal. In one embodiment, a method of
treating kidney damage is provided. In one embodiment, a kidney
biopsy specimen is incubated in the presence of a protease. The
cells liberated from the biopsy specimen by the protease incubation
are collected. The collected cells are cultured on a surface as a
monolayer to expand number of cells, which are then introduced to
the damaged kidney.
[0011] In several embodiments, cells (e.g., stem cells) are
obtained from allogeneic donor tissue, including but not limited to
donated organs having tissue that is at least partially healthy and
harvestable. In some embodiments, cardiac tissue is obtained from
hearts deemed unsuitable for transplantation. Accordingly, in
several embodiments, the methods provided herein are particularly
advantageous because they utilize donor hearts that would otherwise
have been discarded or under-utilized.
[0012] In some embodiments, methods for treating tissue (including
but not limited to a cardiac biopsy specimen) are provided. In
several embodiments, the tissue is incubated in the presence of a
protease. The cells that are liberated from the tissue by the
protease incubation are collected. In some embodiments, the
collected cells are cultured on a surface as a monolayer to expand
number of cells.
[0013] In some embodiments, methods for expanding a population of
cells (including but not limited to cardiac stem cells) are
provided. In several embodiments, one or more bodies (e.g.,
cardiospheres) are disaggregated to individual cells or smaller
aggregates of cells. The individual cells or smaller aggregates of
cells are cultured on a surface as a monolayer, in some
embodiments. In one embodiment, a population of in vitro-expanded
cells in a monolayer is provided. The cells have the capacity to
form cardiospheres in suspension culture. The cells are not,
however, in the form of cardiospheres in some embodiments. In still
another embodiment, a population of cells made by the process of
culturing cells on a surface as a monolayer is provided. In some
embodiments, the cells are obtained from disaggregated
cardiospheres.
[0014] Although several embodiments of the invention are used for
autologous administration, many embodiments are suitable for
allogeneic administration. Allogeneic administration is
advantageous in several embodiments because it is readily available
for immediate administration to patients.
[0015] Use of certain types of cells for cellular therapy may be
hampered by the unwanted differentiation and growth of administered
cells into cell types that are distinct (and in some cases not
functionally complementary) to the target tissue. Teratoma
formation is thus a potential concern with certain types of cell
therapy. To address such concerns, in several embodiments, there is
provided a method for the reduction of teratoma formation following
the delivery of non-self cells to a first subject (e.g., cells
isolated from a tissue source harvested from a second subject,
wherein the second subject is an adult) comprising delivering to a
first subject a population of regenerative cells, wherein the at
least a portion of the regenerative cells engraft into a target
tissue of the first subject after delivery to the subject, wherein
the regenerative cells express one or more factors that reduce
teratoma formation (e.g., in comparison to the delivery of
embryonic cells to a subject). In some embodiments, it is the
engraftment of the regenerative cells that reduces teratoma
formation, at least in part due to the retention of the cells at
the desired target site. In some embodiments, the period of
engraftment reduces teratoma formation. For example, in some
embodiments the period of engraftment is short term (e.g., a few
days to several weeks) and insufficient for teratoma formation to
occur. In some embodiments, whether engrafted or not, death and/or
destruction of the regenerative cells reduces teratoma formation.
In several embodiments, it is the combination of two or more of
engraftment of the cells, the period of engraftment, the
destruction of the cells, and/or the factors released by the cells
that reduces teratoma formation.
[0016] In several embodiments, there is provided a method of
treating a first subject having damaged cardiac tissue with
allogeneic cells from a second subject, the method comprising
obtaining a plurality of regenerative cells (e.g., CDCs) harvested
from the cardiac tissue of a second subject, wherein the
administered CDCs generate one or more cytokines, chemokines or
diffusible factors, wherein, after administration, at least a
portion of the administered CDCs engraft into the cardiac tissue of
the first subject; and wherein the one or more generated cytokines,
chemokines or diffusible factors or the engraftment improves the
function of the damaged cardiac tissue, thereby treating the first
subject. In several embodiments, the cells have been expanded in
culture to yield a population of cardiosphere-derived cells (CDCs).
In one embodiment, the CDCs are not pluripotent and are committed
to differentiating into cardiac tissue, thereby reducing the risk
of producing undesired tissue growth.
[0017] In several embodiments, the engraftment of administered
cells persists for a time period ranging from about 1 week to about
6 weeks. In several embodiments, during the period of engraftment
at least a portion of the regenerative cells are destroyed by the
immune system of the first subject. In several embodiments, the
destruction of cells by the immune system is, at least in part,
responsible for the reduced teratoma formation as a result of the
reduced residence time of the cells.
[0018] In several embodiments, during the period of engraftment,
the regenerative cells induce endogenous cells to express one or
more factors that reduce teratoma formation. Thus, in some
embodiments, the combination of factors generated from the
regenerative cells and the factors induced to be generated by the
endogenous cells is responsible for the reduction in teratoma
formation. In several embodiments, expression of the factors
comprises cell-surface expression. In several embodiments,
expression of the factors comprises release of the factors from the
cells.
[0019] In several embodiments, the delivery of the regenerative
cells is for the purpose of repairing a damaged or diseased tissue
of the first subject. In several such embodiments, the damaged or
diseased tissue of the first subject comprises damaged or diseased
cardiac tissue. In some embodiments, the population of regenerative
cells comprises cardiac stem cells. In several embodiments, the
cardiac stem cells are selected from the group consisting of
cardiospheres, cardiosphere-derived cells, and a subsequent
generation of cardiospheres.
[0020] In several embodiments, the regenerative cells express one
or more stem cell markers selected from the group consisting of
c-kit, CD90, and sca-1. In several embodiments, the regenerative
cells express one or more endothelial cell markers selected from
the group consisting of KDR, flk-1, CD31, von Willebrand factor,
Ve-cadherin, and smooth muscle alpha actin. In several embodiments,
the regenerative cells express one or more of the stem cell markers
or one or more of the endothelial cell markers, but are not
selected for, enriched, purified or otherwise preferentially
obtained based on the expression of the one or more expressed
markers. In several embodiments, the isolated regenerative cells
are expanded in culture prior to delivery. In several embodiments,
the culturing of the cells is performed in order to induce the
expression of one or more of the markers above.
[0021] In several embodiments, the isolated regenerative cells
generate teratoma-reducing factors in culture. In one embodiment,
the method further comprises isolating the teratoma-reducing
factors from the culture. In one embodiment, the method also
comprises delivering the isolated teratoma-reducing factors from
the culture to the first subject. In one embodiment, delivery of
the isolated teratoma-reducing factors is prior to delivery of the
regenerative cells. In one embodiment, delivery of the isolated
teratoma-reducing factors is concurrent with delivery of the
regenerative cells. In one embodiment, delivery of the isolated
teratoma-reducing factors is after delivery of the regenerative
cells. In several embodiments, delivery of the isolated
teratoma-reducing factors is at multiple time points throughout the
period of engraftment of the regenerative cells. In several
embodiments, the delivery of factors isolated in culture
supplements the expressed (and/or the induced) generation of
teratoma-reducing factors.
[0022] In one embodiment, between about 1.times.10.sup.6 and about
100.times.10.sup.6 of the CDCs, or the regenerative cells, are
administered to first subject.
[0023] In several embodiments, there is provided a method of
treating a first subject having diseased or damaged cardiac tissue
with allogeneic regenerative cells obtained from a second subject,
the method comprising obtaining a plurality of regenerative cells
for administration to a first subject and administering at least a
portion of the population of expanded regenerative cells to the
first subject.
[0024] In several embodiments wherein the regenerative cells are
harvested from cardiac tissue obtained from a second subject and
subsequently expanded in culture to yield a population of expanded
regenerative cells, at least a portion of which are suitable for
administration. In several embodiments, after administration, at
least a portion of the administered regenerative cells is destroyed
by the first subject's immune system. However, in some embodiments,
the administered regenerative cells generate one or more paracrine
signals post-administration and prior to the destruction. In
several embodiments, the one or more paracrine signals improve one
or more of the viability or function of the damaged cardiac tissue,
thereby treating the first subject. In one embodiment, the
regenerative cells are cardiosphere-derived cells (CDCs). In one
embodiment, the regenerative cells are cardiospheres. In one
embodiment, a mixture of CDCs and cardiospheres is used.
[0025] In several embodiments, the regenerative cells are harvested
from cardiac tissue obtained from a second subject and subsequently
expanded in culture to yield CDCs. In some embodiments, after
administration, at least a portion of the CDCs is destroyed by the
immune function of the first subject. In several embodiments, the
administered CDCs generate one or more paracrine signals
post-administration and prior to the destruction. In several
embodiments, the one or more paracrine signals improve one or more
of the viability or function of the damaged cardiac tissue, thereby
treating the first subject.
[0026] In several embodiments, the viability or function of the
damaged cardiac tissue is improved directly by the paracrine
signals. In several embodiments, the viability or function of the
damaged cardiac tissue is improved by an indirect mechanism induced
by the paracrine signals. In some embodiments, the indirect
mechanism comprises recruitment of endogenous cells that repair
cardiac tissue. In some embodiments, the indirect mechanism
comprises induction of production of paracrine factors by
endogenous cells. As a result, in some embodiments, a feed forward
repair cascade is initiated, wherein administered cells and their
paracrine signals induce further paracrine signal generation by
endogenous cells, and effect a more robust repair (e.g., viability
or function) of cardiac tissue.
[0027] In several embodiments, the administration of the cells
results in an increase in at least one of left ventricular percent
fractional area and left ventricular ejection fraction. In several
embodiments, increases of at least about 5%, 10%, 15%, or more are
realized. In other embodiments (e.g., myocardial infarction) scar
tissue formation is reduced. In some embodiments, administration of
the cells induces pro-survival paracrine signals that improve the
viability and/or function of the damaged cardiac tissue. Thus, in
such embodiments, the administration induces anti-apoptotic (or
other cell death pathways) signals or cascades that result in
improved viability, despite an injurious event or disease that
affected or is affecting the cardiac tissue. In several
embodiments, the pro-survival paracrine signals decrease apoptosis
in the damaged cardiac tissue. In several embodiments the
pro-survival paracrine signals increase capillary density in the
damaged or diseased cardiac tissue. In some such embodiments, the
increased capillary density increases the flow of oxygenated blood
the regions of the cardiac tissue, thereby improving the viability
of the tissue by reducing periods of ischemia, for example.
[0028] In several embodiments, the paracrine signals comprise one
or more growth factors or cytokines. In one embodiment, the growth
factors or cytokines comprise one or more of VEGF, HGF, and IGFI.
In the alternative or in conjunction with these factors, other
growth factors or cytokines are released (or presented on the
surface) by the cells, in other embodiments.
[0029] In several embodiments the destruction of the cells is
accomplished via phagocytosis. In some embodiments, immune
responses (humoral or complement mediated) act to destroy the
regenerative cells. In several embodiments, natural death of the
administered cells (e.g., apoptosis) occurs, thereby accounting for
destruction of the administered cells.
[0030] In several embodiments, the cells express one or more stem
cell markers selected from the group consisting of: CD105, c-kit,
CD90, and sca-1 and one or more endothelial cell markers selected
from the group consisting of KDR, flk-1, CD31, von Willebrand
factor, Ve-cadherin, and smooth muscle alpha actin. In some
embodiments, other cardiac, vascular, or endothelial markers are
expressed within or on the cells. In some embodiments, the cells
may be selected for by the presence or expression of certain
markers. However, in several embodiments, no selection or
enrichment based on marker selection is made.
[0031] In one embodiment, the diseased or damaged cardiac tissue is
the result of one or more of acute heart failure (e.g., a stroke or
MI) or chronic heart failure (e.g., congestive heart failure). In
several embodiments, about 1.times.10.sup.5 to about
1.times.10.sup.7 of the cells are administered. In several
embodiments, the dose is varied depending on the size and/or age of
a subject receiving the cells. In some embodiments (e.g., those
that induce feed-forward effects in endogenous cells), smaller
numbers of cells are optionally administered. Different routes of
administration are also used, depending on the embodiment. For
example, the regenerative cells may be administered by intravenous,
intra-arterial, intracoronary, or intramyocardial routes (or other
routes) of administration.
[0032] In one embodiment, there is provided a method of treating a
first subject having diseased or damaged cardiac tissue with
allogeneic regenerative cells obtained from a second subject, the
method comprising obtaining a plurality of regenerative cells for
administration to a first subject, administering the expanded
regenerative cells to a first subject having damaged cardiac
tissue, wherein the regenerative cells are harvested from cardiac
tissue obtained from a second subject and subsequently expanded in
culture to yield the population of expanded regenerative cells,
wherein, after administration, at least a portion of the expanded
regenerative cells engraft into the cardiac tissue of the first
subject; wherein the administered regenerative cells generate one
or more paracrine signals; and wherein the one or more paracrine
signals improve one or more of the viability or function of the
damaged cardiac tissue, thereby treating the first subject. In one
embodiment, the regenerative cells comprise cardiosphere-derived
cells (CDCs), wherein the CDCs are about 5 and 20 microns in
diameter. In one embodiment, the CDCs are delivered to the first
subject via intracoronary administration. In some embodiments,
other administration routes are used, for example, intravenous,
direct myocardial injection, etc. Selection of the optimal
administration route is based upon, among other factors, dose of
cells to be delivered, location of the area of damaged tissue,
severity of tissue damage, and the like.
[0033] In addition to the methods disclosed above, there is also
provided a population of allogeneic cells for administration to a
subject for the repair of damaged cardiac tissue, comprising
cardiosphere derived cells (CDCs) isolated from a first subject,
and suitable for administration to a second subject that is
allogeneic with respect to the first subject, and expanded in
culture.
[0034] In several embodiments, there is provided a population of
allogeneic cells isolated from a first subject and suitable for
administration to a second subject for the repair of damaged
cardiac tissue of the second subject comprising cardiosphere
derived cells (CDCs) isolated from a first subject and expanded in
culture, wherein the CDCs express the stem cell markers CD105 and
c-kit, but are not screened, subfractionated, or otherwise selected
based on the expression of the markers, wherein the CDCs express
one or more products that improve the viability or functionality of
the damaged cardiac tissue of the second subject.
[0035] In several embodiments, the CDCs have a diameter of between
about 5 and 20 microns. In some embodiments, the size of the CDCs
is advantageous in that a greater variety of delivery routes are
available with reduced risk of inducing embolization of the
microcirculation upon administration. In several embodiments,
administration of the CDCs to a second subject results in
engraftment of the CDCs in the cardiac tissue of the second subject
for at least about 3 weeks. During that period of engraftment, in
several embodiments, the CDCs generate one or more paracrine
signals (or expressed products) that yield improvements in the
viability or function of the damaged cardiac tissue. In several
embodiments, generation of paracrine factors persists beyond the
period of engraftment (e.g., the administered cells induce a
cascade of events that results in the propagation of paracrine
signal production, even in the absence of some or all of the
originally administered cells). As discussed above, in several
embodiments, the administered cells induce paracrine factor
production in endogenous cells.
[0036] In several embodiments, the CDCs express a variety of
markers that identify the cell types that comprise a CDC
population. In several embodiments, the CDCs express the stem cell
marker CD105. In several embodiments, the CDCs further express one
or more stem cell markers selected from the group consisting of:
c-kit, CD90, and sca-1. In several embodiments the CDCs further
express one or more endothelial cell markers selected from the
group consisting of: KDR, flk-1, CD31, von Willebrand factor,
Ve-cadherin, and smooth muscle alpha actin. As discussed herein, in
several embodiments, while the CDCs express one or more of the stem
cell markers and/or one or more of the endothelial cell markers,
the cells are screened, subfractionated, or otherwise selected for
based on the expression of the one or more expressed markers.
[0037] The beneficial effects of the administration of cells, as
disclosed herein, is attributable to the cells themselves, one or
more of the paracrine factors produced by the cells, or
combinations thereof. In several embodiments, the paracrine signals
comprise one or more growth factors or cytokines. In several
embodiments, the growth factors or cytokines comprise one or more
growth factors or cytokines selected from the group consisting of:
ENA-78, G-CSF, GM-CSF, GRO, GRO-alpha, I-309, IL-1 alpha, IL-1
beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12,
IL-13, IL-15, interferon gamma, MCP-1, MCP-2, MCP-3, M-CSF, MDC,
MIG, MIP-1 beta, MIP-1 delta, RANTES, SCF, SDF-1, TGF-beta 1,
TNF-beta, EGF, IGF-1, angiogenin, oncostatin M, thrombopoeitin,
VEGF, PDGF-BB, leptin, BDNF, BLC, Ck beta 8-1, eotaxin, eotaxin-2,
eotaxin-3, FGF-4, FGF-6, FGF-7, Flt-3 ligand, fractalkine, GCP-2,
GDNF, HGF, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IL-16, IP-10, LIF,
LIGHT, MCP-4, MIP-3 alpha, NAP-2, NT-3, NT-4, osteopontin,
osteoprogenerin, PARC, PIGF, TGF beta 2, TGF beta 3, TIMP-1 and
TIMP-2. In several embodiments, the growth factors or cytokines
comprise one or more of VEGF, HGF, and IGFI.
[0038] The beneficial effects of administration of the population
of allogeneic CDCs are multi-fold, and include both functional
improvements and improvements in viability of the damaged,
diseased, and/or surrounding cardiac tissue. In some embodiments,
administration of the CDCs results in at least a 5%, at least a
10%, or at least a 15% improvement in left ventricular function. In
some embodiments, the improvement in left ventricular function
persists for at least about 6 weeks after administration of the
CDCs. In some embodiments, longer term functional improvement is
achieved (e.g., 2-3 months, 3-5 months, or greater). In some
embodiments, functional improvement is essentially indefinite
(e.g., the treatment methods disclosed herein are one-time methods
in that additional doses of cells are not required). However, in
some embodiments, a serial dosing regimen is preferred. In several
embodiments, administration of the CDCs results in at least a
2-fold improvement cardiac cell viability. In some embodiments,
similar to the functional improvements, the increased viability is
also for longer period (e.g., the damaged cells live longer and/or
endogenous cells live longer). In some embodiments, the increased
viability is due to a reduction in apoptosis. In some embodiments,
apoptosis still occurs naturally, but cells have an altered
threshold for committing to an apoptotic pathway (e.g., a greater
degree of damage or longer period of ischemia is required). As
discussed herein, in several embodiments, the damaged cardiac
tissue of the first subject is a result of myocardial infarction,
chronic ischemia, or congestive heart disease. In some such
embodiments wherein an infarct caused the damage, administration of
the CDCs results in a decrease in infarct size. In some
embodiments, the reduction in infarct size ameliorates the
increased cardiac function (a greater amount of functionally
contractile cardiac tissue) and/or the viability (reduction in scar
size allows for better overall tissue perfusion and better tissue
viability).
[0039] In some instances, damage or disease affects cardiac tissue
to such a degree that cardiac function cannot be maintained at a
level sufficient to support the continued viability of a subject.
There is therefore provided a method of supplementing the cardiac
function of a first subject, the method comprising obtaining a
plurality of cardiac stem cells from a second subject for
administration to a first subject, and administering the cardiac
stem cells to the first subject. In several embodiments, the first
subject has reduced cardiac function due to damaged cardiac tissue.
In some embodiments, the obtained cardiac stem cells are optionally
expanded in culture in order to achieve a certain population
density prior to administration to the first subject. In several
embodiments, the administered cardiac stem cells produce one or
more diffusible factors and the one or more diffusible factors
improve the left ventricular ejection fraction of the heart of the
first subject, thereby, supplementing the cardiac function of the
first subject.
[0040] In some instances, damage or disease affects cardiac tissue
to such a degree that an assist device is required in order to
maintain normal cardiac functionality. Even with an assist device
(e.g., an implanted pacemaker), in some circumstances, cardiac
functionality is still too low for some individuals. Therefore,
there is provided, in several embodiments, a method of
supplementing the function of an implanted left ventricular assist
device (LVAD) in a subject, comprising identifying a subject having
damaged cardiac tissue and an implanted LVAD and delivering a
plurality of cardiac stem cells to the subject, wherein the damaged
cardiac tissue is functionally assisted by the implanted LVAD. In
several embodiments, the plurality of cardiac stem cells is
isolated from healthy donor cardiac tissue, wherein the healthy
donor tissue is either from the subject with the LVAD, or
preferably from a subject that is allogeneic to the subject with
the LVAD. In several embodiments, at least a portion of the
delivered cardiac stem cells engraft into the damaged cardiac
tissue, although, in some embodiments, at least a portion of the
engrafted cardiac stem cells are destroyed by the subject's immune
system. The engrafted cardiac stem cells release one or more
factors prior to the destruction, and as a result, the one or more
factors improve the function of the damaged cardiac tissue, thereby
supplementing the function of the LVAD.
[0041] In several embodiments, the factors comprise one or more of
VEGF, HGF, and IGFI. Other factors, as disclosed herein, are
produced by the cells (either the administered cells or the
endogenous cells, post-administration) in some embodiments.
[0042] In some embodiments, the combination of the direct effect of
the engrafted cells (e.g., new healthy functional cardiac cells)
with the factors serves to increase the LVAD supplementation.
Moreover, in several embodiments, the factors improve the viability
of the damaged cardiac tissue. A greater degree of viable cardiac
tissue also ameliorates the functionality of the cardiac tissue as
a whole, in several embodiments.
[0043] In one embodiment, the donor cardiac tissue is allogeneic
with respect to the subject. In one embodiment, the donor cardiac
tissue is autologous with respect to the subject. In additional
embodiments, combinations of allogeneic and autologous cells are
used. Such combinations may be beneficial in certain patient
populations, such as for example, the extremely immunocompromised.
Allogeneic cells produced by the methods herein induce only a
limited immune response, if any. However, extremely
immunosuppressed individuals may still be susceptible to minor
immune reactions from fully allogeneic cell populations. Thus,
combinations of autologous and allogeneic may be used in such
cases.
[0044] In several embodiments, the cardiac stem cells are selected
from the group consisting of: cardiospheres, cardiosphere-derived
cells (CDCs), and a subsequent generation of cardiospheres. The
choice of which cell type is used may be made based on the target
location. The size of the CDCs versus the cardiospheres may impact
the delivery route and/or the dose delivered. In some embodiments,
about 1.times.10.sup.5 to about 1.times.10.sup.7 cardiac stem cells
per kilogram of body weight of the subject are delivered. In some
embodiments, the regenerative cells are delivered by injection. In
some cases, the regenerative cells are delivered during the process
of implanting an LVAD. In such cases, a simple direct injection may
be used. Also, in such cases, the degree of damage or disease
(which corresponds to the degree of reduced functionality of the
heart) can be used to tailor the dose of cells administered. For
example, moderate damage may be primarily addressed by an LVAD,
with the administration of CDCs minorly supplementing the overall
cardiac function. In contrast, severe damage may require both the
LVAD and the administration of cells in order to maintain
sufficient cardiac function to support the viability of the
recipient.
[0045] In some embodiments, a subject has an implanted LVAD as a
result of at least one prior myocardial infarction. A single
infarction may not require the use of a LVAD, depending on its
severity. However, as discussed above, in several embodiments, even
a minor infarction may optionally be treated with the cells and
methods disclosed herein and/or an LVAD. In some such embodiments,
delivery of the cardiac stem cells reduces the infarct size, which
allows a greater supplementation of LVAD function, as the LVAD is
not forced to assist the same amount of stiff, non-contractile scar
tissue. Moreover, in several embodiments, the delivery of the
cardiac stem cells increase the left ventricular ejection fraction
of the subject by about 15%, thereby supplementing the function of
the LVAD. In some embodiments, greater or lesser increases in left
ventricular function are achieved, which can be tailored to the
amount of assistance provided by the LVAD. In some embodiments,
however, the administration of cells according to several
embodiments herein negates the need for an LVAD, as discussed
below.
[0046] In several embodiments, the damage or disease to a subject's
heart is so severe that the subject is suitable for undergoing a
cardiac transplant. In some embodiments, the cardiac stem cells are
delivered as a bridge to maintain left ventricular function until
the subject receives a cardiac transplant. In other words, such a
great degree of the cardiac tissue is damaged, diseased, or
otherwise compromised, that the administration of the cells
temporarily maintains cardiac viability and function, until such
time as a complete heart transplant can be performed. However, in
cases with less severe damage, the supplementation of the implanted
LVAD negates the need for a cardiac transplant.
[0047] In several embodiments, the engrafted cardiac stem cells
express one or more stem cell markers selected from the group
consisting of CD105, CD90, c-kit, and sca-1. In some embodiments,
the engrafted cardiac stem cells express one or more endothelial
cell markers selected from the group consisting of KDR, flk-1,
CD31, von Willebrand factor, Ve-cadherin, and smooth muscle alpha
actin. Despite the wide variety of markers that may be expressed on
the cells, e.g., one or more of the stem cell markers CD105 and
c-kit, the cells are not screened, subfractionated, or otherwise
selected based on the expression of the markers. In other words the
markers are used to characterize the cells, not selectively choose
the cells. However, in some embodiments, selection based one or
more of such markers is optionally performed.
[0048] In some embodiments, autologous cells are administered to
patients with left ventricular dysfunction and a recent myocardial
infarction with delivery occurring by intracoronary infusion via an
over-the-wire balloon catheter. In other embodiments, allogeneic
cells are administered to patients undergoing ventricular assist
device placement, via intramyocardial injection using a standard
needle and syringe and an epicardial approach during LVAD
placement. In some embodiments, the allogeneic cells produce an
immune response that is similar to the autologous cells.
[0049] In several embodiments, regenerative cells (autologous
and/or allogeneic) are administered to patients via epicardial
injection (or other delivery mechanism) in conjunction with LVAD
implantation.
[0050] Given the widespread use of LVADs to treat reduced cardiac
function, certain subject's that are treated by the methods
disclosed herein may already have an LVAD implanted. However, there
is also provided a method of reducing the dependence of a subject
on an implanted left ventricular assist device (LVAD) comprising
administering to the cardiac tissue of the subject a population of
regenerative cells, wherein, after administration, at least a
portion of the administered regenerative cells are removed from the
subject's cardiac tissue by endogenous mechanisms of the subject,
and wherein, prior to the removal, the administered regenerative
cells generate one or more signals that induce improvement in one
or more of the viability or function of the cardiac tissue of the
subject, thereby reducing the dependence of the subject on the
LVAD.
[0051] As discussed herein, certain subjects present with
widespread and severe cardiac damage and/or decreased functionality
as a result of a cardiac injury (e.g., a myocardial infarction) or
disease (e.g., congestive heart failure). Often, such subjects are
likely candidates for heart transplants. However, the costs, and
complications, associated with transplants mean that such an
approach is not a viable solution for all subjects. There is,
therefore, also provided a method of reducing a first subject's
likelihood of having a cardiac transplant, the method comprising,
obtaining a plurality regenerative cells from a second subject that
are suitable for administration to the first subject, wherein the
regenerative cells are expanded in culture to generate a population
of regenerative cells prior to administration to the first subject,
and administering at least a portion of the population of expanded
regenerative cells to the first subject; wherein the administered
regenerative cells produce one or more diffusible factors, wherein
the administered regenerative cells and the one or more diffusible
factors induce one or more of stimulation of resident cardiac cells
to grow, stimulation of resident cardiac cells to reproduce, or
stimulation of resident cardiac cells to improve functionally,
thereby reducing the likelihood of the first subject requiring a
cardiac transplant.
[0052] In some embodiments, the first subject has reduced cardiac
function and either has an LVAD or is a candidate for a heart
transplant (or both) due to one or more of an acute ischemic event,
chronic ischemia, or congestive heart disease. In several
embodiments, the first subject receives cells isolated from the
first subject's own tissue (e.g., an autologous transplant). In
other embodiments, the first subject is allogeneic with respect to
the donor of the tissue from which the regenerative cells are
isolated. In several embodiments, the administration increases the
function of the cardiac tissue of the first subject thereby
reducing the likelihood of the first subject requiring a cardiac
transplant.
[0053] In several embodiments, the regenerative cells are harvested
from healthy donor cardiac tissue. In several embodiments, the
donor cardiac tissue is allogeneic with respect to the subject.
[0054] In several embodiments, the dose of cells to be administered
may be determined by virtue of the severity of the cardiac damage
or disease, the age of the subject, the subject's overall general
health, and other factors. However, in several embodiments, about
1.times.10.sup.5 to about 1.times.10.sup.7 regenerative cells per
kilogram of body weight of the subject are administered. In other
embodiments, other doses are used. In some embodiments, the
regenerative cells are administered by local intramyocardial
injection. In some embodiments, the regenerative cells are
administered by epicardial injection. Other routes of
administration are used, in some embodiments, for example,
depending on the precise location of a LVAD and/or the damaged
cardiac tissue. In some embodiments, the regenerative cells are
administered during the process of implanting the LVAD. In other
embodiments, the regenerative cells are administered during the
process of explanting an implanted LVAD.
[0055] While various mechanisms, both direct and indirect, may be
involved, in some embodiments, the regenerative cells increase the
incidence of angiogenesis in the subject's cardiac tissue. In some
embodiments, the regenerative cells increase the left ventricular
ejection fraction of the subject by at least about 5%, at least
about 10%, at least about 15%, or more, thereby reducing the
dependence of the subject on the implanted LVAD.
[0056] As discussed herein, in several embodiments, at least a
portion of the administered cells engraft into the target tissue.
Additionally, in several embodiments, at least a portion of the
cells are removed from the target tissue by endogenous mechanisms
of the subject receiving the cells. In one embodiment, the
endogenous mechanisms comprise recruitment of at least a portion of
the subject's immune system. In one embodiment, the endogenous
mechanisms comprise induction of apoptosis of the administered
regenerative cells.
[0057] In several embodiments, the regenerative cells are selected
from the group consisting of: cardiospheres, cardiosphere-derived
cells (CDCs), and a subsequent generation of cardiospheres. In one
embodiment, the regenerative cells are CDCs. In several
embodiments, the regenerative cells express one or more of the stem
cell markers CD105 and c-kit, but are not screened,
subfractionated, or otherwise selected based on the expression of
the markers.
[0058] As a result of acute injury to the heart (e.g., a myocardial
infarction) or long-term damage there may be an associated increase
in cell death as a result, in particular, apoptotic cell death.
Therefore, there is provided a method of decreasing apoptosis in a
heart having been affected by myocardial infarction, comprising
administering a population of cardiac stem cells to a subject
having a heart affected by a myocardial infarction, wherein, after
administration, at least a portion of the administered population
of cardiac stem cells is destroyed by endogenous mechanisms of the
subject, wherein the administered population of cardiac stem cells
generate one or more paracrine signals post-administration and
prior to the destruction, and wherein the one or more paracrine
signals reduce the incidence of apoptosis in the cardiac tissue
affected by the myocardial infarction.
[0059] There is also provided a method of decreasing apoptosis in a
heart having been affected by myocardial infarction, comprising
administering a population of cardiac stem cells to a subject
having a heart affected by a myocardial infarction, wherein the
administered cardiac stem cells generate one or more paracrine
signals, and wherein the one or more paracrine signals act on the
administered cardiac stem cells and resident cardiac stem cells to
increase the viability of the administered cardiac stem cells and
the resident cardiac stem cells, thereby reducing the incidence of
apoptosis in the cardiac tissue affected by the myocardial
infarction.
[0060] In several embodiments the administration reduces the
expression of apoptotic markers on cardiac cells affected by the
myocardial infarction, thereby indicating a reduction in one or
more portions of the apoptotic cascade. For example, in one
embodiment, the decrease in apoptosis is associated with reduced
Caspase 3 expression. In some embodiments, administration reduces
the amount of plasma membrane damage on the cardiac cells affected
by the myocardial infarction. In still additional embodiments,
anti-apoptotic signals or markers are increased. For example, in
one embodiment, the decrease in apoptosis is associated with an
increase in Akt expression. As a result, in several embodiments,
apoptosis is decreased by about 5% abou5 10%, about 15%, about 20%,
or more. In several embodiments, the decrease in apoptosis occurs
within several hours after administration of the cardiac stem
cells. However, in several embodiments, the decrease in apoptosis
occurs within several days after administration of the cardiac stem
cells. In some embodiments, the decrease in apoptosis is associated
with increased cardiac function.
[0061] In some embodiments, damaged or diseased cardiac tissue is
the result of a reduced blood supply to a region of the cardiac
tissue. For example, a myocardial infarction may result from the
partial or total blockage of a vessel providing oxygenated blood to
one or more regions of the heart. In some embodiments, the major
vessels may be affected, while in some embodiments, minor blockages
(e.g., to the arterioles) may also damage the cardiac tissue. Thus,
in order to combat the deleterious effects of reduced blood supply,
there is provided a method for increasing angiogenesis in a heart
having been affected by a myocardial infarction, comprising
administering cardiac stem cells to a subject having a heart
affected by a myocardial infarction, wherein the administered
population of cardiac stem cells generate one or more paracrine
signals post-administration and prior to the destruction, and
wherein the one or more paracrine signals increase the level of
angiogenesis in the cardiac tissue affected by the myocardial
infarction.
[0062] In several embodiments, at least a portion of the
administered population of cardiac stem cells is destroyed by the
subject's immune system. As a result, there may not be long-term
survival of the entire population of administered cells. However,
as a result of the paracrine signals, at least a portion of the
beneficial effects of the administered cells carries on beyond the
time at which the cells are destroyed. In some embodiments, the
administered cells die on their own time frame, and are simply
removed from the tissue by endogenous mechanisms (e.g.,
phagocytosis). In some embodiments, the combination of the
administered cells themselves (a direct repair mechanism) works in
concert with the induced paracrine cascade (either from the
administered cells or from the endogenous cells) to effect the
increased angiogenesis.
[0063] In several embodiments, the increased angiogenesis results
in an increase in vessel density in the cardiac tissue of the
subject. In some embodiments, the vessel density is increased by
about 2-fold, about 3-fold, about 5-fold, or greater. In some
embodiments, a 10% increase, a 15% increase, a 20% increase, or
greater is achieved. In several embodiments, the increased
angiogenesis increases the length of existing blood vessels. In
some embodiments, the vessel length is increased by about 2-fold,
about 3-fold, about 5-fold, or greater. In some embodiments, a 10%
increase, a 15% increase, a 20% increase, or greater is achieved.
In several embodiments, combinations of increased vessel density
length and increased density are achieved. In addition to the
effects on existing vessels, in several embodiments, the increased
angiogenesis increases the formation of new blood vessels. In some
embodiments, a 5%, 10%, 15% or greater increase in new vessels is
achieved. In combination with the positive effects on existing
vessels, blood supply is increased to the region of damaged or
diseased cardiac tissue, which, in several embodiments, provides
increased function and/or viability to the region. In several
embodiments, the increased vessel density, increased vessel length,
and/or the new vessels are associated with improved function of
about 5%, 10%, 15%, or greater in at least one of left ventricular
percent fractional area and left ventricular ejection fraction.
[0064] In several embodiments, the paracrine signal comprises
release of VEGF from the cardiac stem cells. In some embodiments,
additional pro-angiogenic factors are also generated by the cardiac
stem cells, including endogenous cardiac stem cells.
[0065] In several embodiments, the plurality of cardiac stem cells
is harvested from healthy donor cardiac tissue. Depending on the
amount of cells desired or required for administration, the
plurality of the harvested cardiac stem cells is expanded in
culture to yield the population of cardiac stem cells. While in
several embodiments, the cardiac stem cells are allogeneic with
respect to the subject, in other embodiments, the cardiac stem
cells are autologous with respect to the subject.
[0066] In addition to the administration of regenerative cells to a
subject, there is also provided a method of regenerating cardiac
tissue in an individual having damaged cardiac tissue, comprising
isolating a population of regenerative cells from cardiac tissue of
a donor, expanding the population of regenerative cells in culture,
wherein the regenerative cells in culture generate one or more
paracrine factors, isolating the one or more paracrine factors from
the culture, wherein the isolated paracrine factors are suitable
for administration to a damaged heart of an individual, and
wherein, after administration, one or more paracrine factors
facilitate the formation of new cardiac tissue in the individual.
In several embodiments, said damaged cardiac tissue is a result of
myocardial infarction. In some embodiments, administration of said
one or more paracrine factors results in a decrease in infarct
size. In additional embodiments, the one or more paracrine factors
improves one or more of the viability or function of the damaged
cardiac tissue. In one embodiment, administration of one or more
paracrine factors results in at least a 15% improvement in left
ventricular function.
[0067] In several embodiments, there is also provided a method of
improving the cardiac function of an individual having damaged
cardiac tissue, comprising identifying a subject having damaged
cardiac tissue and administering to said subject one or more
paracrine factors, wherein said paracrine factors are obtained from
a population of regenerative cells in culture, wherein said
regenerative cells were isolated from the cardiac tissue of a
donor, wherein said population of regenerative cells comprises
cardiac stem cells and, wherein, after administration, said one or
more paracrine factors induce formation of new functional cardiac
tissue and/or improve the function of the damaged cardiac tissue of
said subject.
[0068] In several embodiments, said cardiac stem cells are selected
from the group consisting of cardiospheres, cardiosphere-derived
cells, and a subsequent generation of cardiospsheres. In one
embodiment, said one or more paracrine factors comprise one or more
of VEGF, HGF, and IGFI.
[0069] Additionally, there is provided a method of increasing the
function of cardiac tissue in an individual having damaged cardiac
tissue, comprising identifying a subject having damaged cardiac
tissue, administering to said subject one or more paracrine factors
selected from the group consisting of VEGF, HGF, and IGFI, wherein
said paracrine factors are obtained from a population of
regenerative cells in culture, wherein said regenerative cells were
isolated from the cardiac tissue of a donor, wherein said
population of regenerative cells comprises cardiac stem cells,
wherein said cardiac stem cells comprise one or more of
cardiospheres, cardiosphere-derived cells, and a subsequent
generation of cardiospsheres, wherein, after administration, said
one or more paracrine factors recruit endogenous cardiac cells to
the damaged cardiac tissue, wherein said recruited cells repair
said damaged cardiac tissue, thereby improving the function of the
damaged cardiac tissue of said subject. In several embodiments, the
one or more paracrine factors induce production of paracrine
factors by endogenous cells at or near the site of administration,
thereby further increasing repair of said damaged cardiac tissue.
In several embodiments, such method result in at least a 15%
improvement in left ventricular function.
[0070] In several embodiments, the donor is allogeneic with respect
to the individual, while in other embodiments, the donor and the
recipient individual are the same. Thus, there are provided herein
both cell-based and cell-free methods of generating and/or
repairing cardiac tissue.
BRIEF DESCRIPTION OF THE FIGURES
[0071] FIGS. 1A-1I depict specimen processing for cardiosphere
growth and Cardiosphere-Derived Cell (CDC) expansion. FIG. 1A
depicts a schematic of the steps involved in certain embodiments of
specimen processing. FIG. 1B depicts a human endomyocardial biopsy
fragment on day 1. FIG. 1C depicts a human explant 3 days after
plating. FIG. 1D depicts the edge of a human explant 13 days after
plating and showing stromal-like and phase-bright cells. FIG. 1E
depicts results of sub-population selection performed using
cardiosphere-forming cells. c-kit.sup.+ cells were 90.0.+-.4.7%
CD105.sup.-, and c-kif.sup.- cells were 94.0.+-.0.8% CD105.sup.+
(n=3). FIG. 1F depicts human cardiospheres on day 25, 12 days after
collection of cardiosphere-forming cells. FIG. 1G depicts human
CDCs during passage 2, plated on fibronectin for expansion. FIG. 1H
depicts cumulative growth curves for 11 specimens from
untransplanted patients over the course of 4 months. FIG. 11
depicts growth for 59 specimens from transplanted patients. Day 0
corresponds to the date the specimen was collected and cell number
on that day is plotted as 1 on the log scale, since no
cardiosphere-forming cells had yet been harvested from the
specimen.
[0072] FIGS. 2A-2C depict cardiosphere and CDC phenotypes. FIG. 2A
depicts expression of c-kit throughout the core of a cardiosphere
and CD105 on the periphery. FIG. 2B depicts expression of cardiac
MHC and TnI primarily on cardiosphere periphery. FIG. 2C depicts
c-kit and CD105 expression levels in CDCs at passage 2 shown for
one representative specimen (n=3 and n=2).
[0073] FIGS. 3A-3E depict engraftment and regeneration. Engraftment
of CDCs (FIG. 3A) or fibroblasts (FIG. 3B) is depicted 20 days
after injection in heart sections double stained for H&E and
beta-galactosidase. Infiltration of CDCs is seen as a distinct
band, while a rare group of a few fibroblasts can be detected in
some sections. Masson's trichrome staining as used to calculate
myocardial regeneration is shown for a representative CDC-injected
mouse (FIG. 3C) and fibroblast-injected mouse (FIG. 3D). FIG. 3E
depicts the percent of viable myocardium found within the infarcted
area in CDC (n=8), PBS (n=4), and fibroblast-injected (n=4) groups
(*p<0.01).
[0074] FIGS. 4A-4F depicts functional cardiac improvement.
Long-axis views from an echocardiogram performed after 20 days in a
CDC-injected mouse are shown. FIG. 4A depicts end-diastole while
FIG. 4B depicts end-systole. Yellow lines trace around the left
ventricular area used for the calculation of LVEF and LVFA. (FIG.
4C and FIG. 4D). FIG. 4E shows the left ventricular ejection
fractions (LVEF) for the three experimental groups after 20 days
(CDC n=8, PBS n=7, Fibroblast n=4; *p<0.01). LVEF was calculated
as
100.times.(LVVolume.sub.diastole-LVVolume.sub.systole)/LVVolume.sub.dias-
tole
[0075] and left ventricular volume (LVVolume) was calculated from
long-axis views assuming a prolate ellipsoid. FIG. 4F depicts left
ventricular percent fractional area (LVFA) for the three
experimental groups after 20 days. *p<0.01. LVFA was calculated
as:
100.times.(LVArea.sub.diastole-LVArea.sub.systole)/LVArea.sub.diastole
[0076] FIGS. 5A-5C depict quantification of cardiac tissue
regeneration. FIG. 5A shows Masson's trichrome staining for a
representative CDC-injected mouse. The total infarct zone is
outlined in yellow in FIG. 5B and FIG. 5C. FIG. 5B depicts areas of
fibrosis in red after image processing. FIG. 5C depicts areas of
viable myocardium in red after image processing. Six sections were
analyzed per animal and an average taken.
[0077] FIGS. 6A-6F depict an engraftment time course. FIG. 6A
depicts the bolus of injected cells on day 0 in an H&E stained
section of cardiac tissue. FIG. 6B depicts engraftment of CDCs 8
days after injection. FIGS. 6C and FIG. 6D depict engraftment of
CDCs 20 days after injection. FIGS. 6E and FIG. 6F depict
corresponding higher magnification views of FIG. 6C and FIG. 6D
demonstrating colocalization of lac-Z-positive CDCs and viable
myocardium.
[0078] FIGS. 7A-7C depict the growth of CDCs generated from 2 whole
hearts and subjected to various growth conditions. FIG. 7A depicts
CDC yield from samples taken from different regions of the heart.
FIG. 7B depicts the effect of tissue storage in cold cardioplegia
for up to 6 days or freezing and thawing. FIG. 7C depicts a
comparison of yield of CDCs from each of the sample hearts grown
under the same conditions.
[0079] FIGS. 8A-8B depict phenotypic identity of human CDCs. FIG.
8A depicts surface immunophenotype by flow cytometry of human CDCs
from multiple patients. Dual-labeled analysis depicting mesenchymal
and progenitor CDC sub-populations is shown in FIG. 8B.
[0080] FIGS. 9A-9C depict characteristics of differentiating CDCs.
FIG. 9A depicts sarcomeric organization in human CDCs co-cultured
with rat neonatal ventricular myocytes. FIG. 9B depicts inwardly
rectifying potassium current recorded form CDCs, which is
consistent with cardiomyocyte ventricular phenotype. FIG. 9C
depicts L-type calcium current I.sub.Ca,L recorded from CDCs
transduced with the beta subunit of the L-type calcium channel,
indicating the presence of the pore-forming alpha subunit.
[0081] FIGS. 10A-10B depict CDCs forming tube matrices in an
angiogenesis assay. Human CDCs (FIG. 10A) form tube-like networks
at early timepoints and undergo complex morphological changes at
late timepoints. This is in comparison to human umbilical vein
endothelial cells (HUVECs) (FIG. 10B) which form distinct tube
networks.
[0082] FIGS. 11A-11B depict engraftment of human CDCS. FIG. 11A
depicts luciferase-labeled cells detected in the heart at 1 day, 4
days, and 1 week post-administration. FIG. 11B depicts peak signals
detected for each of 3 mice.
[0083] FIGS. 12A-12K depict detection of human CDCs in injected
mouse hearts. Mouse hearts excised at different timepoints
following MI and delivery of human CDCs are shown in FIGS. 12A-12C.
Human nuclei are identified at each timepoint by green fluorescence
overlaid on the blue fluorescence of all nuclei (FIGS. 12D-12K).
INF=infarct. BZ=border zone.
[0084] FIG. 13 depicts a summary of echocardiography on mice
injected with CDCs, normal human derived fibroblasts, or phosphate
buffered saline. CDC-injected animals maintained function after MI,
while NHDF- and PBS-injected declined.
[0085] FIG. 14 depicts the effect of various CDC subpopulations on
murine cardiac function. CDC-injected animals outperformed animals
injected with either c-kit or CD90 sub-populations.
[0086] FIGS. 15A-15R depict differentiation of human CDCs at
various time-points in infarcted mice.
[0087] FIGS. 16A-16P depict formation of cardiomyocytes and
non-cardiomyocytes from human CDCs at 6 weeks post-MI. Human nuclei
of interest are outlined in FIGS. 16E-16H. Example cardiomyocyte
nuclei are shown in FIGS. 16M-16P at higher magnification.
[0088] FIG. 17 depicts a schematic of in vitro and in vivo
experimental designs to evaluate functional effects of paracrine
signals released from regenerative cells.
[0089] FIGS. 18A-18C depict protein array analysis of serum free
conditioned media collected from the culture of regenerative cells,
specifically cardiospheres and CDCs.
[0090] FIGS. 19A-19E depict in vitro analysis of VEGF, HGF and IGFI
secretion.
[0091] FIGS. 20A-20F depict the gene expression profiles of various
growth factor receptors on cardiospheres and CDCs.
[0092] FIGS. 21A-21K depict the effects of conditioned media from
regenerative cells on cultured cells. FIGS. 21A-21E depict the
pro-survival effects of conditioned media from regenerative cells
on neonatal rat ventricular myocytes (NVRMs) and pro-angiogenic
(FIGS. 21F-21K) effects of conditioned media from regenerative
cells on cultured HUVEC cells.
[0093] FIG. 22 depicts results of growth factor and cytokine
protein expression analysis in infarcted regenerative cell-injected
mouse hearts.
[0094] FIGS. 23A-23D depict assessment of tissue viability and
cardiac tissue perfusion after administration of regenerative
cells.
[0095] FIGS. 24A-24D depict an evaluation of the amount of
regenerative cell contribution to capillaries and muscle tissue
formed in the border zone of an induced infarction.
[0096] FIGS. 25A-25C depict an analysis of infarct size evaluated
by Masson's trichrome staining.
[0097] FIGS. 26A-26H depict MHC Class I and II expression for human
CDCs before and after interferon stimulation.
[0098] FIGS. 26I-26J depict the fractional change in lymphocyte
proliferation normalized to syngeneic co-culture for allogeneic and
xenogeneic co-cultures.
[0099] FIGS. 26K-26M depict the associated lymphocyte infiltration
in syngeneic, allogeneic, and xenogeneic co-cultures.
[0100] FIG. 26N depicts the index of lymphocyte proliferation
induced by various co-cultures.
[0101] FIG. 26O depicts data related to the quantification of
inflammatory cytokines after co-culture.
[0102] FIG. 27A depicts a graphical experimental scheme employed to
study engraftment and function of CDCs.
[0103] FIG. 27B depicts the experimental and control groups used to
evaluate engraftment and function.
[0104] FIG. 27C depicts data related to 1-week cell engraftment in
various transplant groups.
[0105] FIG. 27D depicts data related to 3-week cell engraftment in
various transplant groups.
[0106] FIGS. 28A-28C depict hematoxylin and eosin staining of
syngeneic, allogeneic, or xenogeneic treated cardiac tissue.
Analysis revealed significant evidence of an immune reaction in
xenogeneic (28A) heart sections, but very little in syngeneic (28B)
or allogeneic (28C) sections. Example images are shown at 3 weeks
post-MI.
[0107] FIGS. 28D-28G depict 1-week and 3-week data related to the
rejection score for various transplant types at various regions
throughout the infarct scar or border zone.
[0108] FIGS. 28H-28J depict immunohistochemistry data related to
cell infiltration post-transplant.
[0109] FIGS. 28K.sub.1-28K.sub.15 depict immunohistochemistry
further defining the types of infiltrating lymphocytes in each
transplant.
[0110] FIGS. 28L-28M depict data related to monocyte infiltration
in each transplant type at 1 and 3 weeks.
[0111] FIGS. 29A-29E represent data and analysis of T-cell, B-cell,
and macrophage infiltration in various treatment groups at 1 and
3-weeks post-MI. Engraftment of GFP-labeled CDCs and the level of T
cell (CD3.sup.+, CD4.sup.+, or CD8.sup.+), B cell (CD45R.sup.+), or
macrophage (CD68.sup.+) infiltration surrounding the cells 3 weeks
post-MI is shown for animals who received syngeneic (29A),
allogeneic (29B), or xenogeneic (29C) CDCs. The number of cells per
high power field is quantified 1 week (29D) and 3 weeks post-MI
(29E).
[0112] FIGS. 30A-30G depict quantification of serum concentrations
of pro-inflammatory cytokines IFN-.gamma., IL-1.beta., KC/GRO,
TNF-.alpha., (FIGS. 30A, 30B, 30F, and 30G, respectively), or the
anti-inflammatory cytokines IL-13, IL-4, and IL-5 (FIGS. 30C, 30D,
and 30E, respectively).
[0113] FIG. 31A depicts morphometric analysis of explanted hearts 3
weeks post infarction in syngeneic (upper left), allogeneic (upper
right), xenogeneic (lower left) and control (lower right)
groups.
[0114] FIG. 31B depicts infarct size in the various transplant
groups while FIG. 31C depicts infarcted wall thickness.
[0115] FIGS. 31D-31G depict fractional area change (31D), ejection
fraction (31E), fractional shortening (31F), and treatment effect
(31G) for the various transplant groups.
[0116] FIGS. 31H-31V depict immunohistochemistry related to the
location of transplanted cells as well as markers for cell cycle,
stem cell markers, and endothelial cell markers.
[0117] FIGS. 31W-31Z depict data at 1 and 3 weeks for,
respectively, Ki67/smooth muscle actin, BrdU expression/smooth
muscle actin expression, cKit expression, and vessel density.
[0118] FIG. 31AA depicts protein analysis of growth factor
secretion at various time points post-MI.
[0119] FIGS. 31BB-31DD depict data for VEGF (31BB), IGF (31CC), and
HGF (31DD) in syngeneic, allogeneic, or control groups.
[0120] FIGS. 32A-32B depict schematics of two study designs
disclosed herein.
[0121] FIG. 33 depicts the percentage of CDCs retained, after
injecting into three different areas of pig heart, as determined by
an in vitro luciferase assay 24 hours after injection.
[0122] FIGS. 34A-34C depict changes in LV function as measured by
ventriculography. Paired analysis of LVEF before injection and 8
weeks later in controls and CDC-injected pigs is shown (34A).
Average LVEF for pigs in the CDC-injected group was significantly
higher than those in the control group 8 weeks after injection
(34B). Treatment effects in the two groups as absolute change in
LVEF (34C).
[0123] FIGS. 35A-35C depict various echocardiographic measurements.
FIG. 35A shows that Emax in the cardiosphere group was
significantly higher than in placebo-injected pigs. Emax in the CDC
group was not significantly higher than placebo-treated animals.
(Levene's test p<0.05, Kruskal-Wallis comparison p=0.003.
Placebo vs. CDC, p=NS; Placebo vs. cardiosphere, p=0.03).
Representative families of PV loops are shown below the graph for
placebo, CDC and cardiosphere-treated animals. FIG. 35B shows the
change in diastolic volume (Final EDV--Baseline EDV) is
significantly lower in the cardiosphere treated group than the CDC
treated group, with a trend to being lower than the placebo group.
(ANOVA p=0.02). FIG. 35C shows that the changes in end-diastolic
pressure measurements demonstrated a significantly higher fall in
end-diastolic pressure in cardiosphere-injected animals compared to
CDC treated animal (ANOVA p<0.01; cardiospheres vs. CDCs
p=0.001). This indicates that, in some embodiments, the ventricles
of cardiosphere-injected hearts are more responsive to treatment
after infarction CDC-treated hearts. However, in some embodiments,
CDC-treated hearts are responsive to an equal or greater
degree.
[0124] FIGS. 36A-36C depict improved regional contractility in
cardiosphere-injected pigs relative to sham-injected controls.
[0125] FIGS. 37A-37B depict two examples of islands of
cardiomyocytes with lacZ-positive nuclei in the periinfarct zone,
one from each animal that received intramyocardial
genetically-labeled CDCs.
[0126] FIGS. 38A-38F depict schematics of various cell processing
and cell banking procedures disclosed herein.
[0127] FIGS. 39A-39B depict correlation of LVEF values measured
independently by two blinded, experienced echocardiographers.
Measurements for each animal from the two readers show good
correlation at baseline (39A) and 3 weeks after treatment
(39B).
[0128] FIGS. 40A-40E depict characteristics of the various stem
cell types evaluated. FIGS. 40A-40D depict phase-bright images of
CDCs (shown in 40A), bone marrow-derived mesenchymal stem cells
(BM-MSC, shown in 40B), adipose tissue-derived mesenchymal stem
cells (AD-MSC, shown in 40C), and bone marrow mononuclear cells
(BM-MNC, shown in 40D) in culture. FIG. 40E displays the expression
of certain surface markers on each cell type.
[0129] FIGS. 41A-41J depict the secretion of a variety of paracrine
factors by various stem cell types. FIGS. 41A-41F display the
concentrations of each factor by each cell type. FIGS. 41G-41K
display a relative paracine factor profile for each cell type.
[0130] FIGS. 42A-42C depict the in vitro production of growth
factors from rat cells. The concentrations of VEGF (42A), IGF-1
(42B), and HGF (42C) measured by ELISA are shown.
[0131] FIGS. 43A-43D depict analysis of in vitro myogenic
differentiation and angiogenesis assay. FIG. 43A shows that
Troponin T, with distinct myocyte-like appearance, was expressed
spontaneously in a fraction of CDCs cultured for 7 days. This
cardiac-specific marker was rarely expressed in BM-MSCs, AD-MSCs,
and BM-MNCs. FIG. 43B depicts quantitative analysis of Troponin T
expression in CDCs (9% of the cells positive), BM-MSCs (0.4%
positive) and AD-MSCs and BM-MNCs (approximately 0.1% positive).
FIG. 43C depicts CDCs, BM-MSCs, and AD-MSC-derived production of
capillary-like tube formations in extracellular matrix. BM-MNCs did
not form similar structures under these conditions. FIG. 43D
depicts quantitation and comparison of tube formation capacity by
the different cell types. Bars=50 um.
[0132] FIGS. 44A-44B depict the in vitro resistance of different
types of human cells to oxidative stress. FIG. 44A shows
representative images of TUNEL-positive cells (red) after 24 hours
exposure to 100 mM H2O2. FIG. 44B depicts quantitative assessment
of apoptotic cells. The number of TUNEL-positive cells was lower in
CDC group compared to BM-MNC group with 100 mM H2O2. Bar=50 mm.
[0133] FIGS. 45A-45B depict in vitro resistance of different cell
types isolated from the same rat, to oxidative stress. FIG. 45A
shows representative images of TUNEL-positive cells (red) after 24
hours' exposure to 100 mM H2O2. FIG. 45B shows quantitative
analysis showed that the number of apoptotic cells was
significantly lower in CDCs, BM-MSCs, and AD-MSCs than in BM-MNCs
with 100 mM H2O2. Bar=50 mm.
[0134] FIGS. 46A-46B depict in vitro myogenic differentiation of
different cell types (all isolated from the same rat). FIG. 46A
shows a fraction of the CDCs positively expressed the cardiac
specific marker Troponin T, with distinct myocyte-like appearance.
Troponin T expression was rarely observed in BM-MSCs, AD-MSCs, and
BM-MNCs. FIG. 46A depicts the quantitative assessment of Troponin T
expression in different cell types is shown. Bar=50 mm.
[0135] FIGS. 47A-47C depict cell engraftment and in vivo myogenic
differentiation. FIG. 47A Immunostaining shows some human CDCs
(green, HNA) expressing a-sarcomeric actin, indicating myogenic
differentiation, 3 weeks after implantation into infarcted mice
hearts. FIG. 47B Quantitation of engraftment (HNA+ cells). C)
Quantitation of cardiomyocytes differentiated from transplanted
human cells (HNA+/aSA+ cells). Bar=20 .mu.m.
[0136] FIGS. 48A-48C depict results of cell apoptosis studies.
FIGS. 48A and 48B are representative images of TUNEL-positive cells
in the infarcted hearts of mice 3 weeks after cell treatment with
CDCs (48A) and PBS (48B). FIG. 48C depicts quantitative assessment
of TUNEL-positive cells in the myocardium of mice treated with
different cell types and control, is shown. Bar=500 .mu.m.
[0137] FIG. 49 depicts cardiac function of mice treated with the
various cell types. LVEF at baseline (4 hrs post-MI) did not differ
among groups, indicating a similar infarct size in animals of all
groups. After 3 weeks, LVEF was higher in mice implanted with CDCs,
compared to animals treated with cells of non-cardiac origin.
Implantation of BM-MSCs also improved cardiac function, compared to
controls injected with saline only. Data are presented as
mean.+-.SEM.
[0138] FIGS. 50A-50H depict Ventricular remodeling after treatment
with various cell types. FIGS. 50A-50F are representative images of
Masson's staining of infarcted mice hearts, after implantation of
different types of human cells or saline injection only.
Quantitative analyses of LV wall thickness (50G) and infarct
perimeter (50H) show that remodeling was attenuated more
efficiently by CDC implantation, compared with BM-MSCs, AD-MSCs,
and BM-MNC treatment, although implantation of BM-MSCs, AD-MSCs,
and BM-MNCs resulted in less remodeling compared to control
treatment with saline injection only.
[0139] FIGS. 51A-51E depict a comparison of purified c-kit.sup.+
stem cells and unsorted CDCs. FIG. 51A shows LVEF 3 weeks after
infarction. LVEF was higher in mice that received unsorted CDCs
than those with c-kit.sup.- cells purified from the same CDCs.
FIGS. 51B-51E show that although the same number of cells was used
for culture, the purified c-kit.sup.+ stem cells released less
VEGF, SDF, IGF-1, and HGF than the unsorted CDCs.
DETAILED DESCRIPTION
[0140] Cell therapy, the introduction of new cells into a tissue in
order to treat a disease, represents a promising new method for
repairing or replacing diseased tissue with healthy tissue.
Therefore, in several embodiments described herein, methods of
isolating, culturing, preparing, and introducing regenerative cells
into a recipient are provided and result in one or more of
treatment of symptoms of a cardiac disease, improvement in cardiac
function, and/or regeneration of cardiac tissue in the recipient.
In several embodiments, the cardiac disease is the result of one or
more of an acute heart failure or chronic heart failure. In some
embodiments, the disease creates damaged to the cardiac tissue due
to one or more of ischemia, reperfusion, or infarction.
[0141] As used herein, the term "regenerative cells" shall be given
its ordinary meaning and shall include mixed cell populations
(e.g., cardiospheres) and derivatives thereof (e.g., CDCs and
secondary generations of cardiospheres (IICSps)), unless explicitly
indicated otherwise. Regenerative cells include cells that directly
repair tissue (e.g., stem cells) and cells that promote tissue
repair (e.g., through paracrine effects or other signaling
events).
General
[0142] Several embodiments disclosed herein provide methods for
expanding populations of resident stem cells from organs, such that
only small initial samples are required. Such small initial samples
can be obtained relatively non-invasively, for example, by a simple
percutaneous entry. Such samples can be obtained using a
percutaneous bioptome, in some embodiments. The bioptome can be
used to access a tissue sample from any organ source, including
heart, kidney, liver, spleen, and pancreas. Particularly suitable
locations within the heart which can be accessed using a bioptome
include, but are not limited to, the crista terminalis, the right
ventricular endocardium, the septal or ventricle wall, and the
atrial appendages. These locations have been found to provide
abundant stem or progenitor cells. Accessing such locations is
facilitated by use of a bioptome which is more flexible than the
standard bioptome used for accessing the right ventricular
endocardium for diagnostic purposes. Preferably the bioptome is
also steerable by an external controller. Such procedures are
enable collection of tissue on an out-patient basis without major
surgery or general anesthesia. While percutaneous biopsy is
employed in some embodiments, in other embodiments, several
embodiments employ explanted tissues (e.g., those removed from a
subject and under evaluation for transplantation).
[0143] One of the advantages of several embodiments disclosed
herein that enables use of a small biopsy sample (or small
non-biopsy samples) as a starting material is the collection of a
cell population which has previously been ignored or discarded. In
one embodiment, this cell population is formed by treating the
sample with a protease and harvesting or collecting the cells that
are liberated from the sample. The use of these liberated cells
enhances the rate of cell population expansion. Non-limiting
examples of proteases which can be employed include collagenase,
matrix metalloproteases, trypsin, and chymotrypsin. This technique
can be applied to any organ from which resident stem cells are
desired, including, for example, heart, kidney, lung, spleen,
pancreas, and liver. In some embodiments, the mass of tissue
collected to isolated resident stem cells is roughly equivalent,
regardless of the manner in which the tissue was obtained. For
example, in some embodiments, the amount of tissue collected ranges
from about 10 mg of tissue to about 1000 mg of tissue. For example,
in some embodiments, the amount of tissue collected ranges from
about 20 mg of tissue to about 500 mg of tissue. In some
embodiments, the amount of tissue collected ranges from about 10-50
mg of tissue, about 50-100 mg of tissue, about 100-150 mg of
tissue, about 150-200 mg of tissue, about 200-250 mg of tissue,
about 250-300 mg of tissue, about 300-350 mg of tissue, about
350-400 mg of tissue, about 400-450 mg of tissue, about 450-500 mg
of tissue, and overlapping ranges thereof. In some embodiments, a
total of no more than 20 mg of tissue is collected. In some
embodiments, a total of no more than 30 mg of tissue is collected.
In some embodiments, a total of no more than 40 mg of tissue is
collected. In some embodiments, a total of no more than 50 mg of
tissue is collected. In some embodiments, a total of no more than
60 mg of tissue is collected. In some embodiments, a total of no
more than 70 mg of tissue is collected. In some embodiments, a
total of no more than 80 mg of tissue is collected. In some
embodiments, a total of no more than 90 mg of tissue is collected.
In some embodiments, a total of no more than 100 mg of tissue is
collected. In several embodiments, the mass of tissue collected
from any single biopsy or collection ranges between about 10-20 mg,
including about 11, 12, 13, 14, 15, 16, 17, 18, and 19 mg. In
several embodiments, the mass of tissue collected from any single
biopsy or collection ranges between about 20-30 mg, including about
21, 22, 23, 24, 25, 26, 27, 28, and 29 mg. In several embodiments,
the mass of tissue collected from any single biopsy or collection
ranges between about 30-40 mg, including about 31, 32, 33, 34, 35,
36, 37, 38, and 39 mg. In several embodiments, the mass of tissue
collected from any single biopsy or collection ranges between about
40-40 mg, including about 41, 42, 43, 44, 45, 46, 47, 48, and 49
mg.
[0144] Resident stem cells are those which are found in a
particular organ. As discussed below in more detail, it is believed
that the stem cells found in a particular organ are not necessarily
pluripotent, but rather, are committed to a particular branch of
differentiation. Thus in the heart, one expects to find cardiac
stem cells, and in the kidney one expects to find kidney stem
cells. Despite this, in several embodiments some of the stem cells
isolation and expanded using the methods disclosed herein are able
to develop into cells of an organ other than the one from which
they were obtained.
[0145] Cardiospheres are self-associating aggregates of cells which
have been shown to display certain properties of cardiomyocytes.
Cardiospheres have been shown to "beat" in vitro. They are
excitable cells and contract in synchrony. In one embodiment, the
cells which form the cardiospheres have been obtained from heart
biopsies. In one embodiment, the cells which form the cardiospheres
have not been obtained from heart biopsies, but rather have been
obtained from a whole heart, or a portion thereof. The
cardiospheres can be disaggregated using standard means known in
the art for separating cell clumps or aggregates, including, but
not limited to trituration, agitation, shaking, blending. In one
embodiment, cardiospheres are disaggregated to single cells. In
several embodiments they are disaggregated to smaller aggregates of
cells. In several embodiments, after disaggregation, the resultant
cells are grown on a solid surface, such as a culture dish, a
vessel wall or bottom, a microtiter dish, a bead, flask, roller
bottle, etc. The surface can be glass or plastic, for example. In
one embodiment, the cells are capable of adhering to the material
of the solid surface. The solid surface is optionally coated with a
substance which encourages adherence in some embodiments. Several
such substances are known in the art and include, without
limitation, fibronectin. hydrogels, polymers, laminin, serum,
collagen, gelatin, and poly-L-lysine. In several embodiments,
growth on the surface is monolayer growth. These cells are
cardiosphere-derived cells (CDCs).
[0146] In several embodiments, after growth of CDCs, they are
directly administered to a mammal in need thereof. In one
embodiment the CDCs are optionally grown under conditions which
favor formation of cardiospheres. In one embodiment, the cells
themselves are not administered, but one or more substances
contacted with or released from the cells are administered.
Repeated cycling between surface growth and suspension growth
(cardiospheres) leads to a rapid and exponential expansion of
desired cells. In one embodiment the cardiosphere phase is
eliminated and CDCs are repeatedly expanded through growth on a
surface without forming cardiospheres at each passage.
[0147] Several embodiments, of the culturing processes disclosed
herein (whether culturing CDCs on surfaces or cardiospheres in
suspension) are performed in the absence of exogenous growth
factors. In one embodiment, fetal bovine serum is used, but other
factors are viewed as expendable. For example the cells of the
present invention are readily cultured in the absence of added EGF,
bFGF, cardiotrophin-1, and thrombin.
[0148] Mammals which can be the donors and recipients of cells are
not limited. Thus, while in several embodiments, humans provide
both the cells and are the recipients, often other mammals will be
useful. Pig cells can be transplanted into humans, for example.
Such cross-species transplantation is known as xenogeneic
transplantation. The transplantation can also be allogeneic,
syngeneic, or autologous, all within a single species. Suitable
mammals for use in such transplants include pets, such as dogs,
cats, rabbits; agricultural animals, such as horses, cows, sheep,
goats, pigs, as well as humans.
[0149] Administration of cells to a mammal can be by any means
known in the art. Cardiac cells can be delivered systemically or
locally to the heart. In several embodiments, the administered
cells are not in the form of cardiospheres, but rather are CDCs. In
some embodiments, cardiospheres are delivered. As discussed above,
they have the capacity to form cardiospheres under suitable
conditions. Local administration can be by catheter or direct
(e.g., during surgery). Systemic administration can be by
intravenous or intraarterial injections, perfusion, or infusion. In
those embodiments, employed systemic administration, the
administered cells migrate to the appropriate organ, e.g., the
heart, if the cells are derived from resident heart stem cells.
[0150] The beneficial effects which are observed upon
administration of the cells to a mammal may be due to the cells per
se in some embodiments. For example, in some embodiments the
engraftment of cells produces a favorable outcome. However, in some
embodiments, the beneficial effects are due to products which are
expressed by the cells (e.g., a surface marker that interacts with
host tissue) or secreted, released or otherwise delivered to the
recipient tissue by the cells. In several embodiments cytokines or
chemokines or other diffusible factors, including but not limited
to paracrine factors (e.g., growth factors) stimulate resident
cells to grow, reproduce, or perform better. In some embodiments,
the combination of the cells and diffusible factors provide the
beneficial effects.
[0151] As discussed below, in some embodiments, an effective dose
of cardiac stem cells will typically be between 1.times.10.sup.6
and 100.times.10.sup.6. In some embodiments, the dose is between
10.times.10.sup.6 and 50.times.10.sup.6. Depending on the size of
the damaged region of the heart, more or less cells may be used.
For example, when treating a larger region of damage, a larger dose
of cells may be used, and a small region of damage may require a
smaller does of cells. On the basis of body weight of the
recipient, an effective dose may be between 1 and 10.times.10.sup.6
per kg of body weight, preferably between 1.times.10.sup.6 and
5.times.10.sup.6 cells per kg of body weight. Patient age, general
condition, and immunological status may be used as factors in
determining the dose administered.
[0152] Diseases which can be treated using the methods and
compositions disclosed herein include acute and chronic heart
disease. For example, hearts having been subjected to an ischemic
incident, subjected of chronic ischemia, or congestive heart
disease are treated in some embodiments. In some embodiments,
patients are candidates for heart transplants or recipients of
heart transplants. In additional embodiments, hearts which are
damaged due to trauma, such as damage induced during surgery or
other accidental damage, are treated according to the methods
disclosed herein.
[0153] The cell populations which are collected, expanded, and/or
administered according to the present invention can optionally be
genetically modified, according to several embodiments. In one
embodiment, they are transfected with a coding sequence for a
protein, for example. The protein can be beneficial for diseased
organs, such as hearts. Non-limiting examples of coding sequences
which can be used include without limitation akt, connexin 43,
other connexins, HIF1-alpha, VEGF, FGF, PDGF, IGF, SCF, myocardin,
cardiotrophin, L-type calcium channel alpha-subunit, L-type calcium
channel beta-subunit, and Nkx2.5. The cells may be conveniently
genetically modified before the cells are administered to a mammal.
Techniques for genetically modifying cells to express known
proteins are well known in the art. As discussed herein, in several
embodiments, genetic modification is not necessary as the
beneficial effects which are observed upon administration of the
cells to a mammal are due to the engraftment of the administered
cells, due to products which are expressed by the cells (e.g., a
surface marker that interacts with host tissue) or secreted,
released or otherwise delivered to the recipient tissue by the
cells (e.g., cytokines or chemokines or other diffusible factors,
such as paracrine factors) or combinations thereof.
[0154] As discussed below, CDCs were easily harvested and readily
expanded from biopsy specimens as well as from transplant-ready
hearts, and after administration regenerated myocardium and improve
function in several acute MI models. 69 of 70 patients had biopsy
specimens that yielded cells by the methods disclosed herein,
making the goal of autologous cellular cardiomyoplasty attainable.
In some embodiments, autologous cells are used, as the cells are a
perfect genetic match and thus present fewer potential safety
concerns than allogeneic cells. However, practical limitations with
the use of autologous cells may arise from the delay from tissue
harvesting to cell transplantation. To avoid the delay, cell banks
can be created of cardiac stem cells from patients with defined
immunological features. These should permit matching of
immunological antigens of donor cells and recipients for use in
allogeneic transplantation. Antigens for matching are known in the
art of transplantation. Likewise, as disclosed below, in several
embodiments allogeneic cells need not be matched, as they present
limited immune responses when administered. In some embodiments,
this is due a short residence time of the cells themselves. In some
embodiments, the choice of cardiospheres or CDCs determines the
residence time. In several embodiments, CDCs reside in host tissues
for about 3-6 weeks, and then are destroyed by host mechanisms.
However, despite the loss of some or all of the originally
administered cells, beneficial effects are still observed, likely
due to paracrine effects set in motion by the cells. In several
embodiments, the relatively short residence time of the
administered cells limits the immune response.
[0155] Previous clinical studies in which bone marrow-derived stem
cells were injected into patients within 2 weeks following acute
MI, resulted in significantly improved LVEF with intracoronary
infusion of 5-80.times.10.sup.6 cells. As such, several million
CDCs may constitute an effective therapeutic dose, in certain
embodiments. From single bioptome or non-biopsy specimens, millions
of CDCs can be derived after just two passages; if biopsies or
sample collection were performed specifically for therapeutic
purposes, the amount of starting material could easily be scaled
upwards by ten-fold or more, further improving the overall cell
yield. In several embodiments, however, other variables, such as
culture conditions, can increase the yield while allowing reduction
in the amount of starting material required.
[0156] In some embodiments, minimizing the number of passages for
expansion will minimize the risk of cancerous transformation of
CDCs, a problem which has been observed in mesenchymal stem cells,
typically after about 6 or more passages. Another prominent risk of
cell transplantation lies in the potential for arrhythmogenicity.
Arrhythmias have not been documented with cardiac stem cells.
Teratoma formation is also a concern, though in several
embodiments, teratoma formation is reduced or non-existent. In one
embodiment, teratoma formation can occur when cells are not
committed to forming a specific type of tissue.
[0157] In several embodiments, CDCs are derived from human biopsies
(or non-biopsy samples) without antigenic selection (including but
not limited to c-kit). In several embodiments, all cells that are
shed from the initial heart specimen and which go on to contribute
to the formation of cardiospheres. Thus, in several embodiments,
cells according to several embodiments of the invention differ
fundamentally from cardiac "stem cells" which have been isolated by
antigenic panning for one or another putative stem cell marker, for
example c-kit. Nevertheless, CDCs include a sizable population of
cells that exhibit stem cell markers, and the observed regenerative
ability in vivo further supports the notion that CDCs include a
number of resident stem cells. In some embodiments, a subfraction
of CDCs suffices to produce the beneficial effects; however,
subfractionation may delay transplantation and raise regulatory
concerns by introducing an artificial selection step. Thus, in
several embodiments CDC or cardiosphere populations that have not
been enriched for any one marker (including but not limited to
c-kit) are particularly advantageous, as the required manipulation
and handling of the cells is reduced and the resultant beneficial
effects are equivalent.
[0158] Adult human cardiac stem cells have been shown to respond to
a limited degree to a state of cardiac hypertrophy by proliferation
and myocardial regeneration and to acute ischemia by mobilization
to the injury border zone and subsequent regeneration, but often
ultimately succumb to apoptosis in a chronic ischemic setting.
Significant progress is currently being made identifying means of
enhancing in vivo survival, mobilization, proliferation, and
subsequent differentiation of CSCs using animal models. The methods
disclosed herein for ex vivo expansion of resident stem cells for
subsequent autologous or allogeneic transplantation may give these
cell populations, the resident and the expanded, the combined
ability to mediate myocardial regeneration to an appreciable
degree. If so, cardiac stem cell therapy may well change the
fundamental approach to the treatment of disorders of cardiac
dysfunction.
Transplant and Regenerative Cell Types
[0159] With respect to the recipient of administered regenerative
cells, several embodiments use allogeneic regenerative cells. In
such a transplant, the donor and recipient are different
individuals within the same species. For example, a first mammal is
the donor of the cells and a second mammal is the recipient of the
cells. In several embodiments, the mammals are humans. In several
embodiments, autologous stem cells (donor and recipient are the
same) are used. In such embodiments, a donor's own regenerative
cells are re-administered to the same individual. In several other
embodiments xenogeneic (donor and recipient are different
individuals from different species) regenerative cells are used. In
some such embodiments, closely related phylogenetic species are
used, such as humans and chimpanzees. In other embodiments,
individuals from more distantly related species may be the donor
and recipient, but immunological compatibility is still possible.
In several embodiments, syngeneic (genetically identical and thus,
immunologically compatible) regenerative cells are used. Although
adult regenerative cells are provided in several embodiments of the
invention, embryonic regenerative cells are used in one embodiment.
However, many embodiments of the invention obviate the need for
embryonic stems cells. In some embodiments, other cell types are
used, for example, myoblasts or peripheral blood-derived
endothelial progenitors may be used. Additionally, induced
pluripotent stem cells are used in some embodiments.
[0160] The Major Histocompatibility Complex (MHC) is large,
gene-dense region of the mammalian genome, which plays and
important role in immune system function. The proteins encoded by
the MHC are expressed on the surface of cells and thus are involved
in antigen presentation as well as lymphocyte recognition. MHC
molecules effectively control the initiation of an immune response
through identification of cells as "self" or "non-self" Thus, MHC
molecules are key targets in transplantation rejection.
[0161] The most well-known genes in the MHC region are the subset
that encode antigen-presenting proteins on the cell surface. In
humans, these genes are referred to as human leukocyte antigen
(HLA) genes. In humans, the MHC is divided into three regions:
Class I, II, and III. HLA class I antigens (A, B, and C) present
peptides from inside the cell (including viral peptides if
present). HLA class II antigens (DP, DM, DOA, DOB, DQ, and DR)
present antigens from outside of the cell to T-lymphocytes. HLA
class III antigens encode components of the complement system.
[0162] While class I and class II MHC molecules are structurally
similar and both present antigens to T-cells, their functions in
the immune response cascade are different. Class I molecules are
found on virtually every cell in the human body while Class II
molecules, in contrast, are only found on B-cells, macrophages and
other antigen-presenting cells. Class I molecules present antigens
to cytotoxic T-cells (CTLs) while class II molecules present
antigen to helper T-cells. This specificity of antigen presentation
leads into another difference, the type of antigen presented. Class
I molecules present endogenous antigens while class II molecules
present exogenous antigens. For example, an endogenous antigen
could be a viral protein fragment or tumor protein. These
endogenous antigens indicate internal cellular alterations that
need to be controlled so that they don't spread throughout the
body. In contrast, exogenous antigens may comprise fragments of
bacterial cells or viruses, e.g., non-self antigens, which are
engulfed and processed by, for example, a macrophage, and then
presented to helper T-cells. The helper T-cells, in turn, activate
B-cells to produce antibody that may lead to the destruction of the
cell. Thus, recognition of a newly administered cell or tissue as
non-self is one aspect of the cascade of events giving rise to
transplant rejection.
[0163] Autologous cell therapies are attractive, and commonly used,
as the "self" immune profile of administered cells/tissues will
rarely elicit an immune response upon re-administration to the
donor/recipient. Autologous transplant with embryonic tissue is not
feasible in the vast majority of cases, as harvesting embryonic
tissue for later use in the same individual is technologically and
temporally challenging. Thus, embryonic cells are typically
allogeneic with respect to the recipient. As a result, rejection of
transplanted embryonic cells may be a significant concern.
Additionally, the pluripotency of embryonic stem cells does not
guarantee differentiation of implanted/administered cells into
cells related to the target tissue. In other words, an embryonic
stem cell implanted into the heart may not necessarily yield heart
tissue, but rather may yield other, unwanted cell types or result
in teratoma formation.
[0164] According to several embodiments of the present invention,
adult stem cells, whether allogeneic, autologous, xenogeneic, or
syngeneic develop into cell types closely related to the
originating tissue type. In other words, adult cardiac regenerative
cells will differentiate into cardiac related cell types, such as
cardiomyocytes or cardiac endothelial cells and vasculature, among
other cardiac cell types. Several embodiments of the invention are
especially advantageous because the risk that adult stem cells will
develop into undesired cell types is less than when embryonic stem
cells are used and can be further reduced by isolating adult stem
cells from the tissue that is to be treated or repaired.
[0165] The determination of whether to use autologous or allogeneic
regenerative cells may not be driven primarily by temporal
difficulties with respect to cell isolation, but by other clinical
implications. Tissue collection for isolation of adult regenerative
cells is commonly accomplished by simple biopsy procedures. In many
cases, the current state of a donor's tissue is a determining
factor on whether to use autologous or allogeneic regenerative
cells. For example, a donor who suffered from extensive tissue
damage may have insufficient or non-ideal tissue from which to
isolate regenerative cells. In such cases, allogeneic regenerative
cells may present a preferred alternative.
[0166] Allogeneic regenerative cells do present the possibility of
immune rejection by the recipient, thereby potentially limiting the
long term survival of the administered regenerative cells. However,
allogeneic regenerative cells also present numerous benefits. They
can be harvested from healthy donors, expanded in culture, and
stored for future use, meaning there is a ready availability of
regenerative cells for use in therapies. In the cardiac context in
particular, this ready supply of stored regenerative cells would
enable administration of cells in the critical post-injury period,
where beneficial therapeutic outcomes may be maximal.
[0167] Additionally, because the donor and recipient are distinct
from one another, allogeneic regenerative cells can be obtained
from healthy tissue of a healthy donor. This may improve the
survival of the regenerative cells in long term storage, as well as
during the post-administration period. In addition, regenerative
cells from a healthy donor may simply induce a more robust and
positive therapeutic effect than regenerative cells taken from a
recipient in a state of compromised health. Additionally, even
though adult regenerative cells can be isolated via a simple biopsy
procedure, allogeneic transplants decrease risk to the recipient,
such as infection risk, as the recipient need not undergo the
tissue isolation procedure. Moreover, these advantages have the
potential to increase the number of recipients that can receive
allogeneic transplants and simultaneously reduce the cost of
providing such therapies.
[0168] Several embodiments described herein provide for methods of
isolating, methods of culturing, methods of preparing, and methods
of introducing regenerative cells into a recipient in need of
amelioration of the symptoms associated with and/or treatment of a
cardiac condition. In several embodiments, the regenerative cells
are cardiac tissue-derived. In certain such embodiments, the
regenerative cells are adult regenerative cells. As used herein,
the term adult shall be given its ordinary meaning and shall also
refer to all stages of life extending from birth to death, (e.g.,
adult cells are non-embryonic/non-fetal cells). Additionally, in
several embodiments, adult cells or adult tissues also refer to
cells or tissues collected after death of an adult individual.
[0169] In several embodiments the regenerative cells are
allogeneic. In some embodiments, allogeneic regenerative cells are
harvested from healthy donors, expanded in culture, and stored for
future use. In certain such embodiments, a pool of allogeneic
regenerative cells for acute therapy is available. Thus, in some
embodiments, an allogeneic source of regenerative cells and
allogeneic transplant reduces overall risk to the recipient. In
some embodiments, the allogeneic regenerative cells are stored in a
manner that allows for rapid preparation and administration to a
recipient in need of cell therapy. In certain embodiments, such
regenerative cells are administered to a recipient as soon as
possible after the recipient has suffered an adverse cardiac event.
However, in other embodiments, the regenerative cells are
administered over a period of time, multiple times, or after a
certain period of time, depending on the severity and nature of an
adverse cardiac event.
[0170] In several allogeneic embodiments, the donor regenerative
cells have not been immunologically matched with respect to the
subject. In other embodiments, donor regenerative cells are known
to be immunologically mismatched with respect to the recipient. In
such embodiments, the donor regenerative cells are mismatched (with
respect to the recipient) at one or more HLA antigens. However, in
certain embodiments, the degree of mismatch does not necessarily
predict a severity of immune response. In other words, in some
embodiments involving a donor and recipient having a larger degree
of immunological mismatch there is a less severe immune response as
compared to a donor and recipient who are more immunologically
similar.
[0171] In several embodiments the regenerative cells are
autologous. In some embodiments, autologous regenerative cells are
harvested from a donor at a point when the donor is healthy,
expanded in culture, and stored for future use. In certain such
embodiments, a pool of autologous regenerative cells for personally
tailored cell therapy is available. In certain embodiments, the
autologous regenerative cells are stored in a manner that allows
for rapid preparation and re-administration to the donor/recipient
when the donor/recipient is in need of cell therapy. In certain
embodiments, such regenerative cells are administered to the
donor/recipient as soon as possible after the recipient has
suffered an adverse cardiac event. However, in other embodiments,
the regenerative cells are administered over a period of time,
multiple times, or after a certain period of time, depending on the
severity and nature of an adverse cardiac event.
[0172] In several embodiments the stem cells are xenogeneic. In
certain such embodiments, a large pool of xenogeneic regenerative
cells isolated from a donor organism is available. In certain
embodiments, the xenogeneic regenerative cells are stored in a
manner that allows for rapid preparation and administration to the
recipient when the recipient is in need of cell therapy. In certain
embodiments, such regenerative cells are available and ready for
administration to the recipient as soon as possible after the
recipient has suffered an adverse cardiac event. However, in other
embodiments, the regenerative cells are administered over a period
of time, multiple times, or after a certain period of time,
depending on the severity and nature of an adverse cardiac
event.
[0173] In several embodiments of the invention, the regenerative
cells are syngeneic. In some embodiments, syngeneic regenerative
cells are harvested from a healthy donor, expanded in culture, and
stored for future use in either the donor (in which case the
transplant would be autologous) or a genetically related recipient.
In certain such embodiments, a pool of syngeneic regenerative cells
for cell therapy tailored to a specific genetic and/or
immunological background is available. In certain embodiments, the
syngeneic regenerative cells are stored in a manner that allows for
rapid preparation and re-administration to the donor/recipient when
the donor/recipient is in need of cell therapy. In certain
embodiments, such regenerative cells are administered to the
donor/recipient as soon as possible after the recipient has
suffered an adverse cardiac event. However, in other embodiments,
the regenerative cells are administered over a period of time,
multiple times, or after a certain period of time, depending on the
severity and nature of an adverse cardiac event.
[0174] As used herein, the term "adverse cardiac event" shall be
given its ordinary meaning and shall also be read to include, but
not be limited to myocardial infarction, ischemic cardiac tissue
damage, congestive heart failure, aneurysm, atherosclerosis-induced
events, cerebrovascular accident (stroke), and coronary artery
disease.
[0175] In several embodiments, the regenerative cells, whether
allogeneic, autologous, xenogeneic, or syngeneic, are multipotent.
In certain embodiments, the regenerative cells advantageously
present a decreased risk of abnormal tissue or teratoma formation,
and depending on the transplant type, a reduced risk of
immunological rejection. Despite these advantages, in certain other
embodiments, pluripotent regenerative cells are used.
[0176] The Role Model Effect--Signals and Cellular Recruitment
Induced by Regenerative Cells
[0177] Depending on the cell types involved, and the type of signal
to be conveyed, multiple varieties of cell signaling are used.
Autocrine signaling involves the generation of a signal that acts
back on the same cell (or same type of cell). In contrast paracrine
signaling involves a target cell which is near, but distinct from
the signal-releasing cell. Among other events, paracrine signaling
is involved in allergic and immune responses, tissue growth and
repair, and blood clotting.
[0178] The beneficial effects which are observed upon
administration of the cells to a mammal may be due to the cells per
se in some embodiments. For example, in some embodiments the
engraftment of cells produces a favorable outcome. However, in some
embodiments, the beneficial effects are due to products which are
expressed by the cells (e.g., a surface marker that interacts with
host tissue) or secreted, released or otherwise delivered to the
recipient tissue by the cells. In several embodiments cytokines or
chemokines or other diffusible factors, including but not limited
to paracrine factors (e.g., growth factors) stimulate resident
cells to grow, reproduce, or perform better. In some embodiments,
the combination of the cells and diffusible factors provide the
beneficial effects.
[0179] In several embodiments, the regenerative cells administered
to a recipient produce paracrine signals that affect the
surrounding target tissue during or after administration. However,
in several embodiments, the direct administration of regenerative
cells is not necessary, in that the culturing of isolated
regenerative cells results in release of the paracrine signaling
molecules into the culture media, which can then be harvested and
administered in place of the regenerative cells. In some
embodiments, regenerative cells and their paracrine signaling
molecule-enriched media are co-administered. In other embodiments,
stem cells and their paracrine signaling molecule-enriched media
are sequentially administered. In still other embodiments,
regenerative cells (in a pharmaceutically acceptable carrier) are
administered alone.
[0180] In several embodiments, paracrine signals from the
administered regenerative cells have multiple positive effects on
the surrounding target tissue. In certain embodiments, the effects
of the paracrine signals persist, even after the administered
regenerative cells are no longer viable. In other words, the
regenerative cells create a "Role Model" effect or a "butterfly
effect", in that they set in motion a cascade of events that
carries on, even when the regenerative cells are no longer present.
In some embodiments, the paracrine signals generated improve the
viability of the surrounding target tissue (e.g. signals are
pro-survival). In certain such embodiments, paracrine signals act
on both damaged and healthy target tissue. In some embodiments,
paracrine signals enhance the recovery of damaged cells in the
target tissue. In some embodiments, paracrine signals enhance the
function of damaged and/or healthy cells in the target tissue. In
some embodiments, paracrine signals induce the regeneration of new
target tissue. In some embodiments, paracrine signals enhance the
recovery of damaged cells in the target tissue.
[0181] In several embodiments, the paracrine signals reduce the
amount of induced programmed cell death (apoptosis). In some
embodiments, reduction in apoptosis is manifest by a reduction in
the expression of certain apoptotic markers in the target tissue.
In other embodiments, anti-apoptotic markers are increased. In some
embodiments, apoptotic markers are simultaneously reduced while
anti-apoptotic markers are increased. In some embodiments,
reduction in apoptosis is manifest by a reduction in the number of
cells in target tissue that are permeable to certain molecules
(i.e. fewer cells have the characteristic plasma membrane damage
associated with apoptosis). In some embodiments, reduction in
apoptosis in the target tissue occurs rapidly after administration
of regenerative cells. In other embodiments, reduction apoptosis in
the target tissue occurs after several hours or days
post-administration of regenerative cells. In some such embodiment,
reductions in apoptosis are detected between about 24 and about 72
hours post-administration. In some embodiments, apoptosis is
reduced after 1 week. In other embodiments, apoptosis remains low
for several weeks. In some embodiments, apoptosis is reduced by up
to 20%. In some embodiments, apoptosis is reduced by about 20%. In
other embodiments, apoptosis is reduced by about 30%. In some
embodiments, apoptosis is reduced by about 35%. In some embodiments
apoptosis is reduced by about 40%. In certain embodiments,
apoptosis is reduced by about 20-30%, including 21, 22, 23, 24, 25,
26, 27, 28, and 29%.
[0182] In several embodiments, paracrine signals induce formation
of new blood vessels, which thereby improves function and/or
survival of the target tissue (e.g., signals are pro-angiogenic).
In some embodiments, new blood vessel formation is manifest by an
increase in the length of existing vessels (e.g., into the
infarcted area). In some embodiments, new blood vessel formation is
manifest by an increase in the density of vessels or in an area of
tissue. In some embodiments, vessel density increases by up to
about 4-fold (as compared to damaged tissue not receiving
regenerative cells). In other embodiments, vessel density increases
by about 2-fold. In some embodiments, vessel density increases by
about 3-fold. In some embodiments, vessel density increases by
about 1.5-fold. In certain embodiments, vessel density increases by
about an amount ranging from about 1.1 to 2.5-fold, including 1.2,
1.4, 1.6, 1.8, 2.2, and 2.4-fold.
[0183] In several embodiments, the paracrine signals generating the
new blood vessels are carried to more remote locations in the
target tissue, and induce positive effects in the remote tissue. In
some embodiments, paracrine signals recruit endogenous stem cells
from the surrounding tissue. In certain other embodiments,
paracrine signals from the administered stem cells initiate a
signaling cascade, causing other local cells to generate additional
paracrine signals. In still other embodiments, paracrine signals
from the administered regenerative cells act both on endogenous
cells in a paracrine manner as well in an autocrine manner on the
stem cells themselves. In several embodiments wherein two or more
paracrine signals are generated, the signals function in a
synergistic manner to generate one or more of the positive effects
described herein. Thus, it shall be appreciated that the
administration of regenerative cells, in several embodiments as
described above, yield positive effects in the target tissue
through either a direct effect (e.g., tissue regeneration),
indirect effect (e.g., increase blood supply to new and endogenous
tissue), or a combination thereof.
[0184] In several embodiments, the type of regenerative cells
administered plays a role in determining one or more of the type,
duration, or intensity of paracrine signals generated. For example,
in certain embodiments, a first type of regenerative cells may
release a particular amount of a first paracrine signal upon
administration, while in other embodiments, a different type of
regenerative cells may release less of the first paracrine signal,
and more of a second (or additional) paracrine signal. It shall be
appreciated that while the types of regenerative cells are
genetically related, their differing structures, compositions,
and/or state of differentiation make it possible that the paracrine
signals released by one type of regenerative cells are quite
different than those released by its derivatives. For example, the
microenvironment surrounding certain stem cells is known to play a
role in the regulation of stem cells. Thus, differentiation of
embryonic stem cells is both spatially and temporally regulated by
the distinct environments, or "cellular niches" created during
development. As discussed above, removal of an embryonic stem cell
and placement of the cell in a distinct niche disrupts the signals
of the native microenvironment, often resulting in teratoma
formation.
[0185] Certain regenerative cells have a multicellular,
3-dimensional structure, thus creating the possibility of numerous
microenvironments or niches within the regenerative cells. For
example, based on the 3-dimensional structure of some regenerative
cells, a gradient of cellular oxygen exists that decreases from the
outer to the inner cell layers. Thus in some embodiments, certain
cells in the inner layers are stimulated to release paracrine
signals by the level of hypoxia the cells experience in their
immediate environment. In some embodiments, the close physical
proximity of certain cell types within the regenerative cells
create a contact-based microenvironment, which subsequently directs
the function and fate of the regenerative cells. In several
embodiments, the paracrine signals produced by the cells comprising
the regenerative cells create a distinct microenvironment which
subsequently directs the function and fate of the regenerative
cells. Thus, in certain embodiments, regenerative cells having a
3-D structure are preferred. However, non-3-dimensional
regenerative cells are used in other embodiments.
[0186] In several embodiments, certain regenerative cells release
more or less of the paracrine signals released by other
regenerative cells, and may release one or more additional
paracrine signals not released by other regenerative cells. In some
such embodiments, each type of regenerative cells has the capacity
to generate any or all of the paracrine signals generated by the
other types (and vice versa) but is not stimulated to do so.
[0187] In several embodiments, the generation of paracrine signals
from regenerative cells varies over time (either during in vitro
culture or in vivo, post-administration). In some embodiments,
generation of paracrine signals continues for at least a week after
administration or culturing begins. In other embodiments,
generation of paracrine signals continues for two, three or four
weeks, several months, or for several years. In several
embodiments, the paracrine signals, either alone or in combination
with the signals generated by endogenous tissue, promote the
engraftment and/or long term survival of the administered
regenerative cells. In other embodiments, the engraftment and
survival are relatively short-lived, but the resultant effects are
long-term. In some embodiments, the survival of administered
regenerative cells is due to their ability to differentiate into
multiple types of cardiac tissue, thus efficiently adapting to the
local environment. In some embodiments, the adaptability of
regenerative cells functions in combination with paracrine signals
to effectuate the survival of the administered cells.
[0188] In several embodiments, the administration of regenerative
cells results in engraftment of the regenerative cells into host
tissue. In some embodiments, as discussed herein, the amount of
detectable engraftment of the regenerative cells changes over time.
In some embodiments, a higher degree of regenerative cells have
engrafted a short period of time after administration as compared
to later times after administration (i.e. engrafted numbers
decrease). In some embodiments, engraftment peaks at an
intermediate time point. In some embodiments, the type of
regenerative cells used (i.e., allogeneic v. autologous, etc.) is a
factor in engraftment. In some embodiments, allogeneic regenerative
cells engraft as well as other types, while in other embodiments,
allogeneic regenerative cells show lesser engraftment. In some
embodiments, engraftment of allogeneic regenerative cells is
equivalent to that of other regenerative cells types at a first
time point, but decreases more rapidly post-administration.
However, in several embodiments, the functional effect of
allogeneic regenerative cells is equivalent, or greater than, that
of other cell types, despite the lesser degree of engraftment. In
certain embodiments, engraftment is correlated with survival of
regenerative cells. In some embodiments, the administered
regenerative cells survive for several days post-administration. In
some embodiments, administered regenerative cells survive for about
a week to about two weeks. In some embodiments, administered
regenerative cells survive for several weeks, or one, two, three or
more months. As discussed above, in certain embodiments, the
effects of the paracrine signals, and in some embodiments the
signals themselves, persist after administered regenerative cells
are no longer viable. In several embodiments, this "butterfly
effect", the persistence of the signals that result from the
engrafted cells, despite the limited term of the engraftment, is
responsible for the long-term beneficial anatomical and functional
recovery of the cardiac tissue.
[0189] In several embodiments, the paracrine signals comprise one
or more growth factors, hormones, cytokines, or other signaling
molecule that are released from the administered regenerative
cells. In certain embodiments the paracrine signals comprise one or
more of the following signaling molecules: ENA-78, G-CSF, GM-CSF,
GRO, GRO-alpha, I-309, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, interferon
gamma, MCP-1, MCP-2, MCP-3, M-CSF, MDC, MIG, MIP-1 beta, MIP-1
delta, RANTES, SCF, SDF-1, TGF-beta 1, TNF-beta, EGF, IGF-1,
angiogenin, oncostatin M, thrombopoeitin, VEGF, PDGF-BB, leptin,
BDNF, BLC, Ck beta 8-1, eotaxin, eotaxin-2, eotaxin-3, FGF-4,
FGF-6, FGF-7, Flt-3 ligand, fractalkine, GCP-2, GDNF, HGF, IGFBP-1,
IGFBP-2, IGFBP-3, IGFBP-4, IL-16, IP-10, LIF, LIGHT, MCP-4, MIP-3
alpha, NAP-2, NT-3, NT-4, osteopontin, osteoprogenerin, PARC, PIGF,
TGF beta 2, TGF beta 3, TIMP-1 and TIMP-2. In several preferred
embodiments, the paracrine signals comprise one or more of VEGF,
HGF, and IGFI. In some embodiments, a single paracrine signal is
responsible for the beneficial therapeutic effects, while in other
embodiments, one or more paracrine signals work synergistically to
produce the effects. As discussed above, several embodiments
described herein reduce the risk of teratoma formation. In certain
embodiments, the paracrine signals from the regenerative cells (or
those induced in the target tissue) reduce, minimize, and/or
eliminate the risk of teratoma formation.
[0190] In several embodiments, various types of regenerative cells
have different paracrine potencies. In other embodiments, the
paracrine potency of a single type of regenerative cells varies
over time. In some embodiments, regenerative cells express one or
more receptors for the paracrine signals generated by the
regenerative cells. As such, in some embodiments the regenerative
cells act in a paracrine manner on other co-administered
regenerative cells. In some embodiments, the regenerative cells act
in a paracrine manner on endogenous tissue. In still other
embodiments, the regenerative cells act in an autocrine manner. In
some embodiments, the regenerative cells express one or more of the
KDR, Met, and IGFI receptors (the receptors for receptors for VEGF,
HGF and IGFI, respectively).
[0191] In several embodiments, endogenous cells are recruited by
the administration of the regenerative cells. In some embodiments,
the presence of the administered regenerative cells induces the
recruitment of endogenous cells. In other embodiments, paracrine
signals released from the regenerative cells induce the recruitment
of endogenous cells. In still other embodiments, the presence of
the regenerative cells and the paracrine signals produced thereby
work in combination to induce the recruitment of endogenous
cells.
[0192] In several embodiments, the recruited endogenous cells
improve the viability of the surrounding target tissue. In certain
embodiments, the recruited cells engraft into the damaged tissue
and generate new, healthy tissue. In certain embodiments, the
recruited endogenous cells generate paracrine signals that act on
both damaged and healthy target tissue. In some embodiments, these
paracrine signals enhance the recovery of damaged cells in the
target tissue. In certain embodiments, these paracrine signals
reduce the amount of programmed cell death. In some embodiments,
paracrine signals from the recruited endogenous cells enhance the
function of damaged and/or healthy cells in the target tissue. In
some embodiments, these paracrine signals induce the regeneration
of new target tissue. In some embodiments, these paracrine signals
enhance the recovery of damaged cells in the target tissue. In some
embodiments, paracrine signals from the recruited endogenous cells
induce formation of new blood vessels, which thereby improves
function and/or survival of the target tissue. In certain such
embodiments, these paracrine signals generating the new blood
vessels are carried to more remote locations in the target tissue,
and induce positive effects in the remote tissue.
[0193] In certain other embodiments, paracrine signals from the
recruited endogenous cells initiate a signaling cascade, causing
other local cells to generate additional paracrine signals. In
still other embodiments, paracrine signals from the recruited
endogenous cells act both on other endogenous cells in a paracrine
manner as well in an autocrine manner on the recruited endogenous
cells themselves. In several embodiments wherein two or more
paracrine signals are generated, the signals function in a
synergistic manner to generate one or more of the positive effects
described herein. Thus, it shall be appreciated that the
recruitment of endogenous cells, in several embodiments as
described above, yield positive effects in the target tissue
through either a direct (e.g., tissue regeneration of increased
function traceable to the endogenous cells), indirect effect (e.g.,
paracrine signals induce increased blood supply to new and
endogenous tissue), or a combination thereof.
[0194] In several embodiments, the regenerative cells are used in
the preparation of a medicament. In some embodiments, the
medicament is suitable for administration to an individual having
damaged or diseased tissue, in particular damaged or diseased
cardiac tissue. In some embodiments, administration of the
medicament results in one or more of alleviation of symptoms of a
cardiac disease, improvement in cardiac function, and/or
regeneration of cardiac tissue in the recipient individual.
Methods of Harvesting Cardiac Tissue and Producing Regenerative
Cells
[0195] It shall be appreciated that the term "regenerative cells"
as used herein refers to each of the mixed cell populations
described herein (cardiospheres) and derivatives thereof (CDCs and
IICSps). In describing the isolation methods, and the methods to
produce derivatives of certain regenerative cells, the individual
type of regenerative cells is used for clarity.
[0196] Donor tissue may be obtained from embryonic or adult
sources. Adult sources are preferred for several embodiments. In
some embodiments, donor cardiac tissue is obtained during a
surgical procedure, such as bypass surgery. In other embodiments,
donor tissue is obtained during a percutaneous endomyocardial
biopsy procedure. In still other embodiments, large quantities of
tissue are obtained from recently deceased organ donors, processed,
and stored for use in allogeneic transplants. While a typical human
adult heart weighs about 200 to 300 g, sufficient amounts of
regenerative cells can be obtained from cardiac tissue samples of
about 1 mg to about 50 mg. In some embodiments, the mass of the
cardiac tissue sample is about 25 mg or less. The tissue sample may
be obtained from a variety of locations in the heart, including but
not limited to, the crista terminalis, the right ventricular
endocardium, the right ventricular septum, the septal or ventricle
wall, the atrioventricular groove and the right and left atrial
appendages.
[0197] In one embodiment, the tissue sample is obtained from donor
hearts that are transplant quality, but are unable to be
transplanted into a human patient (e.g., because of unavailability
of a matched recipient, etc.). In one embodiment, the tissue sample
is obtained from hearts that are not transplant quality because
(e.g., of tissue damage in the donor heart, the age of the heart,
suspected cardiovascular disease in the donor, etc.). In several
embodiments, the entire heart is processed to obtain a tissue
sample for culture. In other embodiments, tissue is extracted from
one or more of the following: atria, atrial appendages and
apex.
[0198] According to several embodiments, percutaneous
endomyocardial biopsy specimens are harvested using the following
procedure. Under local anesthesia, a guide catheter is introduced
into a vein, such as the jugular vein, in the patient's neck if
tissue samples are to be taken from the right ventricle.
Alternatively, the guide catheter can be introduced into an artery
if tissue samples are to be taken from the left ventricle. The
guide catheter is guided to the heart with the aid of visualization
provided by a standard imaging technique, such as fluoroscopy. Once
the guide catheter is in place, a bioptome can be introduced into
the guide catheter and threaded to the heart. Once the bioptome is
within the heart, the flexible distal end of the bioptome can be
manipulated by the surgeon to extract a tissue sample from the
desired location. The bioptome can be removed from the patient so
that the tissue sample can be retrieved and then the bioptome can
be reintroduced so that another sample can be taken from the same
or different location. In another embodiment, the bioptome can
extract multiple samples before being withdrawn, thereby reducing
the time needed to collect the tissue samples.
[0199] In one embodiment, the invention comprises harvesting a
piece of myocardial tissue about 0.25-about 1 cm in length and
width through a catheter placed in the jugular vein of a subject
under local anesthesia. In one embodiment, the weight of the sample
is about 0.25-about 1 gram. The heart biopsy sample is then
cultured over a period of about 3 to about 6 weeks (e.g., about 4
weeks) until approximately 10 to 25 million cells are available for
implantation into the coronary arteries. As disclosed herein, the
biopsy sample may be obtained from a first subject and then
implanted into the same subject. Alternatively, the biopsy sample
may be obtained from a first subject and then implanted into a
different subject.
[0200] In several embodiments, the processing of a biopsy sample
yields regenerative cells that comprise a mixed population of cells
that comprises, for example, stem cells, cardiac cells, and/or
vascular cells, among other cell types. In some embodiments, the
mixed population of cells expresses various stem cell markers. In
some embodiments, stem cells of the mixed population may be
identified by expression of stem cell-related markers including one
or more of CD-105, CD90, CD34, Sca-1, and c-kit, among others. In
certain embodiments, the stem cells do not express one or more of
the stem cell markers identified above. In certain embodiments, the
stem cells are CD45 negative. In some embodiments, the vascular
cells of the mixed cell population express at least one of KDR,
flk-1, CD31, von Willebrand factor, Ve-cadherin, and smooth muscle
alpha actin, among others.
[0201] In vitro, the mixed cell populations are clonogenic and can
give rise to immature cardiomyocytes (heart muscle cells) and
endothelial and smooth muscle cells (blood vessel components). In
addition, in some embodiments, CDCs may be grown on a solid surface
to produce a second (or greater) generation of cardiospheres
(IICSps).
[0202] In one embodiment of the invention, regenerative cells are
isolated and cultured as according to the schematic in FIG. 1.
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. 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.
[0203] 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 us supplemented
with serum, but no additional exogenous growth factors. In yet
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.
[0204] 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.
[0205] 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.
[0206] 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. 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 0.1mM 2-mercaptoethanol.
[0207] 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.
[0208] 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
optionally 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 0.1mM 2-mercaptoethanol. CDCs may
be repeatedly passaged by standard cell culture techniques. In some
embodiments, CDCs are detached from the culturing surface and
plated on poly-D-lysine-coated dishes to form a second generation
of cardiospheres (IICSps). This process of generating cardiospheres
followed by CDCs followed by a subsequent generation of
cardiospheres may be repeated as needed to expand the population of
any of the regenerative cell types.
Administration of Regenerative Cells
[0209] In several embodiments, regenerative cells are administered
to recipients systemically. In some such embodiments, the
systemically administered cells migrate to the recipient's heart,
particularly to the area of damaged tissue. In several embodiments,
regenerative cells are delivered systemically via an intravenous
route. In several embodiments, the regenerative cells are delivered
locally. In certain embodiments, local delivery is achieved by
direct myocardial injection. In certain embodiments, local delivery
is achieved via a biopsy procedure. In some embodiment, delivery is
accomplished during a surgical procedure. Delivery may be
accomplished with specific injection site guidance in certain
embodiments. In one embodiment, NOGA is employed. In certain
embodiments, regenerative cells are delivered alone, while in other
embodiments regenerative cells are delivered in associated with an
additional therapeutic agent. In still other embodiments, the
paracrine agents produced by regenerative cells are administered,
either alone or in conjunction with the regenerative cells.
[0210] In certain embodiments, the regenerative cells are used to
seed a biomaterial or synthetic graft. In certain embodiments, the
graft comprises a biocompatible biomaterial such as hyaluronan,
alginate, or fibrin. In some embodiments, the biomaterial or
synthetic graft is injectable. In other embodiments, the
biomaterial or synthetic graft is painted or directly placed onto
the target tissue.
[0211] In several embodiments, the regenerative cells are delivered
at a dose of about 1.times.10.sup.5 to about 1.times.10.sup.7
regenerative cells per kilogram of body weight of the recipient.
However, in some embodiments, lower numbers of cells may be used,
due to the butterfly effect described herein (e.g., the persistence
of positive effects on the target tissue even after some or all of
the delivered cells are removed by host mechanisms).
Immune Responses and Functional Effects of Regenerative Cells
[0212] As discussed above, the various transplant types, and cells
used in each have advantages and disadvantages. Autologous
transplants are advantageous due to limited risk for immune
rejection of transplanted cells. Thus in some embodiments,
autologous regenerative cells are used. However, in certain
instances, autologous transplants are expensive, and somewhat time
consuming (since tissue must be harvested, processed into
regenerative cells, and re-administered), especially in
circumstances where the donor/recipient requires immediate
administration of therapy (e.g., they are within a critical
post-injury period, typically within a few hours of an adverse
event). Therefore, an ideal cellular therapy would be available
"off the shelf", and preferably would not induce severe immune
responses in the recipient, or at least be able to initiate or
yield a therapeutic response despite an immune response. Thus, in
some embodiments, allogeneic regenerative cells are used.
[0213] Immune responses mounted by a recipient may lead to
rejection of transplanted cells that are immunologically distinct.
Such cells, for example, allogeneic regenerative cells, may be
rejected through a direct or indirect pathway. Direct rejection by
the recipient involves either antigen presenting cells (APCs) that
were transplanted from the donor or their donor APC precursor cells
that have differentiated into APCs after transplantation. When a
recipient T-cell (also known as T-lymphocytes, immune cells that
play a central role in cell-mediated immunity) recognizes a donor
APC as "non-self" (via expression of donor HLA molecules or other
donor-derived antigens), the recipient T-cell becomes activated,
recruits other recipient immune co-stimulatory molecules become
involved (such as CD80 or CD86 with CD28, and CD40 with CD40
ligand), and an immune response is initiated.
[0214] Indirect rejection may occur due to the "shedding" various
antigens from transplanted donor cells or tissues. These donor
antigens are taken up by recipient APCs, and subsequently presented
to recipient T cells. This can result in the activation of
donor-reactive recipient T cells, which then initiate an immune
response.
[0215] In several embodiments, an immune response is initiated
against transplanted allogeneic regenerative cells. In several
embodiments, the grade of any immune response is higher than the
grade of a corresponding autologous or syngeneic transplant,
however, in such embodiments, the severity of the immune response
does not eliminate all of the transplanted regenerative cells, or
their effect on the target tissue. In some embodiments, the grade
of immune response in an allogeneic transplant scenario is
equivalent to or less than that of an autologous or syngeneic
transplant. In certain embodiments, as discussed herein, some of
the administered regenerative cells survive for several days
post-administration. In some embodiments, some of the administered
regenerative cells survive for about a week to about three weeks.
In some embodiments, a local immune effect is initiated against
transplanted regenerative cells. In certain such embodiments, the
local immune response does not destroy or render non-functional the
transplanted regenerative cells until after a series of signal or
events have been initiated that lead to a beneficial therapeutic
effect. In some embodiments, a systemic immune response is
initiated against transplanted regenerative cells. In certain such
embodiments, the systemic immune response does not destroy or
render non-functional the transplanted regenerative cells until
after a series of signal or events have been initiated that lead to
a beneficial therapeutic effect. In still other embodiments, there
is little or no systemic immune response. Thus, in certain
embodiments, the effects initiated by the administration of the
regenerative cells persist after some, or all, of the administered
regenerative cells are no longer viable.
[0216] In several embodiments, the transplant of regenerative cells
(regardless of transplant type) results in a beneficial therapeutic
effect in the recipient. As discussed above, the beneficial
therapeutic effect may comprise one or more of treatment of
symptoms of a cardiac disease, improvement in cardiac function,
and/or regeneration of cardiac tissue in the recipient.
[0217] In several embodiments, transplanted regenerative cells
result in significantly increased fractional area. In some
embodiments, fractional area is increased by about 5%. In some
embodiments, fractional area is increased by about 10%. In some
embodiments, fractional area is increased by about 15%. In some
embodiments, fractional area is increased by about 20%. In some
embodiments, fractional area is increased by about 5-10%, including
6, 7, 8, and 9%. In some embodiments, fractional area is increased
by about 10-20%, including 11, 12, 13, 14, 15, 16, 17, 18, and 19%.
In still additional embodiments, greater increases are
realized.
[0218] In several embodiments, transplanted regenerative cells
result in significantly increased ejection fraction. In some
embodiments, ejection fraction is increased by about 5%. In some
embodiments, ejection fraction is increased by about 10%. In some
embodiments, ejection fraction is increased by about 15%. In some
embodiments, ejection fraction is increased by about 20%. In some
embodiments, ejection fraction is increased by up to about 25%. In
some embodiments, ejection fraction is increased by about 6-12,
12-18, or 19-25% In some embodiments, ejection fraction is
increased by about 5-20%, including 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, and 19%.
[0219] In several embodiments, transplanted regenerative cells
result in a decrease in infarct size (i.e. an increase in viable
tissue in and around the infarct). In some embodiments, infarct
size is reduced by about 5%. In some embodiments, infarct size is
reduced by about 10%. In some embodiments, infarct size is reduced
by about 15%. In some embodiments, infarct size is reduced by about
1-3, 4-7, or 7-11%. In some embodiments, infarct size is reduced by
5-15%, including 6, 7, 8, 9, 10, 11, 12, 13, and 14%.
[0220] In several embodiments, the beneficial therapeutic effect of
transplanting regenerative cells (regardless of the transplant
type) lasts for several weeks. In some embodiments, the beneficial
therapeutic effect last for up to three weeks. In some embodiments,
the beneficial therapeutic effect lasts for a period of time from
about 3 weeks to about 12 weeks. In some embodiments, the
beneficial therapeutic effect lasts for about 3 months to about 1
year. In still other embodiments, the beneficial therapeutic effect
lasts for several years.
[0221] In several embodiments of the invention, a method of
treating an adverse cardiac event is provided, wherein the method
comprises implanting regenerative cells into a recipient patient.
The regenerative cells are obtained from one or more different
donor subjects, and have been obtained by obtaining a cardiac
biopsy sample from said donor(s), and culturing the sample(s) to
obtain the regenerative cells. Such allogeneic transplant
methodology, according to several embodiments, is particularly
beneficial because the regenerative cells do not evoke a
significant chronic immune response that is adverse to the patient.
Instead, the regenerative cells trigger a cascade of therapeutic
signaling effects (e.g., a paracrine effect) prior to destruction
via an acute immune response that destroys the regenerative cells.
Thus, "off-the-shelf" regenerative cells can be produced to treat
patients suffering from cardiac diseases. Further, the patient need
not have healthy tissue from which to harvest his or her own cells
(for an autologous transplant). Moreover, even when a patient has
viable heart tissue for biopsy, the patient need not have to wait
for the culturing process when his or her own tissue is not used.
Instead, the "off-the-shelf" allogeneic cells may be available with
little or no time delay. In one embodiment, the donor cells are
obtained from the recipient patient plus at least one different
donor. In one embodiment, the donor cells are obtained from a
single donor (different than the recipient patient). In one
embodiment, the donor cells are obtained from a two or more
different donor (different than the recipient patient and each
other). The recipient patient and donor(s) may be race, age, sex,
blood-type, and/or HLA-matched in some embodiments. In other
embodiments, the recipient patient and donor(s) need not be matched
in any of the categories identified above. In several embodiments,
the allogeneic cells and/or the patient need not be treated with
immune suppressants prior to (or during or after) implantation of
the regenerative cells. In some embodiments, the allogeneic cells
and/or the patient need not be treated with radiation prior to (or
during or after) implantation of the regenerative cells.
EXAMPLES
[0222] Examples provided below are intended to be non-limiting
embodiments of the invention.
Example 1
Specimen Processing and Cardiosphere Growth
[0223] Following institutional guidelines, and with patient
consent, human biopsy specimens were obtained from patients
undergoing clinically-indicated percutaneous endomyocardial biopsy
and processed as described above, with certain modifications.
Specimens consisted of whole or partial bioptome "bites", stored on
ice in high-potassium cardioplegic solution and processed within
two hours (FIG. 1A, step 1). As discussed, herein, samples, in some
embodiments, are taken from whole donor hearts (e.g., not collected
via biopsy). Samples were cut into fragments from which gross
connective tissue was removed. The fragments were then washed,
partially-digested enzymatically, and the single cells discarded.
In several embodiments, partial digestion is accomplished using
trypsin. In some embodiments, collagenase is used. In some
embodiments, other proteases may be used. The remaining tissue
fragments were cultured as "explants" on dishes coated with
fibronectin (FIG. 1A, step 2). In some embodiments, other surface
coatings may be used (e.g., collagen or other extracellular matrix
proteins. After several days, a layer of stromal-like cells arose
from adherent explants over which small, round, phase-bright cells
migrated. Once confluent, the loosely-adherent cells surrounding
the explants were harvested by gentle enzymatic digestion (FIG. 1A,
step 3). These cells were seeded at 2-3.times.10.sup.4 cells/mL on
poly-D-lysine-coated dishes in media designed for optimal growth of
cardiospheres (FIG. 1A, step 4). Detached cardiospheres were then
plated on fibronectin-coated flasks and expanded as adherent
monolayers (FIG. 1A, step 5), which could be subsequently passaged
by trypsinization. Single cells were counted under phase microscopy
using a hemocytometer as cardiosphere-forming cells and during CDC
passaging to track cell growth for each specimen. Isolation of the
cardiosphere-forming cells was repeated up to 3 more times from the
same specimen.
Sub-Population Selection and Flow Cytometric Analysis
[0224] To characterize the antigenic features of cells that form
cardiospheres, cells obtained during the first harvesting (FIG. 1A,
step 3) were sub-selected by magnetic-activated cell separation
with an APC-conjugated monoclonal antibody against c-kit, followed
by labeling with a microbead-conjugated anti-APC, followed by
separation using OctoMACS. CD105.sup.+ populations were then
sub-selected with a second antibody directly conjugated to a
microbead. As discussed herein, in several embodiments, selection
is used for analysis of the cell population only, and the cells
that are administered have not been selected for expression of a
particular marker, including, but not limited to c-kit.
[0225] CDCs were passaged two times as adherent monolayers and then
used for flow cytometry experiments. c-kit-APC, CD105-PE, and
similarly conjugated isotype-matched control monoclonal antibodies
were utilized. Gates were established by 7-AAD fluorescence and
forward scatter. Data were collected using a FACScalibur
cytofluorometer with CellQuest software.
Adenovirus Creation and Cell Transduction
[0226] The E. coli beta-galactosidase (lacZ) gene was cloned into
an adenoviral shuttle vector pAd-Lox to generate pAd-Lox-LacZ by
Cre-Lox recombination in Cre-4 293HEK cells as described. CDCs were
passaged two times and transduced with virus as adherent
monolayers. Transduction efficiencies of 90% were achieved with an
MOI of 20 for 12 hours.
Myocardial Infarction and Cell Injection
[0227] Adenovirally-transduced CDCs were injected into adult male
SCID-beige mice 10-16 weeks of age. Myocardial infarction (MI) was
created by ligation of the mid-left anterior descending coronary
artery and cells or vehicle injected under direct visualization at
two peri-infarct sites. As disclosed herein, other delivery routes
(e.g., intracoronary, IV, etc.) are used in some embodiments. CDCs
(10.sup.5) were injected in a volume of 10 .mu.L of PBS (5 .mu.L at
each site), with 10.sup.5 primary human skin fibroblasts or 10
.mu.L of PBS as controls. All mice underwent echocardiography prior
to surgery (baseline) and again 20 days post-surgery. Ejection
fractions (EFs) were calculated using V1.3.8 software from 2D
long-axis views taken through the infarcted area. Mice were then
euthanized at 0, 8, or 20 days, and the excised hearts prepared for
histology.
Immunostaining, Immunohistochemistry, and Microscopy
[0228] Cardiospheres were collected for immunostaining when they
had reached 100-1000 cells in size. Primary antibodies against
c-kit, CD105, cardiac myosin heavy chain (cMHC), and cardiac
troponin I (cTnI) were used for immunostaining. Secondary
antibodies conjugated with Alexa fluorochromes were utilized.
Immunostaining was performed as previously described. Confocal
fluorescence imaging was performed on an Eclipse TE2000-U equipped
with a krypton/argon laser using UltraVIEW software.
[0229] Mouse hearts were excised, embedded in OCT compound, frozen,
and sectioned in 5 .mu.m slices. Tissue sections were stained with
hematoxylin-eosin and b-galactosidase reagent or Masson's
trichrome. Tissue viability within the infarct zone was calculated
from Masson's trichrome stained sections by tracing the infarct
borders manually and then using ImageJ software to calculate the
percent of viable myocardium within the overall infarcted area.
Statistics
[0230] All results are presented as means.+-.SEM. The significance
of differences between any two groups was determined by the
Student's t-test. Multiple groups were compared using GB-Stat
software using one-way ANOVA and group pairs compared by the
Bonferroni-Dunn method if a significant F value was obtained. A
value of p<0.05 was considered significant.
[0231] The generalized estimation equation (GEE) approach was
employed to identify parameters that were independently associated
with high cell yield. Data from patients who donated multiple
specimens were treated as repeated measures. Those parameters that
were significant (p.ltoreq.0.1) in the univariate models were
included in the final, multivariate models. The analysis was
performed with the use of SAS software. A final value of p<0.05
was considered significant. All p-values reported are 2-sided.
Statistics
[0232] Product manufacturers, recipes, and reagents used in several
embodiments are shown in Table 1.
TABLE-US-00001 TABLE 1 Products and Manufacturers and Media Recipes
Explant and CDC media IMDM 20% FBS 1% penicillin-streptomycin 1%
L-glutamine 0.1 mM 2-mercaptoethanol Cardiosphere media 35% IMDM
and 65% DMEM/F-12 Mix 3.5% FBS 1% penicillin-streptomycin 1%
L-glutamine 0.1 mM 2-mercaptoethanol Thrombin, B-27, bFGF, EGF and
Cardiotrophin-1 at final working concentrations Product: Working
concentration: Manufacturer: IMDM Invitrogen DMEM/F-12 Mix
Invitrogen Thrombin 1 unit/mL Sigma B-27 1:50 Invitrogen bFGF 80
ng/mL PeproTech EGF 25 ng/mL PeproTech Cardiotrophin-1 4 ng/mL
PeproTech Fibronectin 25 .mu.g/mL BD Biosciences Poly-D-lysine 20
.mu.g/mL BD Biosciences c-Kit-APC 1:10 BD Pharmingen CD105
MicroBeads 1:5 Miltenyi Biotec Anti-APC MultiSort 1:4 Miltenyi
Biotec CD105-PE 1:10 R&D Systems 7-AAD 20 .mu.g/mL Calbiochem
SCID-beige mice Harlan Dermal fibroblasts ATCC c-Kit pAb 1:100
Abcam CD105 mAb 1:50 R&D Systems cMHCpAb 1:100 (6) Rome, Italy
cTaImAb 1:200 Chemicon Alexa 488, 568 1:400 Invitrogen OCT VWR
Scientific Equipment and Software: Manufacturer: OctoMACS Miltenyi
Biotec FACScalibur BD Biosciences Vevo 660 Echo VisualSonics
Eclipse TE2000-U Nikon CellQuest BD Biosciences V.1.3.8 software
VisualSonics UltraVIEW software Perkin Elmer ImageJ software NIH
GB-Stat v10 Dynamic Microsystems Inc. SAS software v9.1 SAS
Institute Inc.
Example 2
Specimen Processing and Cardiosphere-Forming Sub-Populations
[0233] FIG. 1B shows a typical explant, after mincing and partial
enzymatic digestion, on the day it was obtained and also on days 3
(FIG. 1C) and 13 (FIG. 1D), immediately prior to first harvest.
Harvesting of cardiosphere-forming cells (FIG. 1A, step 3) was
initially performed 8 or more days after obtaining a specimen and
at 4-12 day intervals thereafter. Panel E summarizes the results of
sub-population selection experiments performed using cells
harvested from 3 different patient specimens. The large majority of
the cells that generate cardiospheres are CD105.sup.+, those that
are c-kit.sup.+ and those that are c-kit.sup.-. Typical
cardiospheres are shown in FIG. 1F, 12 days after harvest. Floating
cardiospheres were plated for expansion (FIG. 1A, step 5) 4-28 days
after step 3 and passaged at 2-7 day intervals thereafter. FIG. 1G
shows CDCs plated on fibronectin during expansion at passage 2,
when those cells were harvested for injection.
Example 3
Patient Specimens and Cardiosphere Growth
[0234] 83 patient specimens (21.0.+-.1.9 mg) were obtained for
analysis. 72 of the specimens were obtained from patients who had
received a heart transplant and 11 were from patients awaiting
transplant. Nine transplanted patients donated multiple specimens.
78 of 83 specimens were processed, and 4 of those specimens never
harvested were from repeat patients, yielding growth data from 69
of 70 patients. Cumulative growth curves for each specimen are
depicted in FIG. 1, Panels H and I. The growth curves from patients
awaiting transplant (FIG. 1H) are similar to those from
transplanted patients (FIG. 1I), showing a wide range of growth
potential among specimens. Patient parameters are summarized in
Table 2 for the non-transplanted and transplanted groups. A GEE
analysis involving all patient parameters listed in the table
revealed no independent predictors for high cell yield within the
non-transplanted group. Within the transplanted group, specimens
from patients with a higher EF tended to yield more cells, but the
effect was weak (final R.sup.2 estimate=0.04, p<0.05).
TABLE-US-00002 TABLE 2 Patient Population Summary Non-transplanted
Transplanted Patients: Patients: Patient age 47.2 .+-. 3.7 years
53.6 .+-. 1.7 years Patient sex 63% male, 37% 73% male, 27% female
female Patient ejection fraction 36.9 .+-. 4.7% 61.9 .+-. 0.8%
Donor age 31.4 .+-. 1.6 years Donor sex 69% male, 31% female Time
out from transplant 4.5 .+-. 0.6 years Donor ischemic time 173.9
.+-. 7.8 minutes Pathological rejection 0.5 .+-. 0.1 level*
Immunosuppressive level** 31% normal, 43% low, 26% high *grade 0 =
0, grade 1A = 0.5, grade 1B = 1, grade 2 = 2, grade 3A = 3
**considered for Cyclosporine and FK506 (.+-.Rapamycin) relative to
time out from transplant (24, 25)
Example 4
Cardiosphere and Cardiosphere-Derived Cell Phenotypes
[0235] Part of the rationale for using CDCs lies in the unique
biology of cardiospheres and their cell progeny. The
self-organizing cardiospheres create a niche environment favoring
the expression of stem cell antigens (e.g., c-kit and CD105, FIG.
2A) and frequently manifest a surface phenotype marked by mature
cardiac-specific antigens (cMHC and cTnI, FIG. 2B) with retention
of internal "stemness". In fact, c-kit and CD105 were present in
all cardiospheres examined (10 or more from each of 10 patients),
with c-kit either localized to the core or expressed throughout the
sphere, and CD105 typically localized to the periphery or expressed
throughout. CDCs after two passages retain high levels of c-kit and
CD105 antigen expression (FIG. 2C, representative of expression
profiles of CDCs from 3 and 2 different patients respectively).
Example 5
Cardiosphere-Derived Cell Engraftment, Regeneration, and Functional
Improvement
[0236] CDCs from 4 different patients were utilized for in vivo
experiments. To assess engraftment and cell migration, mice were
injected with lac-Z-expressing CDCs and sacrificed at each of 3
time points (0, 8, and 20 days following injection). At day 0, CDCs
were located at injection sites in the border zone, but at day 8
and day 20 injected cells were distributed mainly within the MI
area, forming islands or continuous bands of .beta.-galactosidase
positive tissue (see e.g., FIG. 5).
[0237] Eight mice were injected with CDCs and followed for 20 days;
11 mice served as controls (4 with fibroblasts, and 7 with PBS).
FIG. 3A shows a typical beta-galactosidase staining pattern
indicating the distribution of injected human cells after 20 days
in vivo. Note the band of blue cells infiltrating the infarct zone,
which was not apparent in the fibroblast-injected mice (FIG. 3B) or
the PBS-injected mice. Masson's trichrome-stained sections were
used to quantify regeneration (FIG. 3, C and D). Panel C, from a
CDC-injected heart, shows a number of obvious red regions within
the blue infarct zone; fewer such regions are evident in the
fibroblast-injected heart (FIG. 3D). CDC-injected mice had a higher
fraction of viable fuchsin-positive tissue within the MI zone
(24.9.+-.1.1%) compared to fibroblast-injected mice (17.7.+-.1.8%,
p<0.01) or PBS-injected mice (13.7.+-.0.7%, p<0.01), but the
overall total infarct area was similar to that in the two control
groups (60.6.+-.6.4 CDC, 76.9.+-.7.0 fibroblast, 75.7.+-.2.7 PBS,
units in 10.sup.4 pixels; p=NS). The differences between the CDC
group and each of the control groups in percent viable myocardium
within the MI zone, 7.2% and 11.2%, represent the extents of
myocardial regeneration attributable to the CDCs.
[0238] Echocardiograms were performed for all groups at 20 days;
FIG. 4 shows examples from the CDC and fibroblast-treated groups at
end-diastole and end-systole. Pooled data for left ventricular EF
(LVEF, FIG. 4E) and left ventricular fractional area (LVFA, FIG.
4F) reveal a higher LVEF in the CDC-treated group (38.8.+-.1.7%) as
compared to either the fibroblast-treated (24.5.+-.1.8%, p<0.01)
or the PBS-treated group (26.4.+-.3.0%, p<0.01), but the two
control groups were indistinguishable. There was no difference
among the LVEFs at baseline.
Example 6
Process for the Isolation of Cardiac Stem Cells from Cardiac Biopsy
Specimens
[0239] Pluripotent stem cells may be isolated from cardiac biopsy
specimens or other cardiac tissue using a multi-step process (see
FIG. 1A for schematic). First, cardiac tissue is obtained via
percutaneous endomyocardial biopsy or via sterile dissection of the
heart. Once obtained, tissue specimens are stored on ice in a
high-potassium cardioplegic solution (containing 5% dextrose, 68.6
mmol/L mannitol, 12.5 meq potassium chloride, and 12.5 meq sodium
bicarbonate, with the addition of 10 units/mL of heparin) until
they are processed (up to 12 hours later). For processing,
specimens are cut into 1-2 mm.sup.3 pieces using sterile forceps
and scissors; any gross connective tissue is removed. The fragments
are then washed with Ca.sup.++Mg.sup.++ free phosphate buffered
saline (PBS) and typically digested for 5 min at room temperature
with 0.05% trypsin-EDTA. Alternatively the tissue fragments may be
digested in type IV collagenase (1 mg/mL) for 30 minutes at 37
degrees C. Preliminary experiments have shown that cellular yield
is greater per mg of explant tissue when collagenase is used.
[0240] Once digestion is complete, the remaining tissue fragments
are washed with "Complete Explant Medium" (CEM) containing 20%
heat-inactivated fetal calf serum, 100 Units/mL penicillin G, 100
.mu.g/mL streptomycin, 2 mmol/L L-glutamine, and 0.1 mmol/L
2-mercaptoethanol in Iscove's modified Dulbecco medium to quench
the digestion process. The tissue fragments are minced again with
sterile forceps and scissors and then transferred to
fibronectin-coated (25 .mu.g/mL for at least 1 hour) tissue culture
plates, where they are placed, evenly spaced, across the surface of
the plate. A minimal amount of CEM is added to the plate, after
which it is incubated at 37.degree. C. and 5% CO.sub.2 for 30
minutes to allow the tissue fragments, now referred to as
"explants", to attach to the plate (FIG. 1B). Once the explants
have attached, enough CEM is added to the plate to cover the
explants, and the plates are returned to the incubator.
[0241] After a period of 8 or more days, a layer of stromal-like
cells begins to arise from adherent explants, covering the surface
of the plate surrounding the explant. Over this layer a population
of small, round, phase-bright cells is seen (FIG. 1C, 1D). Once the
stromal cell layer becomes confluent and there is a large
population of bright phase cells, the loosely-adherent cells
surrounding the explants are harvested. This is performed by first
washing the plate with Ca.sup.++Mg.sup.++-free PBS, then with 0.48
mmol/L EDTA (for 1-2 min) and finally with 0.05% trypsin-EDTA (for
2-3 min). All washes are performed at room temperature under visual
control to determine when the loosely adherent cells have become
detached. After each step the wash fluid is collected and pooled
with that from the other steps. After the final wash, the explants
are covered again with CEM and returned to the incubator. Each
plate of explants may be harvested in this manner for up to four
times at 5-10 day intervals. The pooled wash fluid is then
centrifuged at 1000 rpm for 6-8 minutes, forming a cellular pellet.
When centrifugation is complete, the supernatant is removed, the
pellet is resuspended, and the cells are counted using a
hemacytometer. The cells are then plated in poly-d-lysine coated
24-well tissue culture plates at a density ranging from
3-5.times.10.sup.4 cells/well (depending on the species) and
returned to the incubator. The cells may be grown in either
"Cardiosphere Growth Media" (CGM) consisting of 65% Dulbeco's
Modified Eagle Media 1:1 with Ham's F-12 supplement and 35% CEM
with 2% B27, 25 ng/mL epidermal growth factor, 80 ng/mL basic
fibroblast growth factor, 4 ng/mL Cardiotrophin-1 and 1 Unit/mL
thrombin, or in CEM alone.
[0242] In either media, after a period of 4-28 days, multicellular
clusters ("cardiospheres") will form, detach from the tissue
culture surface and begin to grow in suspension (FIG. 1E, 1F). When
sufficient in size and number, these free-floating cardiospheres
are then harvested by aspiration of their media, and the resulting
suspension is transferred to fibronectin-coated tissue culture
flasks in CEM (cells remaining adherent to the poly-D-lysine-coated
dishes are not expanded further). In the presence of fibronectin,
cardiospheres attach and form adherent monolayers of CDCs (FIG.
1G). These cells will grow to confluence and then may be repeatedly
passaged and expanded as CDCs, or returned to poly-d-lysine coated
plates, where they will again form cardiospheres. Grown as CDCs,
millions of cells can be grown within 4-6 weeks of the time cardiac
tissue is obtained, whether the origin of the tissue is human (FIG.
1I), porcine or from rodents (data not shown). When collagenase is
used, the initial increase in cells harvested per mass of explant
tissue results in faster production of large numbers of CDCs.
Example 7
Evaluation of Processing and Culture Conditions
[0243] Optionally, changes in processing or culture methods and or
reagents disclosed herein may be made in order to promote
generation of cardiospheres, CDCs, or IICSps in sufficient numbers
and having sufficient viability for use in allogeneic (or
autologous) therapies
[0244] In several embodiments a commercially-available cardioplegic
solution is used to store the biopsy or donor tissue until
processing begins. In some embodiments, the initial biopsy
digestion step is modified through the used of with collagenase
rather than trypsin. In some embodiments, the number of mincing
steps is minimized.
[0245] In several embodiments the presence of recombinant human
proteins and/or cytokines in cardiosphere medium are eliminated,
and fetal bovine serum used in their place. However, in some
embodiments, recombinant human proteins and/or cytokines are used
in combination with fetal bovine serum. In several embodiments,
gentamicin is used as an antibiotic. In some embodiments,
gentamicin is preferred as compared to penicillin and streptomycin,
or combinations thereof. In several embodiments, antibiotics are
removed from the culture media in the final phase of CDC culture.
In several embodiments, trypsin is replaced with TrypLE Select
during the harvesting of cardiosphere-forming cells and passaging
of CDCs.
[0246] Tissue collected sites were also compared for CDC yield. The
sites were the right ventricular septal wall (RVS), the atria, the
apex, the right ventricular epicardium (RVE), and the left
ventricular epicardium (LVE). As shown in FIG. 7A, yields from the
atria and RVE are somewhat greater than yields from the other
regions. Thus in some embodiments these sites are preferred
(whether from biopsy or whole heart). However, these data also show
that CDCs can reliably be produced using tissue from each of the
collection regions tested. In some embodiments, other regions of
the heart are used.
[0247] Because several embodiments involve the use of a heart that
is in condition for transplant, there may be a delay from the
collection of the heart itself and the initiation of processing the
heart to generate CDCs. Therefore, the effect of tissue storage on
CDC yield was examined. Specimens were taken from two transplant
quality human hearts. Specimens processed immediately after
collection were compared to those stored for 3 or 6 days in cold
cardioplegia solution or to those cryopreserved and subsequently
thawed. For each of the two hearts, 6-12 specimens were processed
following each storage condition. Four out of 12 specimens taken
from the first heart and stored for 6 days did not yield CDCs, and
therefore, in some embodiments, tissue is preferably stored for
less than 6 days (e.g., 1, 2, 3, 4, or 5 days). However, in several
embodiments, storage for 6 days is acceptable, as the possibility
of generating CDCs from such a sample exists.
[0248] Data demonstrate a slight effect of 3 days of cold storage
in terms of the time required to achieve a similar CDC yield, and a
larger effect seen after freezing and thawing the specimen (FIG.
7B). Freezing and thawing decreased the average yield and increased
processing time required. However, following either 3 days of cold
storage or cryopreservation CDCs were reliably generated. Thus in
several embodiments, tissue is processed for CDCs immediately,
while in some embodiments, a storage time of 1-2 or 2-3 days (or
longer) is allowed. In some embodiments, cold storage is used,
while in other embodiments, cryopreservation is used.
[0249] Tissue samples from the RVS and cultured in conditions such
as those described above are used, in some embodiments, to generate
a master cell bank (MCB). CDCs were passaged to P1 to create the
MCB. A fraction of CDCs underwent further passaging to P6 in order
to generate a working cell bank (WCB). The yields presented for the
WCB (Table 3) represent the potential yield extrapolated from the
growth seen with the fraction of cells expanded. In some
embodiments, greater passage numbers are used for either the MCB or
the WCB. For example, the MCB is optionally generated at P2, P3,
P4, P5, or P6. Likewise, the WCB is optionally generated at P7, P8,
P9, P10, P11, P12, or more. In some embodiments, CDCs are expanded
just prior to the point of to the point of in vitro senescence).
The WCB is then split into multiple doses, e.g., single
cryopreserved doses.
[0250] These data taken as a whole, demonstrate the feasibility of
generating CDCs and cell banks from the CDCs from
transplant-quality hearts.
TABLE-US-00003 TABLE 3 Cell Banks Generated from Whole Hearts
Source MCB Yield WCB Yield Donor Heart 1 116 million 8.72 billion
Donor Heart 2 185 million 42.0 billion
Example 8
Characterization of Human CDCs
[0251] The markers expressed on CDCs, in several embodiments, are
related to the "sternness" of the cells. In some embodiments,
markers are used to select, screen, isolate, or enrich a sample for
cells bearing one or more markers. In some embodiments, however,
cells are isolated without preference to a given marker or set of
markers. The vast majority of CDCs (derived from 13-27 different
preclinical patient endomyocardial biopsy specimens) were
CD105.sup.+, with significant pluralities that were CD90.sup.+ and
c-kit.sup.+ (FIG. 8A). CDCs were largely negative for CD45
(0.1.+-.0.1%). FIG. 8B demonstrates that CDCs contain distinct
sub-populations of cells that are CD105.sup.+CD90.sup.+c-kit.sup.-.
These particular profiles, in some embodiments are suggestive of
cardiac mesenchymal cells or fibroblasts. CDCs also, in some
embodiments, contain distinct cardiac progenitor populations
(c-kit.sup.+CD90.sup.-). In several embodiments, all populations
express CD105, the regulatory component of the TGF-.beta. receptor
complex important in angiogenesis and hematopoiesis. In several
embodiments all populations also lack CD45. In some embodiments,
CD45 is used to screen for contaminating blood-derived cells to
ensure purity of the CDCs to be administered.
[0252] Data presented in Example 10 illustrate the potency of the
sub-populations in comparison to the total population in an animal
model.
Example 9
In Vitro Differentiation of Human CDCs
Cardiomyocyte Differentiation
[0253] In order to examine the ability of human CDCs to
differentiate fully into functional cardiomyocytes, an in vitro
co-culture system was utilized. DiI-labeled or
lentivirally-transduced GFP.sup.+ CDCs were identified in
co-cultures with neonatal rat ventricular myocytes (NRVMs) which
spontaneously contract in culture. Cocultures were subjected to
immunostaining or whole-cell patch clamp in order to record
voltage-sensitive currents. Co-cultured CDCs demonstrated
biophysical features characteristic of cardiomyocytes, including:
contractions as early as 24 hours after the start of co-culture,
sarcomeric organization (FIG. 9A), expression of the
inwardly-rectifying potassium current IKi which is consistent with
a cardiomyocyte ventricular phenotype (FIG. 9B), and when
transduced with the .beta.-subunit of the L-type calcium channel
expression of the calcium current I.sub.Ca,L indicating the
presence of the pore-forming a-subunit (FIG. 9C). Thus, in several
embodiments, CDCs differentiate into cardiomyocytes having normal
functional characteristics. Therefore, such CDCs, upon
administration, are used in some embodiments to effectuate cardiac
repair.
Endothelial Differentiation
[0254] Human CDCs were also challenged to an endothelial
tube-forming assay by culturing them on MATRIGEL.TM. in endothelial
differentiation media. Within 4-6 hours, human CDCs formed complex
tube networks (FIG. 10A) resembling those created by human
umbilical vein endothelial cells (HUVECs) (FIG. 10B). Unexpectedly,
CDCs display a unique morphology between about 24 and about 72
hours after the start of the assay. CDCs contract toward one
another within about 24 hours and then begin to migrate into the
underlying gel substrate within 72 hours. This response could be
reversibly inhibited by including HERCEPTIN.RTM. in the endothelial
media (data not shown), a factor known to inhibit angiogenesis.
Thus, in several embodiments, CDCs are capable of forming
endothelial cells, and in some embodiments, such endothelial cells
are related to angiogenesis. These data demonstrate the potential
for administered CDCs to generate not only cardiomyocytes, but also
to generate supporting endothelial cells and, in some embodiments,
new vasculature to supply newly generated cardiomyocytes with
blood.
Example 10
Mouse Model of Allogeneic Cardiac Repair
Protocols for Testing Human CDCs in Mouse Model
[0255] As a supplement to the Examples described above, myocardial
infarction was created in adult male SCID-beige mice by permanent
ligation of the left anterior descending (LAD) coronary artery, as
described above. Cells were delivered intramyocardially by direct
injection at two peri-infarct sites immediately following ligation.
CDCs (10.sup.5) 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 controls. During the course of the study, mice underwent
echocardiography prior to surgery (baseline), and at 2 days, 3
weeks, and 6 weeks post-surgery. Left ventricular ejection fraction
was evaluated by manual planimetry of the endocardial border in
end-diastolic and end-systolic frames. Mice were euthanized at the
end of the study for histology. Human cells were identified in
histological sections using a human-specific monoclonal antibody
and a human-specific DNA probe.
Time Course of Engraftment of Human CDCs in Infarcted Mice
[0256] In order to track the time course of migration of human CDCs
injected into infarcted mice, an in vivo bioluminescence study was
performed, with treated animals being sacrificed at various time
points for histology. To investigate acute cell retention in the
heart and to visualize the biodistribution of the cells over the
short term, CDCs were transduced with a lentivirus containing the
luciferase gene. Animals were given an injection of D-luciferin
intraperotineally and subjected to optical imaging on an IVIS.RTM.
SPECTRUM (Xenogen) 1 day, 4 days, and 1 week after cell delivery.
Images were acquired every 4 minutes with a 1 minute exposure time
until the peak signal was obtained and the signal began to decline.
FIG. 11A depicts a strong luminescent signal present in the heart
out to 1 week in a representative animal (n=3). Cells were not
detectable in other organs. This approach demonstrates acute
survival and localization of the delivered cells. Peak luminescence
values normalized to the day 1 value detected in the hearts of each
animal are shown in the graph in FIG. 11B.
[0257] Regardless of the apparent decrease in the number of
detectable cells by about 1 week, overall morphological and
functional improvement in the CDC-treated group persists for at
least 6 weeks post-MI (see e.g., FIGS. 12A-12K and FIG. 13). These
data suggest that the therapeutic effects of the CDCs are initiated
at an early post-administration time point. Thus, these data
support the concept of a persistent effect of the administered
cells that is not dependent on the continued presence of viable
cells and perhaps recruits endogenous signaling cascades and/or
endogenous cells to maintain the effect. To that end, in several
embodiments, transient engraftment of the cells into recipient
cardiac tissue is sufficient to yield anatomical and/or functional
benefits.
[0258] Histologically, human cells were detected by immunostaining
using an antibody against a human nuclear antigen (shown below as
green fluorescent nuclei among the blue fluorescence of all
nuclei). A representative Masson's Trichrome stained section shows
the extent of the infarct at each timepoint examined (FIG. 12A-C).
Low magnification images demonstrate the presence of human CDCs in
the infarct border zone injection site at 2 days post-MI (FIGS. 12D
and 12E are from the boxed area in FIG. 12A) and throughout the
border zone and infarct itself at 1 week post-MI (FIGS. 12F and 12G
are from the boxed area in FIG. 12B). Over the course of the 6 week
study period, CDCs distributed throughout the infarct, border zone,
and eventually the remote myocardium (FIGS. 12H-12K are from the
boxed areas in FIG. 12C). The majority of CDCs could be found
engrafted throughout the infarct region (57.+-.3% of the total
engrafted) and the immediate border zone (30.+-.5%), but stable
engraftment also existed in the remote myocardium (13.+-.3%).
Engraftment of CDCs into murine cardiac tissue was also confirmed
by western blot using human-specific antibodies (see FIG. 22).
Human CDCs reconstituted large portions of the mouse hearts 6 weeks
after delivery.
[0259] These data therefore demonstrate that, in several
embodiments, CDCs administered to a subject having damaged or
diseased cardiac tissue are capable of being retained in damaged or
diseased cardiac tissue for both short term and longer time frames.
In some embodiments, cells are retained for between about 1-10
days, 2-9 days, 3-8 days, or 4, 5, or 6 days, or overlapping ranges
thereof. In some embodiments, there is detectable cell loss (e.g.,
loss of retention of the cells in the tissue and/or loss of
viability of the cells) over the short term. In some embodiments,
no or limited cell loss occurs, and administer cells are retained
for about 1 week, about 4 weeks, about 6 weeks, or longer. In some
embodiments, cells are retained for between about 1-2 weeks, 2-3
weeks, 3-4 weeks, 4-6 weeks, 6-10 weeks, 10-15 weeks, and
overlapping ranges thereof. In some embodiments, cells are
permanently retained (e.g., for the lifetime of the recipient). As
discussed more fully below, in some embodiments, the long-term
retention of cells within a target site is not critical to the
successful regeneration of cardiac tissue (or improvement in
cardiac function).
Efficacy of Human CDCs in Mouse Model
[0260] To assess efficacy, CDCs from 21 different randomly-selected
preclinical patients were utilized for functional experiments.
Twenty-one mice were subject to experimental MI (as described
above) were injected with CDCs. Thirty-five mice served as controls
(17 injected with normal human neonatal dermal fibroblasts (NHDFs),
and 18 with PBS). Echocardiograms performed in normal mice prior to
MI revealed a left ventricular ejection fraction (LVEF) of
.about.80%. Two days after MI, there were no significant
differences among the groups in terms of LVEF, although all had a
substantial decline in function indicating a successful MI that was
uniform in severity in all groups. Data for all animals summarized
in FIG. 13. CDC-injected animals showed no significant
deterioration of LV function from 2 days post-MI to 3 weeks.
Moreover, CDC-injected animals showed no decrease in function at 6
weeks post-MI. Given the relatively long-term time point for a
mouse model (>10% of the typical murine lifespan of .about.1
year), persistence CDC of the benefit was unexpected. At 3 and 6
weeks, LVEF was significantly higher in the CDC-treated group than
in either the NHDF treated or the PBS-treated group. LVEF in the
two control groups were indistinguishable from each other.
[0261] In several embodiments, administration of CDCs prevents
further loss of cardiac function due to MI. In some embodiments,
loss of cardiac function is less than about 45% to about 40%, less
than about 40% to about 35%, less than about 35% to about 30%, less
than about 30% to about 25%, less than about 25% to about 20%, less
than about 20% to about 15%, less than about 15% to about 10%, less
than about 10% to about 5%, less than about 5% to about 1%, and
overlapping ranges thereof. In some embodiments, loss of function
is reduced compared to non-CDC treatments. In some embodiments,
cardiac function is increased over time, based on the
administration of CDCs.
[0262] In several embodiments, administration of CDCs induces
increases in cardiac function due post-MI. In some embodiments,
cardiac function is increased by up to about 5%, up to about 10%,
up to about 15%, up to about 20%, up to about 25%, up to about 30%,
up to about 35%, up to about 40%, and overlapping ranges thereof.
In some embodiments, cardiac function is increased to levels beyond
those levels existing pre-MI.
[0263] In several embodiments, CDC administration provides an
initial short term benefit. In some embodiments, this initial
benefit continues for a longer time period. In some embodiments,
the time frame ranges from about 3-6 weeks, or longer. In several
embodiments, the benefit is realized for about 1-2 weeks, 2-3
weeks, 3-4 weeks, 4-6 weeks, 6-10 weeks, 10-15 weeks, and
overlapping ranges thereof. In some embodiments, the CDC-derived
benefit is realized for the lifetime of the recipient.
[0264] A morphometric analysis was also conducted at the 6 week
study endpoint to assess the effects of CDC treatment on infarct
remodeling. Mouse hearts were excised, washed in PBS, arrested in
diastole with ice-cold KCl, frozen and sectioned transversely in
5-8 .mu.m slices. For a morphometric analysis, tissue sections were
selected from the largest extent of the infarct area and stained
using Masson's Trichrome (representative results are shown in FIG.
12). Photographs encompassing the entire section were acquired.
Infarct wall thicknesses and tissue viability within the infarct
zone were calculated from Masson's Trichrome-stained sections by
tracing borders manually and then using ImageJ software (NIH) to
make measurements. Three to six sections were analyzed per animal
and values averaged. In several embodiments, administration of CDCs
results in a larger tissue thickness in the infarct area. In turn,
in some embodiments, the larger thickness results in an increased
percentage of viable myocardium. In several embodiments, percentage
of viable myocardium in the infarct area ranges from about 15% to
about 20%, from about 20% to about 25%, from about 25% to about
30%, from about 30% to about 35%, from about 35% to about 40%, and
overlapping ranges thereof. In some embodiments, viable myocardium
in the infarct zone is greater than 40%. In some embodiments, these
increases are also associated with increased cardiac function.
[0265] In addition, whole hearts and Masson's Trichrome-stained
tissue sections were examined for evidence of tumor formation. No
tumors were detected in the hearts of any animal. Thus, in several
embodiments allogeneic administration of CDCs serves accomplish one
or more of increasing cardiac function, increasing cardiac tissue
thickness in the infarct zone, increase the percentage of viable
myocardium in the infarct zone. In some embodiments, the above are
accomplished in the absence of tumor (e.g., teratoma)
formation.
Efficacy of CDC Sub populations in Mouse Model
[0266] The efficacy of the cardiac mesenchymal cell and the cardiac
progenitor cell sub-populations alone were tested in comparison to
the total CDC population. A magnetic-activated cell sorting
technique was utilized to enrich the sub-populations of interest,
CD90.sup.+ cardiac mesenchymal cells and c-kit.sup.+ cardiac
progenitor cells. CDCs were sorted using the CELLection Pan Mouse
IgG Kit (Invitrogen) and a Dynal Magnetic Particle Concentrator-15
(Invitrogen). The following monoclonal antibodies were utilized for
the first labeling step: CD9O-FITC (1:10, Dianova), c-kit-APC
(1:10, BD Pharmingen), and CD105-PE (1:10, R&D Systems). After
staining and washing, cells were labeled with prewashed CELLection
Dynabeads conjugated via a DNA linker to a secondary antimouse IgG
antibody. After staining and diluting, the labeled cell solution
was placed into the MPC-15 and the unlabeled cell fraction
aspirated and discarded. The labeled cell fraction was resuspended
in releasing buffer to allow for cleavage of the DNA linker and
release of the Dynabeads from the cells. The cell and Dynabead
solution was placed into the MPC-15, the cell fraction was
collected and the Dynabeads were discarded. After another wash,
cells were resuspended in media for culture overnight (prior to in
vivo delivery).
[0267] Enriched CDC sub-populations were then tested in the same
mouse MI model described above. CDCs enriched for CD105, present on
97% of all CDCs, were used as a control. The cardiac function (as
measured by LVEF) of mice with a CD-105-enriched population was
comparable to CDC-injected mice. See FIG. 14. These data indicate
that the sorting protocol did not itself significantly impair the
therapeutic potential of CDCs. Thus, in several embodiments, cell
sorting for one or more particular markers present on (or absent
from) CDCs is used to enrich a population of CDCs for one or more
particular markers.
[0268] LVEF for c-kit- and CD90-injected mice were
indistinguishable from one another. Both of these groups were
significantly outperformed by the CD105-injected mice and the
CDC-injected mice. All groups were then compared to mice treated
with fibroblasts and mice treated with PBS. c-kit-injected mice had
significantly greater LVEF than both fibroblast- and PBS-injected
mice. The CD90-injected group approached significance when compared
with the fibroblast and PBS groups. While the therapeutic
mechanisms of action of these two distinct sub-populations may
differ, both offer similar global functional benefits to those
enriched for CD-105 and unenriched CDCs. Thus, in some embodiments,
cell sorting/enrichment is used to prepare a sub-population for use
in allogeneic therapies. However, in several embodiments, use of a
CDC population that has not been enriched for any one marker
(including but not limited to c-kit) is particularly advantageous,
as the required manipulation and handling of the cells is reduced.
Thus, the generation of a population of CDCs for allogeneic
therapies is simpler, more rapid, and less likely to be affected by
contamination.
Differentiation of Human CDCs in Infarcted Mice
[0269] The extent to which CDCs proliferated and formed new
cardiomyocytes over the study time period (e.g., the 6 weeks
post-MI) was investigated. Mitotically-active CDCs were identified
by expression of a human-reactive Ki67 (FIG. 15A-15C). The
percentage of Ki67.sup.+ CDCs increased from 2 days to 4 days
post-MI from 4% to 6% (7 of 112 counted), after which time it
became difficult to detect proliferative CDCs (see FIG. 15D-15F).
As many as 25% (28 of 111 counted) of CDCs were cardiac-committed
at 2 days post-MI as evidenced by expression of Nkx2.5 (FIG.
15G-15I). Human CDCs did not express cTnI at 2 days post-MI (FIG.
15M-15O), but could be found lodged within regions of dead and
dying cardiomyocytes (evidenced by loss of sarcomeric organization
and anucleation). By 1 week post-MI, infiltration of mouse
progenitors into the infarct was apparent. Clusters of Nkx2.5.sup.+
cells of both human (yellow arrows) and mouse (white arrows) origin
were prominent. After the early proliferative CDC response had
largely resolved, cardiomyocyte differentiation appeared to
commence, as newly forming cardiomyocytes of both human and mouse
origin could be identified within the infarct. These newly forming
cardiomyocytes were identified as such due diffuse cytoplasmic
expression of cTnI (see FIG. 15P-15R).
[0270] At the end of the 6 week period, CDCs had formed not only
cardiomyocytes, but also non-cardiomyocytes throughout the heart,
as demonstrated in FIG. 16. FIG. 16 shows fluorescence in situ
hybridization using a human-specific centromeric probe and the red
fluorescence of cardiac troponin I (FIGS. 16A-16D). Human nuclei of
interest are outlined in FIGS. 16E-16H. Example cardiomyocyte
nuclei are shown in FIGS. 16M-16P at higher magnification.
[0271] These data indicate that, in several embodiments, CDCs not
only have the capacity to proliferate, but CDCs also have the
capacity to differentiate and repopulate damaged myocardium by
forming cardiomyocytes as well as non-cardiomyocytes. Moreover, in
several embodiments, administered CDCs, also attract endogenous
cardiac progenitor cells. Taken together, in several embodiments,
one or more of these characteristics of CDCs are responsible for
the resultant increase in cardiac tissue viability, regeneration,
and/or overall function.
In Vitro Analysis of Paracrine Factors from Regenerative Cells
[0272] In order to characterize the paracrine signals that, in
several embodiments provide a beneficial therapeutic effect, the
cytokines and growth factors released from regenerative cells were
screened. Also assessed was whether the released cytokines and
growth factors yield favorable biological effects on neonatal rat
ventricular myocytes (NRVMs) and human umbilical vein endothelial
cells (HUVECs).
[0273] Human cardiospheres and CDCs were obtained from percutaneous
septal endomyocardial biopsies from 21 different patients as
described above.
[0274] Media were conditioned for 48 hours by cardiospheres and
IICSps after 4-5 days of culture on poly-D-Lysine, or by CDCs and
NHDFs when they were approximately 90% confluent. Media for
conditioning comprised 2.5% FBS complete explant medium (CEM), or
glucose-free FBS-free basal medium (BM): Medium 199, 10 mmol/L
HEPES, 0.1mmol/L MEM non-essential amino acids, 2 mmol/L
L-glutamine, 0.8 .mu.g/mL vitamin B12, 2 unit/mL penicillin.
Conditioned media (CM) were stored at -80.degree. C. until used. In
some experiments, CMs were pre-incubated on a shaker for 1 hour at
room temperature with anti-VEGF and/or anti-HGF neutralizing
antibodies. As discussed above, changes to the culturing protocol
(e.g., % oxygen, media changes, and the like) are used in some
embodiments.
[0275] Serum-free CMs from and CDCs were screened for secreted
factors using a protein array according to the manufacturer's
protocol (Ray Biotech).
[0276] Neonatal rat ventricular myocytes (NRVM) were isolated by
standard procedures known in the art. Culture plates were incubated
in humidified 2% 0.sub.2 atmosphere for 24 or 72 hours with CM or
FBS-free BM (lower portion of FIG. 19). A portion of the cells were
then collected by trypsinization, labeled with Annexin V-FITC and
7AAD and analyzed by flow cytometry for indications of
apoptosis.
[0277] Human umbilical vascular endothelial cells (HUVEC) were
plated on pre-cast, matrix-coated 96-well plates. They were plated
with either endothelial cell media (ECM), as a positive control, or
with CM, or with FBS-free BM, as the negative control (see FIG. 17
for a protocol outline). After 18 hours, total tube length formed
was measured microscopically.
[0278] Cell cultures were lysed in lysis buffer (20 mmol/L TrisHCl,
5 mmol/L EDTA, 50 mmol/L NaCl, 1% SDS) with proteinase inhibitors
cocktail (Sigma), and homogenized by sonication. Tissue samples
were lysed in lysis buffer with proteinase inhibitors cocktail
(Roche) and homogenized with a rotor-stator homogenizer.
Homogenates were spun at 12,000 rcf for 15 minutes at 4.degree. C.
Supernatants were then collected and stored at -80.degree. C.,
after quantification by Lowry assay of the protein content
(BioRad). Western blots were performed as described above. Primary
antibodies used were as follows: hVEGF and pan-GAPDH (Abcam), hHGF
and hIGFI (R&D Systems), hGAPDH (LabFrontier), Akt (Cell
Signaling Technology), Caspase 3 (Csp3; Santa Cruz). Human
specificity was confirmed. Membranes were washed in TBST, incubated
with HRP-conjugated secondary antibodies (Pierce; Santa Cruz), and
developed with ECL (Amersham) or West-Femto substrate (pierce).
Western blots on media were performed with the Nupage system,
loading 30 .mu.l of media per lane. After blocking, membranes were
incubated with HRP-conjugated anti-mouse IgG antibody (Santa Cruz).
Densitometric analysis was performed with ImageJ software and
plotted as ratios to the GAPDH signal.
[0279] RNA from cells and tissue samples was extracted with
column-based kits (Qiagen). Reverse transcription was performed on
lug starting RNA (Stratagene) in a 20 .mu.l reaction, and 2 .mu.l
of cDNA product were then subjected to PCR (Invitrogen) with human
specific or pan-specific primers for 35 thermal cycles.
[0280] In preparation for immunofluorescence and histology,
cardiospheres and CDCs were fixed for 10 minutes with
ethanol-acetone 50:50% at 4.degree. C. Hearts were cut in 5 .mu.m
sections. After deparaffinization and rehydration of the tissue
sections, slides were washed and permeabilized with 0.1% Triton
X-100 in PBS with 1% BSA, then blocked in 10% goat serum and
incubated overnight at 4.degree. C. in 1% goat serum with primary
antibodies: anti h VEGF and human nuclear antigen (HNA; Chemicon),
hHGF, hIGFI and CD 105 (R&D Systems), FN, KDR and c-kit
(Abeam), Met and nkx2.5 (Santa Cruz), IGFI-R (Upstate). Slides were
then washed and incubated with Alexa Fluor 488 or 568-conjugated
secondary antibodies (Invitrogen). Incubation with secondary
antibodies alone did not give any detectable background signal.
[0281] For capillary staining, tissue sections were incubated for 2
hours with FITC-conjugated Isolectin B4 (Lab Frontier) and
Alexa-S68-Phalloidin (Invitrogen); a total of 26700 nuclei were
analyzed overall on multiple sections of the border zone, of which
3300 for the assessment of CDC contribution. Co-incubation of
isolectin B4 with 500 mM galactose was used as a negative
control.
[0282] TUNEL staining was performed according to the manufacturer's
instructions (In situ Cell Death Detection Kit, TMR red, Roche) and
quantified on a total of 20500 nuclei in the border zone.
[0283] Confocal fluorescence imaging was performed on an Eclipse
TE2000-U equipped with a krypton/argon laser using UltraVIEW
software (Perkin Elmer). For image analysis of capillary and TUNEL
slides, the ImageJ software was used for binary threshold of
fluorescent images for each channel and consequent particle count.
Masson's Trichrome staining was performed by standard methods.
Briefly, staining was performed according to the kit manufacturer
instructions (Sigma). High resolution images were acquired and
processed with ImageJ software: color channels were split and the
infarct area was manually traced on the blue channel. Threshold
adjustment and area measurement functions allowed automatic
calculation of the collagen-stained fraction within the defined
infarct region.
Protein Array Screening of Conditioned Media
[0284] Analysis of serum free conditioned media by protein array
analysis yielded 79 spots corresponding to various cytokines and
growth factors. FIG. 18 shows two representative blots (18A) from
cardiospheres and CDCs derived from the same patient sample,
together with the corresponding densitogram (18B and 18C), showing
the cardiosphere/CDC optical density ratios for each factor. Three
candidates were selected for further analysis, VEGF, HGF and IGFI.
This was based on their identity as non immune-modulatory factors,
the high CSp/CDC ratio (suggesting enhanced secretion in
three-dimensional culture) and established roles in cardiac
pathophysiology, particularly in myocardial infarction and heart
failure.
Regenerative Cells Release Growth Factors In Vitro
[0285] The various types of regenerative cells were analyzed to
compare their relative paracrine potencies. Quantification of VEGF,
HGF and IGF-1 protein levels (by standard ELISA techniques) in the
conditioned media from cardiospheres, CDCs, and IICSps indicate
that all three regenerative cell types secrete significant amounts
of these growth factors. See FIG. 19A. During 48 hours of
conditioning in low serum, cardiospheres released VEGF, HGF and
IGFI. By contrast, CDCs and IICSps released only VEGF in measurable
amounts, although secretion by IICSps was at the level measured in
cardiospheres. In several embodiments, VEGF, HGF, and IGFI are all
released, while in some embodiments, one or more are released. In
some embodiments, release of growth factors is time-dependent
(e.g., one or more are released at an early time-point, while one
or more are released at a later time-point).
[0286] Although different culture serum concentrations and
different cell densities were also analyzed, IGFI secretion was not
detected in CDCs or IICSps. CDCs are capable of releasing HGF,
under certain culture conditions. CDCs cultured in basal media
release detectable amounts of HGF as detected by ELISA (see FIG.
19B).
[0287] Immunofluorescent analysis of cardiospheres revealed ample
VEGF, HGF and IGFI (FIG. 19C-19D). Reverse transcription PCR on RNA
isolated from cardiospheres, CDCs and IICSps also confirmed the
expression of VEGF, HGF and IGFI mRNAs (FIG. 19E). No growth
factors were detected by ELISA in normal human dermal fibroblast
(NHDF)-CM in any of the conditions tested, although the
corresponding mRNA was detected by PCR. NHDFs were also grown as
spheres on poly-D-lysine, but cell-associated growth factors were
not detected by immunofluorescence (data not shown) nor were
secreted growth factors detected in CM by ELISA (FIG. 19A).
[0288] Cardiospheres and CDCs also express the receptors for VEGF,
HGF and IGFI (respectively KDR, Met, IGFI-R), as assessed by
immunofluorescence and RT-PCR (see FIG. 20).
Effects of Regenerative Cells Conditioned Media on Cardiac Cell
Viability
[0289] Conditioned media from CDCs and NHDF cells were collected in
fetal bovine serum-free basal media and used in place of NRVM
media. After 24 or 72 hours in 2% hypoxia, the percentage of early
apoptotic NRVMs was assessed by Annexin V/7-AAD labeling. No
differences were detected after 24 hours, however after 72 hours
the percentage of early apoptotic NRVMs was dramatically lower in
the CDC-CM (FIG. 21A) compared to the NHDF-CM (FIG. 21B) and the
control FBS-free BM (FIG. 21C). The summary FACS data is
represented in FIG. 21D. When CDC-CM was pre-incubated with
neutralizing anti-VEGF and HGF antibodies and used for culturing
NRVMs for 72 hours, the apoptosis-reducing effect was significantly
reduced (FIG. 21E). The neutralization of both secreted growth
factors resulted in an excess 23% early apoptotic NRVMs compared to
plain CDC-CM. Significance, in all examples, was evaluated using
standard statistical techniques and significant differences are
represented by p values<0.05, unless otherwise specified.
Effects of Regenerative Cells Conditioned Media on Angiogenesis
[0290] To determine if paracrine signals from the CDCs may be
mechanistically involved in the blood vessel formation, HUVECs were
cultured in either endothelial cell media (ECM; the normal media
used to culture HUVECs), fetal bovine serum-free basal media, or
CDC-CM (see FIGS. 21F, 21G, and 21H, respectively). The ability of
HUVECs to form complex tube networks was lost in BM, but when
cultured in CDC-CM this ability was almost completely recovered
(compare 21G with 21H). The tube-forming ability of HUVECs cultured
in CDC-CM was significantly greater than that of HUVECs cultured in
NHDF-CM (FIG. 21J). Pre-incubation with anti-VEGF or both anti-VEGF
and anti-HGF neutralizing antibodies caused a slight but
significant reduction of the total tube length per well (FIG.
21K).
[0291] In summary, the preliminary screening of human regenerative
cells and their conditioned media revealed that these cell
populations are capable of releasing many different cytokines and
growth factors. Cardiospheres were found to spontaneously release
significant and higher amounts of VEGF, HGF and IGFI in vitro as
compared to CDCs. CDCs secrete only VEGF and HGF, although they
maintain the transcription of all their mRNAs. However, IICSps were
able to secrete VEGF at levels comparable to cardiospheres,
suggesting the 3D structure of cardiospheres may influence release
of VEGF. This may be due to hypoxic stimulation of the cells
residing in the interior layers of the cardiospheres. These results
suggest that: a) the primary CSp is the stage at which paracrine
abilities are maximal in vitro; b) VEGF release is affected by the
3D structure; c) HGF and IGFI release fades with progressive time
in culture. Furthermore, the expression of receptors for these
growth factors on regenerative cells suggests a possible autocrine
feedback effect.
[0292] Although these results suggest that cardiospheres are more
potent in terms of paracrine signal release in vitro, other cell
types may be equally, or more potent in a the more complex
physiological environment in vivo. To this end, functional in vitro
analysis was performed to test the effects of CDC-CM on cell
viability and angiogenesis. The present experiments demonstrated
that CDC-CM reduces the frequency of apoptosis initiation in
ventricular myocytes. This pro-survival effect is partially due to
synergy between secreted VEGF and HGF, as NRVM viability was
reduced after pre-incubation of the CDC-CM with both anti-VEGF and
HGF neutralizing antibodies. CDC-CM has also potent pro-angiogenic
effects, as demonstrated by promoting formation of complex tube
networks by HUVECs.
[0293] These data indicate that regenerative cells secrete growth
factors that positively impact the survival of ventricular myocyte
cells and the angiogenic capacity of endothelial cells. Thus, in
several embodiments, these secreted growth factors play a role in
the in vivo repair of damaged myocardial tissue by enhancing the
survival of endogenous cells and/or increasing angiogenesis in the
myocardial tissue, among other possible mechanisms.
Detection of Human Growth Factors in Mouse Model
[0294] Western blot analysis using human-specific antibodies was
used to detected engraftment of CDCs that were injected into murine
cardiac tissue as described above. Additionally, western blot was
used to demonstrate the presence of human growth factors within the
infarcted mouse heart. In several embodiments, growth factors from
the administered CDCs, such as VEGF, HGF, IGF-1 contribute to the
functional benefits observed. Animals were sacrificed 1 day, 1 week
or 3 weeks after cell delivery. Regional tissue samples, ranging
from 15 to 20 mg on average, were taken from infarct (INF), border
zone (BZ), right ventricle (RV) and septum (SEP) areas for WB.
Tissue samples were lysed in lysis buffer with proteinase
inhibitors cocktail, homogenized, and spun. Supernatants were then
collected and stored, after quantification by Lowry assay of the
protein content. Lysates were loaded on 4-12% Bis-Tris gels
(Invitrogen), and blots 50 .mu.g of protein per lane was performed
with the Nupage mini-gels system (Invitrogen). Primary antibodies
against human VEGF and pan-GAPDH (Abcam), human HGF and human IGF1
(R&D Systems), and human GAPDH (LabFrontier) were used.
Membranes were washed and incubated with HRP-conjugated secondary
antibodies. Human growth factors were detectable with human
specific antibodies only in lysates from CDC-injected animals,
although hGAPDH was evident in the INF and BZ of both CDC- and
NHDF-injected animals. These data indicate the cells survive for at
least three weeks in CDC-injected and for at least 1 week in
NHDF-injected hearts (see FIG. 22) Growth factors were also
detectable in remote areas, like the RV and SEP, 1 day after cell
delivery. After 3 weeks, bands for hHGF and hIGF-1 were faint, but
detectable, although hVEGF could no longer be found detected. The
absence of a hGAPDH band in the BZ after 24 hours can be
attributed, at least in part, to the difficulty in distinguishing
the infarct from the peri-infarct zone at such an early time
point.
[0295] Despite the apparent reduction in expression of human growth
factors over time, CDCs had clearly engrafted and survived, as
evidenced by the detection of hGAPDH in tissue lysates. These data
suggest that, in several embodiments, CDCs exert an effect on the
recipient tissue early after transplantation, which has long
lasting effects. In some embodiments, the engraftment of the CDCs
itself triggers this effect. However, in several embodiments,
paracrine effects are responsible for initiating a cascade of
events that lead to functional improvements at later time point. In
some embodiments, the initially administered CDCs are no longer
present, yet the paracrine effects and the ensuing cascade are
still active in repairing and/or regenerating cardiac tissue. In
several embodiments, CDCs benefit the injured heart by direct
myocardial regeneration with a large portion of their effect being
due to the secretion of paracrine factors that stimulate endogenous
repair pathways. Based on a quantitative analysis of the
contribution of engrafted human cells (discussed in more detail
below), it can be estimated that the paracrine effect accounts for
at least 50% of the total effect, in one embodiment. In several
embodiments, the paracrine effect is responsible for about 50% to
about 60% of the total effect, for about 60% to about 70% of the
total effect, for about 70% to about 80% of the total effect , or
greater that 80% of the total effect. In some embodiments, the
paracrine effect is entirely responsible for the myocardial
regeneration and/or increased cardiac function.
CDC-Injected Tissue Displays Higher Tissue Viability and Capillary
Density
[0296] As discussed above, one week after cell delivery, human
growth factor levels remained high in the heart of mice injected
with CDCs (see FIG. 22). Lysates from CDC-injected mice contained
higher levels of Akt protein compared to NHDF-injected animals, as
shown by WB and relative densitometric analysis (FIG. 23A).
Moreover, active Csp3 expression was reduced in CDC-injected hearts
relative to controls (FIG. 23B). These results correlate with a
reduced apoptotic rate (FIG. 23C) and higher capillary density in
the border zone of CDC-injected mice (FIG. 23D), compared to
controls, as assessed by TUNEL and isolectin B4 staining,
respectively. Taken together, these data indicate that CDCs
suppress post-ischemic apoptosis and improve blood supply. Thus, in
several embodiments, reduction in apoptosis accounts for at least a
portion of the higher tissue viability. In several embodiments,
increased angiogenesis, alone or in combination, with reduced
apoptosis, accounts for the increased tissue viability.
Direct Versus Indirect Regenerative Contribution
[0297] As a means of quantifying how much of the functional
improvement due to CDC administration is related to direct
regeneration versus indirect paracrine effects, the relative
contribution of human CDCs to the capillary density in the tissue
areas where CDCs were detectable after 1 week was calculated.
Despite an overall doubling of capillary density in CDC-injected
mice, only 9.6.+-.2.7% of the total capillaries were found to be of
human origin (FIG. 24A and 24B). This amounts to .about.20% of the
enhanced angiogenesis; thus, the angiogenic effect reflects both
direct regeneration and paracrine effects, with latter
predominating.
[0298] With respect to the cardiomyogenic effect of CDC
transplantation, after 1 week the infarct area of CDC-injected mice
contained a higher percentage of viable myocardium (FIG. 25A), as
assessed by Masson's trichrome staining. Within those viable areas,
11.8.+-.4.5% of the MHC-expressing cells were of human origin (FIG.
24C and 24D). While significant, this explains only half of the
.about.20% increase in relative tissue viability revealed by
Masson's trichrome staining, and only .about.25% of the overall
doubling in MHC.sup.+ nuclei (FIG. 24C). Thus, both direct
regeneration and paracrine effects underlie the cardiomyogenic
effects of CDC transplantation. No human capillaries or
MHC-positive cells were detectable in the NHDF-injected hearts.
[0299] This Example has therefore demonstrated that when injected
intramyocardially in infarcted SCID mice, CDCs release, among other
growth factors, VEGF, HGF and IGFI. These growth factors are
detectable for at least 1 week after cell. Growth factor release is
not simply a result of cell administration or engraftment, as both
CDCs and NHDF cells engrafted into cardiac tissue, rather growth
factors secreted by transplanted cells were detectable only in
CDC-injected animals. Additionally, only CDC-injected animals
demonstrated a significant functional improvement.
[0300] Growth factors were detected in remote areas of the heart 24
hours after cell injection. Remote areas did not appear to contain
growth factors after 1 week, suggesting that diffusion of growth
factors occurs at the earlier time point. This may be due to the
relatively intact vascular system at the earlier time, which loses
functionality in the more mature infarct scar 1 week after surgery,
thus restraining human growth factors to tissue more local to the
CDC engraftment zone. Alternatively, the early spread into
neighboring areas may simply reflect a greater intensity of growth
factor production soon after injection. This may be due to a higher
production rate by newly-injected cells, or may be due to the
presence of more cells at early post-injection time points.
[0301] Cell survival, particularly in an unfavorable environment
(such as ischemic cardiac tissue), mostly depends on the cells'
ability to overcome death triggers and secondarily to promote
angiogenesis. In this respect, the in vitro anti-apoptotic and
pro-angiogenic effects demonstrated in the prior example correlate
with the in vivo observation that, 1 week after cell delivery, Akt
was up-regulated in tissue samples from CDC-injected animals.
Contemporaneously, the active form of the apoptotic effector Csp3
was less expressed in CDC- compared to NHDF-injected hearts (FIG.
23B). Furthermore, the rate of TUNEL-positive cells was
significantly reduced in the border zone of CDC-injected mice,
which also displayed higher capillary density compared to controls
(FIG. 23C-23D). Overall, these results imply that the injection of
CDCs in infarcted SCID mouse hearts favors higher tissue viability
in the infarct and peri-infarct areas, and consistently correlates
with reduced apoptosis and improved blood supply. This conclusion
is consistent with previous histological data showing a higher
percentage of viable myocardium in the infarct area of
CDC-injected, as compared to NHDF-injected mice (which was also
confirmed at the 1 week time point in the present study).
[0302] While injected CDCs and/or their progeny persist for at
least three weeks post-administration, in several embodiments the
persistence of CDCs reflects their multilineage differentiation. In
several embodiments, the persistence of CDCs reflects a survival
advantage conferred by paracrine effects such as those described
here. In several embodiments a combination of both mechanisms is at
play. The present study demonstrates that CDCs directly contribute
to approximately 10% of the overall capillary density in the areas
of CDC engraftment. However, there remains a statistically
significant difference between the capillary density of the CDC and
the NHDF groups, confirming the major role of indirect paracrine
induction by CDCs.
[0303] With respect to their contribution to cardiomyogenesis,
direct regeneration and paracrine mechanisms supporting
regeneration appeared to be more equivalent in their effects. Given
that long-term engraftment of transplanted cells is low, it seems
reasonable to focus on enhanced engraftment as a prime strategy to
boost the overall efficacy of cell therapy, even if the final
benefit reflects both direct and indirect regeneration.
[0304] While the use of cytokines and growth factors as cardiac
therapeutic tools has been carefully investigated in the past few
years, especially by means of gene therapy, many such protocols
involve risks such as pathologic angiogenesis, severe inflammatory
reactions and arrhythmias. The possibility of growth factor release
under biological and physiological control through regenerative
cell therapy might offer an ideal combination of strategies in that
regenerative cells are able to differentiate directly and to
concurrently secrete beneficial molecules and harness endogenous
repair.
[0305] Direct injection of HGF and IGF-1 into the myocardium has
been shown to successfully mobilize endogenous cardiac stem cells.
Given that CDC transplantation produces these same growth factors,
as shown herein, this study suggests that recruitment of endogenous
stem cells is a likely contributor to the functional improvement
demonstrated in this study.
[0306] These data, taken together, demonstrate that regenerative
cells secrete significant amounts of pro-survival and
pro-angiogenic growth factors in vitro, and in an in vivo cardiac
cell therapy model. This Role Model Effect appears to be a key
mechanism, together with the natural propensity of regenerative
cells for cardiac differentiation, contributing to the regenerative
potential and therapeutic effects of regenerative cells in the
post-infarction period.
Efficacy of CDC Cell Banks in Mouse Model
[0307] Based on the discussion above regarding the generation of
cell banks, which are used in some embodiments, the following sets
forth an example of a contemplated experiment using allogeneic
cells generated and banked prior to use. The same mouse MI model
will be employed. CDCs having gone through the manufacturing
process described above, including intermediate banking, will be
suspended and delivered in a cryopreservation solution
(CRYOSTOR.RTM. CS5). Also as discussed above, CRYOSTOR.RTM. CS5
used, in some embodiments as an excipient that is combined with the
CDCs. Control groups will consist of mice injected with CRYOSTOR
CS5 alone, mice injected with CDCs suspended in PBS, and mice
injected with PBS alone. In addition to measuring function 3 weeks
post-MI, histology will be performed to assess the degree of local
tissue damage that may occur due to the 5% dimethylsulfoxide (DMSO)
contained in the cryopreservation solution.
Example 11
Immunologic Properties of Regenerative Cells
[0308] In order to understand the potential for cardiac tissue
repair via allogeneic transplant of regenerative cells, the immune
responses initiated against allogeneic regenerative cells was
characterized. Both in vitro and in vivo analyses were performed
using an established rat model. CDCs were isolated from two rat
strains with different MEW haplotypes (Wistar Kyoto and Brown
Norway). Cross-transplantation of organs from these mismatched
strains has been used as a model for allograft rejection. CDCs were
isolated from Wistar Kyoto rats and ex vivo expanded, as described
above. These CDCs were intramyocardially injected into either
Wistar Kyoto rats (syngeneic/autologous model) or Brown Norway rats
(allogeneic model). In some rats, human CDCs were injected
(xenogeneic model). Expanded CDCs (or PBS for control groups) were
injected immediately after MI was simulated via LAD ligation, as
described above. Injections comprised 2.times.10.sup.6 CDCs in 100
.mu.L total injection volume (50 .mu.L at each of two peri-infarct
sites). In all transplants, male Wistar-Kyoto rats were the donors
and female rats were the recipients, thus enabling mRNA isolated
from rats post-injection to be used in quantitative y-chromosome
real time PCR reactions to determine engraftment of cells into the
target tissue.
[0309] Rats underwent echocardiography to assess efficacy, were
subjected to blood draws to assess the level of pro- and
anti-inflammatory cytokines present in the serum, and were
euthanized at the end of the study for histology to examine the
degree of immune rejection evident or PCR to quantify the
percentage of CDCs engrafted.
In Vitro Immunologic Properties of Allogeneic CDCs
[0310] The immunologic properties of rat and human CDCs (rCDCs and
hCDCs, respectively) were first examined in vitro. Established flow
cytometry methods were used to assess the expression of MHC Class I
and Class II antigens and before and after stimulation with
interferon-y, a known immunostimulatory molecule, for 1 day or for
5 days. With regard to immune antigens, both rCDCs and hCDCs
express MHC I but not MHC class II surface antigens (FIG. 26B and
26F). Incubation with interferon-y upregulated both MHC I and MHC
II expression in a time-dependent manner (FIG. 26C/D and 26G/H).
CD80/CD86 are expressed on antigen presenting cells and provide
co-stimulatory signals needed for T cell activation and survival.
Expression of both CD80 and CD86 was relatively low in both rCDCs
and hCDCs at baseline. Stimulation with interferon-.gamma. did not
significantly induce expression of these co-stimulatory molecules
(see e.g., FIG. 26I). Several other markers did not differ in
expression levels between rCDCs and hCDCs (see e.g., FIG. 26J).
[0311] The observed baseline immunophenotype of CDCs is beneficial
for allogeneic applications. In several embodiments, the higher
baseline levels of MHC class I antigen expression are beneficial,
as this antigen, at least in part, protects the cells from
natural-killer cell-mediated destruction. Similarly, the low level
expression of MHC class II antigens (potent activators of the
immune system) allows, in several embodiments, allogeneic CDCs to
escape direct recognition from CD4+ T helper cells. While MHC class
I antigens may activate effector T cells in certain cases, the low
quantities of costimulatory molecules (CD80/CD86) would induce an
interruption in the signaling pathway, thereby likely leaving T
cells inactive.
[0312] Lymphocyte proliferation was measured by BrdU incorporation.
Well-known methods were used to evaluate immunogenicity, which are
described in brief. Spleens were harvested aseptically from
euthanized WKY and BN rats, mechanically dissociated and filtered
through a 100 .mu.m nylon mesh. Erythrocytes were lysed with 0.83%
ammonium chloride, cells were washed in RPMI 1640, dead cells were
removed by density centrifugation and cell viability was assessed
by trypan blue dye exclusion. Stimulating rCDCs and hCDCs were
mitotically inactivated with 50 .mu.g/ml mitomycin C
(Sigma-Aldrich) in the dark at 37.degree. C. for 30 minutes and
washed three times with RPMI 1640. 10.sup.4 stimulating CDCs were
cocultured with 10.sup.5 responder lymphocytes in 200 .mu.l of
culture medium (RPMI 1640 supplemented with 10% FBS) in 96-well
flat-bottom plates for 5 days. The following experimental
conditions were tested in quadruplicates: a) rCDCs cocultured with
WKY lymphocytes (syngeneic coculture); b) rCDCs cocultured with BN
lymphocytes (allogeneic coculture); c) hCDCs cocultured with BN
lymphocytes (xenogeneic coculture). All appropriate controls were
also tested. BrdU was added to the cocultures for the last 24 hours
and responder cell proliferation was assessed by the Cell
Proliferation Biotrak ELISA System (GE Healthcare) according to the
manufacturer's instructions. Absorbance was measured with a
microplate reader (Bio-Rad) at 450 nm. Alloreactive and
xenoreactive lymphocyte proliferation is presented as relative
proliferative response, normalized to syngeneic coculture
proliferation (stimulation index). The cell-free supernatant of the
cocultures was collected and the levels of secreted IFN-g, IL-1b,
IL-13, IL-4, IL-5, KC/GRO and TNF-.alpha. were measured by
electrochemiluminescence. The levels of secreted IL-2 were measured
using enzyme-linked immunosorbent assay (ELISA) kits, according to
the manufacturer's protocols (R&D Systems).
[0313] Co-culture of rat CDCs with allogeneic lymphocytes induced
negligible lymphocyte proliferation, which was comparable to that
induced by syngeneic CDCs and less than that of xenogeneic human
CDCs (FIG. 26K-26M). The lymphocyte proliferation induced by
allogeneic CDCs was not significantly different than that induced
by syngeneic CDCs (syngeneic stimulation index: 1.4.+-.0.2; p=ns
versus allogeneic). In contrast, xenogeneic hCDCs induce a strong
proliferative response (stimulation index: 2.6.+-.0.6; p<0.01
vs. syngeneic or allogeneic cocultures; FIG. 26N). Quantification
of inflammatory cytokines in the coculture supernatants by
electrochemiluminescence and ELISA demonstrated comparable levels
of pro-inflammatory (IFN-.gamma., TNF-a, IL1b, IL2, KC/GRO) and
anti-inflammatory (IL5, IL13, IL4) cytokines in syngeneic and
allogeneic cocultures. Again in contrast, in the xenogeneic
cultures, secretion of all inflammatory cytokines was markedly
increased, indicating significant activation of responder
lymphocytes (FIG. 26O). Consistent with these in vitro results, the
in vivo immune response (discussed in more detail below), was not
significantly different between allogeneic and syngeneic cell
transplants.
Engraftment of Allogeneic CDCs in Rat Model
[0314] To enable measurements of cell engraftment in vivo, CDCs
were transduced with a lentiviral construct expressing GFP.
Quantitative PCR was performed 1 week and 3 weeks post cell
injection in order to monitor transplanted cell engraftment after
syngeneic, allogeneic and xenogeneic cell transplantation. Cells
isolated from male donor WKY rats and male human biopsies were
injected into the myocardium of female recipients and quantified
absolute cell engraftment by real-time PCR using the (rat and human
respectively) SRY gene located on the Y chromosome as target. In
brief, the recipient heart was explanted, weighted, homogenized and
genomic DNA was isolated using the DNA Easy minikit (Qiagen),
according to the manufacturer's protocol. The TaqMan.RTM. assay
(Applied Biosystems) was used to quantify the number of
transplanted cells with the rat (for syngeneic and allogeneic
transplantation) and human (for xenogeneic transplantation) SRY
gene as template. A standard curve was constructed with samples
derived from multiple log dilutions of genomic DNA, isolated from
male rat hearts and samples of male human myocardium, spiked with
50ng of female rat genomic DNA as control. The copy number of the
SRY gene at each point of the standard curve was calculated based
on the amount of DNA in each sample and the total mass of the rat
genome per diploid cell. All samples were tested in triplicates.
For each reaction, 50 ng of template DNA was used. The result from
each reaction, copies of the SRY gene in 50 ng of genomic DNA, was
expressed as the number of engrafted cells/heart by extrapolation
to the total DNA content of each heart, taking into account that
there is one copy of the SRY gene per transplanted cell. FIG. 27A
depicts the experimental scheme employed to study engraftment and
function. FIG. 27B describes the experimental and control groups
used to evaluate engraftment and function.
[0315] Two million male syngeneic, allogeneic or xenogeneic CDCs
were implanted into the ischemic myocardium of female rats,
immediately after LAD ligation. Similar to the immunogenicity
results discussed above (e.g., the limited differences in immune
induction between syngeneic and allogeneic cultures) engraftment of
allogeneic and syngeneic CDCs was not significantly different
between allogeneic and syngeneic transplants at 1 week post MI
(absolute cell number: 117,587.+-.94181 vs. 122,662.+-.68,637;
p=ns). In contrast, the majority of xenogeneic CDCs fail to engraft
within 1 week of transplantation (absolute cell number:
10,535.+-.4012; p<0.05 compared to syn, allo groups). Data
related to 1-week engraftment are shown in FIG. 27C.
[0316] Three weeks after experimental infarction and CDC delivery,
cell engraftment decreased markedly (to <1% of cells
transplanted) in both syngeneic and allogeneic groups. However, the
residual number of engrafted cells is higher after syngeneic
transplantation (absolute cell number: 13,343.+-.6427 vs.
3169.+-.4012; p<0.05). This likely reflects gradual destruction
and clearance of the allogeneic cells by the host immune system. No
engrafted xenogeneic cells were detected after 3 weeks. Data are
presented in FIG. 27D. These results indicate, that, in some
embodiments, allogeneic CDCs are cleared more rapidly than
syngeneic CDCs between days 8 and 21 post-delivery. In some such
embodiments, the beneficial effects of allogeneic CDCs have already
been realized (or put in motion), such that the survival and
residency of the delivered cells is no longer necessary. Moreover,
based on the immunogenicity data discussed above, these data
indicate that the immune response is not solely responsible for the
decreased number of engrafted cells. Endogenous mechanisms to
remove transplanted cells, and/or the natural lifespan of the
administered cells may account for the decrease.
Immune Response to Allogeneic CDCs in Rat Model
[0317] Consistent with the limited in vitro induction of
pro-inflammatory cytokines by allogeneic CDCs, administration of
allogeneic CDCs induced only a mild local immune reaction in the
heart. In fact, the immune reaction is barely visible using a
standard H&E stain (example figures shown at 3 weeks post-MI,
FIG. 28B-28C). Significant infiltration is seen in the xenogeneic
transplant (FIG. 28A). Further, using an established pathological
assessment scale, the ISHLT grading system, which is used in
clinical practice to diagnose rejection, the level of rejection
seen in H&E sections were scored at 1 week, 3 weeks, and 6
months post-MI. Four to five animals and 48-60 sections were scored
in each group (Grade 0=No rejection, Grade 1R=Interstitial and/or
perivascular infiltrate with up to one focus of myocyte damage,
Grade 2R=Two or more foci of infiltrate with associated myocyte
damage, Grade 3R=Diffuse infiltrate with multifocal myocyte
damage). The analysis was conducted by a blinded cardiac
pathologist. No significant immune rejection could be detected in
the allogeneic setting at any time point (see 28B and 28D, data for
6 weeks not shown). In contrast, xenogeneic cell transplantation
resulted in significant mononuclear infiltration that could be
detected in the infarct scar and border zone 1 week (FIG. 28D) and
3 weeks (FIG. 28F) post treatment. As shown in FIG. 28A, the
infiltrating cells were located at interstitial and perivascular
spaces, but notably, no foci of myocyte damage could be detected.
The lack of foci suggests that immune rejection of xenogeneic CDCs
did not inflict additional damage to the myocardium. The immune
response was resolved in all groups at 6 months post-MI.
[0318] Despite its utility in the clinical assessment of transplant
rejection, detection of small foci of rejection by H&E staining
is complicated in a post-MI setting due to the natural inflammatory
response to the ischemic insult. In order to corroborate the
histochemical data, immunostaining against a variety of immune cell
markers was performed for each time point. This approach allows a
characterization/identification of the infiltrating inflammatory
cells. After allogeneic transplants, immunohistochemistry revealed
rare events of rejection. A few small and sparse infiltrates around
some transplanted cells were detected 3 weeks post-treatment. See
for example FIG. 28H, 28I, and 28J. These infiltrates comprised
mainly CD3+ T lymphocytes (with equal contributions of CD8+ T
cytotoxic and CD4+ T helper subpopulations) and to a lesser extent
CD45R+ B lymphocytes and CD11c+ dendritic cells (see FIG.
28K.sub.1-28K.sub.15 and also 29A-29C, which depict syngeneic,
allogeneic, and xenogeneic transplants, respectively). Based on the
similar quantities of CD4+ and CD8+ T lymphocytes in the graft
area, as well as the presence of dendritic cells, it is possible
that an indirect pathway of allorecognition plays a greater role in
the immune rejection of transplanted cells. For example, antigens
shed by apoptotic donor CDCs may be phagocytosed by host antigen
presenting cells (like dendritic cells) and subsequently presented
to CD4+ cells, thus activating the immune cascade. However, in some
cases, participation of the direct pathway of allorecognition also
likely plays a role. Notably, the increased lymphohistiocytic
infiltration was significantly lower than that seen with xenogeneic
transplantation (FIGS. 28L and 28M; and also FIGS. 29D and 29E),
and had completely resided by 6 months (data not shown). The higher
infiltration of CD68+ macrophages (which in general did not
localize within the infiltrates but were evenly dispersed along the
infarct) detected at 1 and 3 weeks post MI in the xenogeneic and
control groups was consistent with the larger infarct size observed
in those groups.
[0319] Furthermore, there were no significant signs of systemic
immunogenicity (FIGS. 30A-30G) in the animals who received
allogeneic CDCs as evidenced by serum concentrations of
pro-inflammatory cytokines IFN-.gamma., IL-1.beta., KC/GRO,
TNF-.alpha., (FIGS. 30A, 30B, 30F, and 30G, respectively), or the
anti-inflammatory cytokines IL-13, IL-4, and IL-5 (FIGS. 30C, 30D,
and 30E, respectively). In contrast, in the xenogeneic transplants,
the circulating levels of IFN-.gamma., IL1.beta., IL13 and IL4 were
markedly increased. Taken together, these data indicate that the
systemic inflammatory response observed after xenogeneic
transplantation did not occur in the allogeneic setting. Thus, in
some embodiments, allogeneic transplants provide the unexpectedly
beneficial combination of functional and morphological improvements
in damaged cardiac tissue, without the expected immune response.
For these reasons, among others disclosed herein, allogeneic cells
are particularly advantageous in some embodiments.
Allogeneic CDCs Elicit a Cellular but Not a Humoral Response
Memory
[0320] In order to assess the development of cellular memory immune
response after allogeneic CDC transplantation, the alloreactivity
of lymphocytes isolated from spleens of allogeneic recipients 3
weeks post-transplantation was assessed by one-way mixed lymphocyte
reactions. Lymphocytes from sensitized animals exhibited higher
proliferation after coculture with allogeneic CDCs (stimulator
index 2.32.+-.0.52), compared to naive lymphocytes (p<0.05) or
syngeneic cocultures (p<0.01). In addition, markedly increased
levels of inflammatory cytokines in the supernatants of sensitized
lymphocyte cocultures were detected by electrochemiluminescence and
ELISA. These results are indicative of a T cell memory response and
are in accordance with the immunohistochemistry data discussed
below, which shows a predominant role of T cells in the sparse
mononuclear infiltrates observed 3 weeks post allogeneic
transplantation (FIG. 28C).
[0321] In order to assess the development of a humoral memory
response, recipient rat sera obtained 1 and 3 weeks
post-transplantation were screened for circulating anti-donor
antibodies. No alloreactive antibodies were detected in any
recipients of allogeneic CDCs at any timepoint. This finding is in
contrast to the xenogeneic transplants in which high titers of
xenoreactive IgM antibodies were detected 1 and 3 weeks post
transplantation. Additionally, a progressive increase of
xenoreactive IgG antibodies was observed from week 1 to week 3. The
development of anti-donor antibodies in xenogeneic recipients, but
not in allogeneic recipients, is consistent with the significantly
higher (.about.8 fold) B cell myocardial infiltration observed in
the xenogeneic setting (FIGS. 28L-28M).
Efficacy of Allogeneic CDCs in Rat Model
[0322] Morphometric analysis of explanted hearts 3 weeks post
infarction showed severe LV chamber dilatation and infarct wall
thinning in animals in the xenogeneic and control groups (FIG. 31A,
bottom row). In contrast, the syngeneic and allogeneic groups
exhibited smaller scar size, increased infarcted wall thickness and
attenuation of LV remodeling (FIGS. 31A). Scar size and infarcted
wall thickness did not differ among animals treated with syngeneic
or allogeneic CDCs, which indicates, in some embodiments, that
similar physical (e.g., treatment effects are obtained whether
autologous or allogeneic cells are used. (FIGS. 31B-31C).
[0323] To assess functional benefit of CDC transplantation, global
cardiac function was assessed by echocardiography, quantifying
fractional area change (FAC), left ventricular ejection fraction
(LVEF), and fractional shortening (FS). At baseline (D0), FAC, LVEF
and FS did not differ among treatment groups, indicating a similar
degree of initial injury. Over the first 3 weeks after infarction,
indices of function failed to improve in the xenogeneic and control
groups, whereas FAC, LVEF and FS all rose significantly in both the
allogeneic and syngeneic groups (FIGS. 31D-31F). Notably, the
functional benefit observed at 3 weeks persisted out to 6 months
post infarction in the allogeneic and autologous groups, but not in
xenogeneic or control group. A treatment effect summary is shown in
FIG. 31G, which show that, despite their lower engraftment at 3
weeks, allogeneic cells are equivalent to syngeneic (which are
modeling autologous cells in this experiment) in terms of providing
functional repair to damaged cardiac tissue
[0324] Both syngeneic and allogeneic CDCs led to similar
improvements in cardiac function in the rat model 3 weeks, 12
weeks, and 6 months post-MI (FIG. 31). Both syngeneic and
allogeneic groups differed significantly from control animals at
each timepoint. The xenogeneic group showed a decline in function
at 3 weeks post-MI that is significantly different from the result
seen in the syngeneic and allogeneic groups. This study indicates
that allogeneic CDC transplantation without immunosuppression is
safe and improves heart function in a rat model without the need
for persistent cell engraftment.
[0325] To further investigate possible mechanisms of the benefits
provided by allogeneic transplants, the fate of the transplanted
cells themselves as well as indirect mechanisms of benefit were
evaluated. Immunohistochemistry revealed that syngeneic and
allogeneic CDCs primarily resided in the border zone and infarct
scar. A subset of administered cells were found have reentered the
cell cycle at 1 and 3 weeks post-MI, as indicated by Ki-67
positivity and BrdU incorporation. Rare events of cardiomyogenic
(GFP+/.alpha.SA+ cells) and angiogenic (GFP+/vWf+ cells)
differentiation of surviving CDCs could be detected in both the
syngeneic and the allogeneic setting. While the majority of
GFP+/.alpha.SA+ cells were small and exhibited an immature
cardiomyocyte phenotype (FIG. 31H), mature GFP/.alpha.SA+ cells
structurally integrated into the host myocardium could also be
detected (FIG. 31I, white arrow). In addition, GFP+/vWf+ cells were
found to be incorporated in microvessels in the risk region (FIG.
31J, white arrows). These results demonstrate the multilineage
potential of CDCs, e.g., that CDCs can generate the various cell
types needed for complete cardiac repair. However, direct
differentiation of administered cells was low, and thus, in some
embodiments, unlikely to fully account for the observed robust
functional benefit of allogeneic cell transplant. Thus, in some
embodiments, the direct effect of administered cells is only
partially responsible for the observed benefits (e.g., physical and
functional cardiac repair).
[0326] Endogenous cardiac regeneration is another possible
mechanism and was therefore evaluated. Endogenous regeneration may
involve one or more of endogenous cardiomyocyte cell cycle
re-entry, recruitment of endogenous progenitor cells to the site of
cell transplantation, and/or enhanced angiogenesis. Both syngeneic
and allogeneic CDC therapy stimulated resident cardiomyocyte
cell-cycle re-entry. The number of cycling host cardiomyocytes
(GFP-/.alpha.SA+/Ki67+ and GFP-/.alpha.SA+/BrdU+ cells) was
markedly increased in CDC-treated hearts compared to controls
(FIGS. 31K-31M, 31N-31P, 31W, and 31X) at 1 and 3 weeks post MI.
However, the number of GFP-/.alpha.SA+/Ki67+ and
GFP-/.alpha.SA+/BrdU+ cells significantly decreased from 1 week to
3 weeks, dropping to nearly undetectable levels at 6 months,
suggesting that, in some embodiments, the re-entry of endogenous
cardiac cells into the cell cycle is perhaps an acute effect. In
other embodiments, however, this effect may be longer-lasting.
Syngeneic and allogeneic CDC transplantation also recruited
endogenous stem cells (FIG. 31Q-31S and 31Y) as evidenced by the
increased number of GFP-/c-Kit.sup.+ in CDC-treated hearts compared
to controls at 1 week and 3 weeks post MI. As with resident cycling
myocytes, the number of endogenous progenitors significantly
decreased as a function of time.
[0327] Finally, it was determined that both syngeneic and
allogeneic CDC transplantation enhanced angiogenesis in the infarct
border zone. Vessel density, identified by immunostaining for vWf,
was markedly increased 3 weeks after cell therapy compared to
controls (FIGS. 31T-31V and 31Z). While control hearts did display
some activity in these endogenous reparative mechanisms, both
syngeneic and allogeneic showed a greater magnitude than that of
control cells.
[0328] These data thus indicate that either syngeneic or allogeneic
transplants yield functional improvements, while control groups
lose cardiac function over time. Additionally, in vivo data
evaluating infarct size indicate that infarct size is significantly
smaller in both cell therapy groups compared to controls. See,
e.g., FIG. 25. Additionally, the data presented herein suggests
that exogenous CDC administration stimulates activation of
endogenous repair and/or regeneration pathways. Thus, indirect
mechanisms may be, in some embodiments, largely responsible for the
observed benefit following CDC therapy.
[0329] To further investigate the indirect effects, myocardial
levels of beneficial paracrine factors in the infarct border zone
were analyzed. Western Blot analysis revealed increased secretion
of VEGF, IGF-1 and HGF in hearts treated with syngeneic and
allogeneic CDCs, compared to controls, at day 1, day 4 and day 7
post MI (FIG. 31AA-DD). 3 weeks post MI, no difference in secretion
of these factors could be observed among groups. This indicates
that syngeneic and allogeneic CDCs are equivalent in terms of their
generated paracrine effects, both in magnitude and in time course.
In some embodiments, the factors evaluated above are primary
players in the indirect repair effect, however, in some
embodiments, other factors (described elsewhere herein) may also
play a role.
[0330] Both rat and human CDCs show similar patterns of MHC
expression at baseline and after IFN stimulation. As with the human
CDCs, IFN stimulation induces upregulation of rat CDC MHC I, II
molecules. Co-culture of rat CDCs with allogeneic lymphocytes
induces lymphocyte proliferation and secretion of pro-inflammatory
cytokines. However, the level of immune response is significantly
lower when compared to xenogeneic co-culture.
[0331] In vivo, rat syngeneic and allogeneic CDCs demonstrate
similar survival rates at Day 8 after experimentally induced MI. At
Day 21, cell survival after syngeneic transplantation is
significantly higher. This suggests that the pro-immunogenic
characteristics that CDCs display in vitro, induce an in vivo
immune response between day 9 and day 21. Regardless, overall cell
survival is poor in both syngeneic and allogeneic groups. It is
possible, that more than one mechanism of cell death is involved,
depending on the group. For example, higher levels of apoptosis may
be involved in reducing the overall survival of the allogeneic
cells while a lesser amount of apoptosis couples with increased
necrosis may account for cell loss in the syngeneic group.
[0332] Despite the significantly reduced number of cells present
after 21 days, both syngeneic and allogeneic CDC transplantation
led to significant improvement of LV function after MI, as compared
to controls. The treatment effect is similar between the two types
of transplants at day 21. Moreover, infarct size is reduced in the
two groups as compared to controls. This suggests that, as
discussed above, an early, but long-lasting effect is induced by
the transplant of cells into damaged myocardium. It is possible
that the paracrine effects described in the prior Examples are
responsible for inducing a cascade of events that serve to improve
cardiac function by repairing damaged tissue or generating new,
functional tissue. It is also possible that these early signals
recruit endogenous cardiac stem cells that augment cardiac function
and induce tissue repair. In either case, the data presented here
indicate that the presence of viable transplanted cells is not a
necessary precursor to improved cardiac function and/or tissue
repair. It appears that the transplanted cells function as a
trigger, and thus, in some embodiments, induce and/or recruit
repair mechanisms, then are removed by various cell death pathways,
including, but not limited to phagocytosis, autophagy, apoptosis,
enzymatic degradation, among others. In this light, the allogeneic
cell transplant becomes even more attractive, not only because of
the practical advantages discussed above, but because the life span
of the transplanted cells appears to be short enough that the
induced immune responses are not significant enough to interfere
with functional repair of the damaged tissue.
Example 12
Pre-Clinical Trials Using Autologous Cells
[0333] Cardiosphere-derived cells (CDCs) and their 3-dimensional
precursors, cardiospheres were tested, according to several
embodiments of the invention, for cellular cardiomyoplasty in a
mini-pig model of heart failure post-myocardial infarction (MI).
Although porcine studies were conducted, data can be extrapolated
to human patients according to several embodiments of the
invention.
[0334] According to one embodiment, autologous cardiospheres or
CDCs grown from endomyocardial biopsies were injected via
thoracotomy four weeks post-anteroseptal MI. Engraftment
optimization with luciferase-labeled CDCs guided the choice of cell
dose (0.5M cells/site) and target tissue (20 periinfarct sites).
Pigs were randomized to placebo (n=11), cardiospheres (n=8) or CDCs
(n=10). Functional data were acquired before injection and again 8
weeks later, after which organs were harvested for histopathology.
Beyond the immediate perioperative period, all animals survived to
protocol completion. Ejection fraction was equivalent at baseline
but, at 8 weeks, was higher than placebo in both of the
cell-treated groups (placebo vs. CDC p=0.01; placebo vs.
cardiospheres p=0.01). Echocardiographic and hemodynamic indices of
efficacy improved disproportionately with cardiospheres. Likewise,
adverse remodeling was more attenuated with cardiospheres than with
CDCs. Provocative electrophysiologic testing showed no differences
among groups, and no tumors were found.
[0335] Thus, according to several embodiments, dosage-optimized
direct injection of cardiospheres or CDCs is safe and effective in
preserving ventricular function in ischemic cardiomyopathy. In one
embodiment, CDCs and cardiospheres have equivalent effects on LVEF.
In some embodiments, cardiospheres are superior in improving
hemodynamics and regional function, and in attenuating ventricular
remodeling.
[0336] In several embodiments of the invention, the regenerative
cells delivered to patients are cardiospheres. In other
embodiments, the regenerative cells are CDCs. In yet other
embodiments, the regenerative cells are a combination of
cardiospheres and CDCs. In some embodiments, regenerative cells are
useful for treating dysfunction (e.g., left ventricular
dysfunction) post-MI.
[0337] In some embodiments, CDCs delivered to subjects (e.g.,
non-human mammals, human patients) are a heterogeneous mix of cells
expanded from cardiac tissue, with formation of self-assembling
spherical clusters of heart-derived cells (cardiospheres) as an
intermediate processing step. In several embodiments, CDCs are
clonogenic and exhibit multi-lineage potential, thus fulfilling key
criteria for cardiac stem cells, and they can be readily and
reliably expanded from tiny specimens of heart muscle. According to
one embodiment, approximately 20 mg samples yield about 1.5 million
CDCs on average within 45 days.
[0338] In one embodiment, cardiospheres having a size of about
50-200 .mu.m in diameter are used. In one embodiment, delivery
mechanisms other than intracoronary administration are used to
reduce the risk of embolization at the arteriolar level. For
example, in one embodiment, cardiospheres are delivered by
intramyocardial injection. In one embodiment, cardiospheres having
a size of about 50-150 .mu.m in diameter are used. In several
embodiments, CDCs are particularly advantageous due to their size
being less than that of cardiospheres. In some embodiments, CDCs
are of a size that is associated with little to no risk of
embolization of at the arteriolar level. For example, in several
embodiments, CDCs are less than about 50 .mu.m in diameter. In some
embodiments, CDCs are less than about 40 .mu.m in diameter, less
than about 30 .mu.m in diameter, less than about 20 .mu.m in
diameter, and less than about 10 .mu.m in diameter. In some
embodiments, CDCs range from about 5-10 .mu.m in diameter, about
6-11 .mu.m in diameter, about 7-12 .mu.m in diameter, about 8-13
.mu.m in diameter, about 9-14 .mu.m in diameter, about 10-15 .mu.m
in diameter, about 11-16 .mu.m in diameter, about 12-17 .mu.m in
diameter, about 13-18 .mu.m in diameter, about 14-19 .mu.m in
diameter, about 15-20 .mu.m in diameter, and overlapping ranges
thereof In some embodiments, CDCs are less than about 75% of the
size of a cardiosphere. In some embodiments, CDCs are less than
about 50% of the size of a cardiosphere. In some embodiments, CDCs
are less than about 25% of the size of a cardiosphere. In some
embodiments, CDCs range from about 75% to about 70% of the size of
a cardiosphere, from about 70% to about 65% of the size of a
cardiosphere, from about 65% to about 60% of the size of a
cardiosphere, from about 60% to about 55% of the size of a
cardiosphere, from about 55% to about 50% of the size of a
cardiosphere, from about 50% to about 45% of the size of a
cardiosphere, from about 45% to about 40% of the size of a
cardiosphere, from about 40% to about 35% of the size of a
cardiosphere, from about 35% to about 30% of the size of a
cardiosphere, from about 30% to about 25% of the size of a
cardiosphere, from about 25% to about 20% of the size of a
cardiosphere, from about 20% to about 15% of the size of a
cardiosphere, from about 15% to about 10% of the size of a
cardiosphere, from about 10% to about 5% of the size of a
cardiosphere, from about 5% to about 1% of the size of a
cardiosphere, and overlapping ranges thereof. In several
embodiments, CDCs are particularly advantageous because of their
size, which, being less than that of cardiospheres, enables a
greater degree of engraftment, which, in some embodiments,
increases the beneficial effects of CDC administration, whether due
to the engraftment itself or an increased paracrine effect related
thereto. It shall be appreciated, that the source of the CDCs, be
it an autologous, xenogeneic, or allogeneic source, does not
substantially impact the advantages realized in certain embodiments
due to the smaller size of CDCs.
[0339] Two studies were performed to examine regenerative cells
according to several embodiments of the invention. Study 1 (FIG.
32A) consisted of open-label experiments to quantify engraftment 24
hours after intramyocardial CDC injection in the porcine MI model.
The engraftment data were used to inform the dosage and target
tissue of injection of CDCs for Study 2 which was a pivotal
placebo-controlled, blinded randomized study of safety and efficacy
of either cardiosphere or CDC direct intramyocardial injection.
(FIG. 32B).
[0340] Animals were randomized to receive either placebo or about
10 million cells in cardiosphere or CDC form, administered as 20
injections of 0.5 million per site. Other cell and/or injection
numbers are used in other embodiments. The dose of 10 million cells
was selected on the basis of the engraftment data from study 1,
which had demonstrated, according to one embodiment, that the
highest percentage engraftment of cells occurred when a smaller
number was injected at each site in the periinfarct border zone.
(FIG. 33). Cells or placebo were injected under direct
visualization by open chest surgery performed 4 weeks after MI. The
pigs were then followed for eight more weeks, to assess safety and
efficacy. Thus, in one embodiment, injections are made in the
periinfarct border zone. In other embodiments, injections are made
outside of said zone.
[0341] General anesthesia was induced in adult female Yucatan
mini-pigs. Endotracheal intubation was then performed and
anesthesia maintained. The mini-pigs were subjected to an
anteroseptal MI by inflation of an angioplasty balloon in the
mid-LAD to cause coronary occlusion for 2.5 hours. Catheters were
inserted via the left carotid artery. After reperfusion, during the
same episode of general anesthesia, 4-6 right ventricular biopsies
were obtained using a standard clinical cardiac bioptome introduced
via the right internal jugular vein. The biopsies were immediately
placed into ice-cold cardioplegia solution (e.g., Ca.sup.++ and
Mg.sup.++ free PBS with 5% dextrose, mannitol 68.6 mmol/L, KCl 1.6
mmol/L, NaHCO.sub.3 3.1 mmol/L and heparin) and cardiospheres or
CDCs were grown from these biopsy samples.
[0342] According to several embodiments of the invention, cardiac
biopsy specimens (10-40mg) were minced, and subjected to
collagenase IV digestion. These explants were plated onto
fibronectin-coated plastic plates with cardiac explant medium (IMDM
(Invitrogen), 20% FBS, 1% penicillin-streptomycin, 1% L-glutamine,
0.1mM 2-mercaptoethanol). A monolayer of adherent cells grew out
from the biopsy, which was harvested after 1-2 weeks. The harvested
outgrowth was re-plated onto poly-D-lysine coated wells. Under
these conditions, within 3-5 days the majority of the cells gave
rise to free-floating clusters of cells (e.g., cardiospheres). In a
third phase, the adherent cells were discarded, while the floating
cardiospheres were collected and plated once again onto
fibronectin-coated cellware. The cardiospheres adhered and
flattened to form a monolayer of cells referred to as CDCs, which
were passaged as they became confluent. So called "secondary
cardiospheres" were used in the in vivo experiments, meaning that
an equivalent number of CDCs (e.g., about 10 million) were
harvested and counted, then plated back into poly-D-lysine coated
wells where they formed cardiospheres for a second time, which were
injected into the animals.
[0343] Cultured cells were transduced at the outgrowth stage with a
lentiviral vector encoding the firefly luciferase gene, and further
processed to create CDCs. Seven animals that had been subjected to
the MI and RV biopsy protocols, received intramyocardial injection
of 0.5, 2.0 or 10 million CDCs per injection site in intrainfarct,
periinfarct (borderzone) or remote normal ventricular locations.
(See FIG. 33) Other dosages may be used according to other
embodiments of the invention. The animals were sacrificed 24 hours
later for assessment of cell engraftment.
[0344] Thirty-three pigs had general anesthesia induced a second
time, 4 weeks after MI, with the same drugs. Intramyocardial direct
injection was performed by open chest surgery under sterile
conditions. Sternotomy was performed and the pericardium opened to
expose the heart. Twenty intramyocardial injection of either
cardiospheres (0.5 million cells suspended in 0.1 mL per
injection), CDCs (0.5 million cells suspended in 0.1 mL per
injection) or placebo (0.1 mL of medium alone) were performed into
the beating heart, using a 1 mL tuberculin syringe and 26 gauge
needle. The injections were spaced around the perimeter of the
macroscopically visible infarct scar, approximately lcm from the
gross border. Other injection sites are used in according with
other embodiments of the invention. 11 pigs were allocated to
receive placebo (vehicle alone), 8 allocated to receive
cardiosphere injections, and 10 allocated to receive CDC
injections.
[0345] About 0.5M cells/site was administered per site in the
periinfarct zone for the study 2. Quantification of off-target
expression at 24 hrs revealed no measurable cells in liver, spleen
or kidney, but 0.9% of injected CDCs could be detected in the lungs
(see Table 4.) In several embodiments, the percentage retained in
the heart can be increased by iron-loading cells and applying an
apical magnet, as described in PCT Application No.
PCT/US2010/054358, the disclosure of which is herein incorporated
by reference.
TABLE-US-00004 TABLE 4 Cell distribution in non-target tissues. Pig
1 Pig 2 Pig 3 Pig 4 Pig 5 Pig 6 Pig 7 Lung 1.8% 0.3% 2.4% 0.6% 0.6%
0.3% 0.4% Liver -- -- -- -- -- -- -- Spleen -- -- -- -- -- -- --
Kidney -- -- -- -- -- -- --
[0346] On the basis of the 24 hour engraftment data, a dosage of
0.5M CDCs (or an equivalent cell number of cardiospheres) per site
was selected, and direct intramyocardial injections performed in 20
periinfarct sites, giving a total cell dosage of 10 million CDCs.
FIGS. 34A shows the LVEF data derived from contrast
ventriculography. LVEF at baseline was equivalent in the three
groups. FIG. 34B also demonstrates that, eight weeks
post-injection, LVEF was significantly higher than placebo in both
of the cell treated groups, while there was no significant
difference in final LVEF between the CDC and the
cardiosphere-treated groups. FIG. 34C shows that the treatment
effect (final minus baseline LVEF) was significantly higher than
placebo in both of the cell treated groups. Thus, according to some
embodiments of the invention, cardiac function is improved when
CDCs are administered. In some embodiments, cardiospheres improve
cardiac function.
[0347] Echocardiographic measurement of LVEF yielded qualitatively
similar differences in final LVEF and delta LVEF measurements,
though the differences between the groups were not statistically
significant by this modality (Table 5). Echocardiographic
measurement did, however, demonstrate progressive ventricular
dilatation in placebo and CDC groups, which was attenuated in the
cardiosphere-treated animals (FIG. 35B and Table 5). Baseline
systolic and diastolic LV volume measurements were randomly lower
in the CDC-treated animals (Table 5).
TABLE-US-00005 TABLE 5 Echocardiographic Indices Placebo CDC CSph p
values: Placebo Placebo CDC (n = 9) (n = 9) (n = 5) ANOVA vs. CDC
vs. CSph vs. CSph Ejection fraction Baseline 43 .+-. 7 44 .+-. 12
43 .+-. 5 0.98* -- -- -- Final 40 .+-. 7 47 .+-. 5 44 .+-. 5 0.07
-- -- -- Treatment effect (delta) -3 .+-. 11 +3 .+-. 10 +1 .+-. 5
0.39 -- -- -- systolic volume, mL Baseline 29.5 .+-. 4.8 24.7 .+-.
5.0 31.4 .+-. 3.9 0.04 0.04 0.49 0.02 Final 40.5 .+-. 11.8 34.7
.+-. 7.231.8 .+-. 5.6 0.21 -- -- -- -- Treatment effect (delta)
+10.9 .+-. 13.2 +10.0 .+-. 6.2 +0.4 .+-. 5.4 0.13 -- -- --
Diastolic volume, mL Baseline 52.0 .+-. 9.4 44.2 .+-. 5.3 55.6 .+-.
9.8 0.04 0.054 0.44 0.02 Final 66.1 .+-. 12.9 65.0 .+-. 10.7 56.2
.+-. 7.8 0.27 -- -- -- Treatment effect (delta) +14.0 .+-. 15.0
+20.8 .+-. 10.1 +0.7 .+-. 8.5 0.02 0.25 0.06 <0.01 Abbreviation:
CSph, cardiospheres *Baseline Ejection fraction did not exhibit
homogeneity of variance between groups, so the Kruskal-Wallis test
was performed instead of ANOVA
[0348] In addition, echocardiography revealed that final
measurements of LV septal wall thickness were increased in both of
the cell-injected groups relative to placebo (FIG. 36B). The
thickening fraction of the apical septum was also increased in
cardiosphere-injected pigs. (FIG. 36C). Thus, in one embodiment
cardiospheres are particularly efficacious. According to some
embodiments, the invention provides improve morphology and function
in the infarct region with autologous CDC or cardiosphere
injections. In other embodiments, the invention provides improve
morphology and function in the infarct region with allogeneic CDC
or cardiosphere injections.
[0349] Table 6 and FIG. 35A outline the results of LV
pressure-volume loop analysis. Most measurements were made at
steady state. However one important measurement, end-systolic
elastance (Emax) was derived, by definition, as the slope of the
end-systolic pressure-volume relationship from the family of loops
produced during balloon occlusion of the inferior vena cava. (FIG.
35A). Emax is a rigorous load-independent measure of contractility.
Final Emax in the cardiosphere group was higher than in
placebo-treated pigs, indicating improved left ventricular
contractility in these animals (placebo 1.03.+-.0.29, CDC
1.66.+-.0.45, cardiosphere 3.16.+-.1.32 mmHg/mL. Kruskal-Wallis
p=0.003. Placebo vs. CDC, p=NS; Placebo vs. cardiosphere, p=0.03;
Cardiosphere vs. CDC, p=NS). Emax in the CDC group tended to
increase but was not significantly higher than in placebo-treated
animals.
TABLE-US-00006 TABLE 6 Pressure-Volume Loop derived Indices Placebo
CDC Cardiosphere (n = 11) (n = 10) (n = 7) ANOVAp Heart rate, bpm
Baseline 121 .+-. 12 119 .+-. 12 117 .+-. 17 0.72 Final 112 .+-. 17
109 .+-. 13 117 .+-. 19 0.65 Treatment effect -9 .+-. 16 -9 .+-. 12
-1 .+-. 36 0.72** (delta) P.sub.max mmHg Baseline 97.0 .+-. 11.5
86.9 .+-. 6.9 93.8 .+-. 14.6 0.13 Final 87.1 .+-. 11.4 88.4 .+-.
10.4 86.2 .+-. 8.1 0.90 Treatment effect -9.9 .+-. 12.9 +1.5 .+-.
11.2 =7.9 .+-. 16.0 0.14 (delta) LVEDP, mmHg Baseline 14.6 .+-. 3.1
14.5 .+-. 5.1 18.0 .+-. 5.3 0.20 Final 12.6 .+-. 3.7 16.0 .+-. 6.2
12.1 .+-. 4.1 0.19 Treatment effect -1.9 .+-. 3.4 +1.5 .+-. 4.4
-5.8 .+-. 1.7 0.01* (delta) dP/mt max Baseline 1967 .+-. 370 17709
.+-. 278 1784 .+-. 693 0.56 Final 1589 .+-. 446 1430 .+-. 370 1422
.+-. 333 0.58 Treatment effect -378 .+-. 462 -340 .+-. 280 -382
.+-. 876 0.93 (delta) dP/dt min Baseline -1984 .+-. 480 -1584 .+-.
346 -1784 .+-. 394 0.11 Final -1516 .+-. 291 -1527 .+-. 357 -1546
.+-. 444 0.99 Treatment effect 468 .+-. 600 56 .+-. 501 247 .+-.
520 0.25 (delta) tau, seconds Baseline 39.84 .+-. 4.87 38.93 .+-.
5.51 41.28 .+-. 7.21 0.70 Final 41.25 .+-. 7.21 40.92 .+-. 4.57
41.88 .+-. 3.82 0.94 Treatment effect +1.41 .+-. 8.39 +1.98 .+-.
6.12 -0.03 .+-. 5.42 0.84 (delta) Abbreviations: P.sub.max, the
maximum pressure generated by the left ventricle during the cardiac
cycle; LVEDP, left ventricular end-diastolic pressure; dP/dt max,
the maximum rate of rise of left ventricular pressure; tau, a
measure of left ventricular relaxation. *For further details about
post-hoc comparisons of delta LVDDP between the three groups,
please refer to FIG. 35C. **The delta heart rate variable did not
exhibit homogeneity of variance between groups, so the
Kruskal-Wallis test was performed instead of ANOVA.
[0350] Steady state hemodynamics showed few differences (Table 6)
except for a greater fall in LV end-diastolic pressure in the
cardiosphere-treated group (FIG. 34C). Taken together with the
lesser increase of end-diastolic volume in this group, cardiosphere
injected animals experience disproportionate benefit with regard to
attenuation of adverse ventricular remodeling relative to the other
two groups (CDCs or placebo).
[0351] Ventricular tachycardia was readily inducible by application
of programmed extra-stimuli in all animals prior to sacrifice,
consistent with previous reports. However, there were no deaths
(sudden or otherwise) in either group after the immediate
periprocedural period. Necropsy, with gross analysis as well as
histology of heart, brain, kidney, lung, liver and spleen (Table 3)
detected no tumors eight weeks after intramyocardial injection of
CDCs or cardiospheres.
[0352] Fluorescence immuno-histochemistry in the two animals with
lacZ.sup.+ CDCs, revealed the presence of labeled cells 8 weeks
after injection. FIGS. 37A-37B shows two examples of islands of
cardiomyocytes with lacZ-positive nuclei in the periinfarct zone,
one from each animal that received intramyocardial
genetically-labeled CDCs. Thus, in one embodiment, a proportion of
injected autologous CDCs, or their progeny which will also be
labeled by this integrating vector, persist for 8 weeks within the
border zone of infarcted myocardium. In some embodiments, human
cardiospheres have improved engraftment as compared to human CDCs
when injected into SCID mouse hearts under certain conditions and
delivery mechanisms.
[0353] In several embodiments, direct surgical injection of
autologous cardiospheres or CDCs effectively halts the
deterioration in LVEF after a large myocardial infarction, compared
to a 7% absolute reduction in LVEF over eight weeks of observation
in placebo-treated animals. Cardiospheres increased end-systolic
elastance and attenuated the ventricular dilatation associated with
myocardial infarction. Although not demonstrated in the study,
CDC's may also exhibit similar effects under certain
conditions.
[0354] According to several embodiments, short-term engraftment of
about 8% regardless of injected cell dose in remote normal
myocardium is feasible. In some embodiments, in the infarct border
zone, the percent survival at 24 hrs decreases progressively from
.about.8% to <1% as dosage escalates. Thus, in one embodiment,
the proportional engraftment of injected cells is improved by
injection of lower cell doses at each injection site. In one
embodiment, survival in the border zone may be limited by the
tenuously-perfused, substrate limited, peri-infarct environment. In
this scenario, while the absolute number of injected cells able to
survive remains about the same, percentage survival of injected
cells is greater with injection of lower numbers of cells per
site.
[0355] In some embodiments, engraftment rates are from 5%-10%,
10%-20%, 20-50%, 50-75% and higher. In several embodiments, the
following dosages of CDCs or cardiospheres per site are used:
0.1-0.2M, 0.2-0.3M, 0.3-0.4M, 0.4-0.5M, 0.5-0.6M, 0.6-0.7M,
0.7-0.8M, 0.8-0.9M, 0.9-1.0M, 1.0-1.5M, 1.5-2.0M, and higher, or
overlapping ranges thereof. The following number of injection sites
are used in some embodiments: 1, 1-5, 5-10, 10-15, 15-20, 20-25,
25-50, and more sites, and overlapping rages thereof.
[0356] In some embodiments, the preservation of global LVEF in
cell-treated animals, compared to the deterioration in the placebo
group is provided with intramyocardial injection. In several
embodiments, cardiospheres are used to provide hemodynamic benefit
and/or attenuation of adverse remodeling.
[0357] In some embodiments, CDCs comprise a natural mixture of
progenitor and support cells expanded from myocardial biopsy
specimens, with clonogenicity and multi-lineage potential. In one
embodiment, CDCs provide significant functional improvements even
when engraftments rates are low. In one embodiment, functional
benefit involves indirect effects to boost angiogenesis and
cardiomyogenesis.
[0358] In some embodiments. regenerative cells described herein
(e.g., allogeneic or autologous cardiospheres and CDCs,) can be
administered in procedures where open chest surgery is performed
(e.g., for other therapy, such as implantation of cardiac devices).
In some embodiments, less invasive methods of intramyocardial
administration, such as trans-endocardial catheter-mediated
delivery are used. In one embodiment, regenerative cells are
administered via an intracoronary route.
Example 13
Clinical Trials Using Autologous Cells
[0359] The preliminary safety and efficacy of autologous CDCs in
patients with ischemic left ventricular dysfunction and a recent
myocardial infarction have been evaluated in a Phase I clinical
trial (CADUCEUS, NCT00893360, which is incorporated by reference
herein). Twenty-four patients (at least 18 years of age with recent
myocardial infarction and ischemic left ventricular dysfunction)
have or are scheduled to undergo a cardiac biopsy to obtain tissue
for generating the cell product. Autologous cardiosphere-derived
stem cells, with a low dose of 12.5 million and a high dose of 25
million, are generated. Within 8 weeks of biopsy, and on average
within 4 weeks, patients receive CDCs by intracoronary infusion in
the infarct-related artery.
[0360] A total of 14 subjects have completed the 6 month follow up.
In the 12.5 million cell group, 4 subjects who received cells, 4
control subjects and 1 intention to treat subject have completed
their 6 month visit. In the 25 million cell group, 3 subjects who
received cells, 1 control subject, and 1 intention-to-treat subject
have completed their 6 month visit.
[0361] Magnetic resonance imaging (MRI) is being used to assess
secondary efficacy endpoints, including: changes in MM-assessment
of function in the region which received CDC therapy; changes in
MRI assessment of infarct size expressed in absolute value (grams)
and as a percent of LV mass; changes in MRI assessment of perfusion
in the region which received CDC therapy; changes in MM assessment
of global LV function; and changes in MRI assessment of LV
end-diastolic and end-systolic volumes.
[0362] In terms of efficacy, the data sets are incomplete. Results
to date have been summarized in tabular format below. Six-month
follow-up MRIs have been completed for a subset of the study
subjects: all 4 patients randomized to receive 12.5 M CDCs and 5
controls.
[0363] Although preliminary, the results to date are encouraging,
in that the relative changes in infarct size by % LV infarcted are
already showing differences between CDCs and controls. At baseline,
i.e. before cell infusion, infarct size was equivalent in the two
groups (CDC 23.22% vs. Control 24.78%, p=0.84). By six months,
those values trended in opposite directions, CDCs getting smaller,
as expected from the preclinical data (please refer to IND #13930
for details), and controls creeping upwards (CDC 17.14% vs. Control
28.97%, p=0.12). Comparison of the deltas (baseline minus 6 months)
reveals a significant difference (CDC -6.08% vs. Control+4.2%,
p=0.021).
[0364] The p values for absolute infarct mass are now nearly
significant when comparing infarct mass between the groups at 6
months, and for the comparison of the change in infarct mass:
Infarct Mass (g) Baseline: CDC 26.24 g vs. Control 28.95 g, p=0.78
6 months: CDC 19.34 g vs. Control 32.07 g, p=0.0684 Delta: CDC -6.9
g vs. Control+3.12 g, p=0.0676
Example 14
Protocols for Manufacturing of CDCs
Cell Processing
[0365] A schematic showing one embodiment of cell processing is
shown in FIG. 38A-38F. In one embodiment, hearts will be collected
from donors and transported to the manufacturing facility. As
described above the tissue will go through three stages: 1)
explants (EXP) stage: cell outgrowth from the tissue; 2)
cardiospheres (CSP) stage: enrichment of cardiac stem cells by
culturing; and 3) cardiosphere-derived cells (CDCs) stage:
expansion of cells. A master cell bank (MCB) and working cell bank
(WCB) of CDCs will be created as described below. In several
embodiments, aliquots of the WCB equivalent to a single drug dosage
will be formulated as the final composition to be administered.
Cell Banking System
[0366] In several embodiments, a cell banking system is provided
comprising a plurality of cryogenically preserved, single use,
populations of CDCs for administration. In some embodiments, a
master cell bank is provided. In some embodiments, a working cell
bank is additionally provided (with CDCs that have undergone a
greater number of passages). The disclosure herein details an
example of a contemplated cell banking system.
[0367] In several embodiments, endomyocardial biopsies will be used
to generate the cell population for banking. In other embodiments,
donor-quality hearts will be dissected and biopsy-sized pieces are
collected from the right ventricular septal wall, the atria, the
apex, the right ventricular epicardium, or the left ventricular
epicardium. Data demonstrate that CDCs can be produced using tissue
from all regions reliably (discussed above). In some embodiments,
specimens will be processed immediately after collection, while in
other embodiments, they will be processed after up to about 6 days
of storage in cold cardioplegia solution, and in still other
embodiments after cryopreservation and subsequent thawing. In some
embodiments, a delay (from collection to processing) of about 3
days is the preferred maximal delay. As discussed above, data
demonstrate that the above mentioned tissue storage delays affect
resultant CDC yield in an acceptable manner.
[0368] In several embodiments, a master cell bank (MCB) will be
created from the source material and a working cell bank (WCB) will
be created from the MCB. The WCB will be aliquoted into single drug
dosages which are the final product. In some embodiments, the MCB
will comprise cells that have gone through the three stages of the
culture process detailed above: 1) the explants (EXP) stage: cell
outgrowth from the tissue; 2) cardiospheres (CSP) stage: enrichment
of cardiac stem cells; 3) cardiosphere-derived cells (CDCs) stage:
expansion of cells. In several embodiments, CDCs will be
cryopreserved at 5 million cells per milliliter and stored in a
liquid nitrogen tank located adjacent to the manufacturing
facility. Other concentrations of CDCs may also be used, for
example, a range of about 2-3 million cells per milliliter, about
3-4 million cells per milliliter, about 4-5 million cells per
milliliter, and overlapping ranges thereof. In some embodiments,
concentrations above 5 million cells per milliliter will be used,
as the higher concentration help to ensure that, even if a portion
of the cells die after cryopreservation, sufficient numbers are
available in the dose for use in therapy.
[0369] Genetic and phenotypic stability of the MCB will be assessed
after cryopreservation. Viability of the MCB will be assessed prior
to cryopreservation and post-thaw.
[0370] The WCB will consist of CDCs from the MCB that undergo
additional passages. Genetic and phenotypic stability of the WCB
will be assessed prior to cryopreservation. Viability of the WCB
will be assessed prior to cryopreservation and post-thaw. The final
product consists of those cells cryopreserved from the WCB. In
several embodiments, a single WCB aliquot will be removed from the
freezer for administration to the patient. The post-thaw WCB
aliquot represents the final product to be delivered as the
therapeutic. Stability of a cryopreserved product is demonstrated
above and will be confirmed for each lot of final product produced.
Efficacy of the final product will be verified in a mouse model of
myocardial infarction (see Examples for study plan).
[0371] As detailed above, a proof of concept banking system was
generated. CDCs were passaged to P1 to create the MCB and subjected
to testing as summarized below. A fraction of CDCs underwent
further passaging to P6 in order to generate a WCB. The yield
presented for the WCB is the potential yield extrapolated from the
growth seen with the fraction of cells expanded. CDCs will be taken
up to P6 to generate the MCB and up to P12 for the WCB used in one
embodiment. Testing to be employed on the MCB, WCB, and final
product is described in detail above.
Reagents and Excipients
[0372] In several embodiments, several reagents used in the
processing, culturing, cryopreservation, and administration of CDCs
are animal origin free (except for reagents such as serum and/or
fibronectin, which are of inherently animal origin). In some
embodiments, the cultured cells are treated to remove any products
of animal origin. In one embodiment, fetal bovine serum is removed
by washing the cells. In one embodiment, the cells are washed with
phosphate buffered saline. In several embodiments, washing of cells
removes fetal bovine serum such that the concentration remaining is
less than about 0.05%. In one embodiment, less than 0.0005 percent
fetal bovine serum remains.
[0373] In several embodiments, dedicated protocols for collecting,
manufacturing, and storing CDCs dedicated to allogeneic therapies
are used, which are detailed above. While in some embodiments,
endomyocardial biopsies may be used as a tissue source, in several
embodiments, transplant-quality hearts to generate allogeneic CDCs.
In some embodiments, a master cell bank is generated and in some
embodiments, a working cell bank is generated. Thus, in some
embodiments, allogeneic CDCs are cryopreserved prior to
administration to the patients. Depending on whether CDCs are
cryopreserved (certain allogeneic embodiments) or administered to a
patient without cryopreservation (certain autologous and certain
allogeneic embodiments) the final excipients in the administered
composition will vary. The single excipient in cryopreserved
products is CRYOSTOR.RTM. CSS, a cryoprotectant solution.
CRYOSTOR.RTM.CS5 is GMP manufactured, made with USP grade
components, serum-free, protein-free, and animal-origin free.
Example 15
Clinical Trials Using Allogeneic Cells
[0374] Cardiosphere-derived cells were used in patients with left
ventricular dysfunction and a recent myocardial infarction with
product delivery occurring by intracoronary infusion via an
over-the-wire balloon catheter. The following sets forth an example
of a contemplated trial using allogeneic cells.
[0375] In several embodiments, allogeneic cells would be used in
patients with damaged heart tissue. Damaged heart tissue includes
heart tissue with sub-optimal function. In one embodiment, the
patients have one or more of the following: left ventricular
dysfunction, prior myocardial infarction and LVAD placement.
Patient may be receiving LVAD placement either as a bridge to
transplantation or as destination therapy. In one embodiment, the
patients will be undergoing LVAD placement simultaneously with the
administration of allogeneic cells. In some embodiments, the use of
allogeneic cells as an adjunct to another therapy (e.g., LVAD) will
work synergistically with said therapy. In one embodiment, the
delivery of regenerative cells (autologous or allogeneic) according
to several embodiments herein is used prior, during and/or after
heart transplant surgery.
[0376] Several delivery approaches may be used. For example,
intramyocardial injection using a standard needle and syringe and
an epicardial approach during LVAD placement is used in some
embodiments. In one embodiment, intramyocardial injection using a
dosage of about 5-15 million (e.g., 10 million) CDCs is used. Other
agents, such as preservatives, carriers and recipients may be
delivered with the cells, including but not limited to DMSO. In one
embodiment, 2 mLs of solution containing 5% DMSO (100 .mu.Ls of
DMSO) will be administered by intramyocardial injection. In one
embodiment, patients will receive a single dose of 10 million CDCs
delivered with a standard needle and syringe. Each injection will
consist of 100 microliters containing 0.5 million CDCs. A total of
20 epicardial injections will be made during LVAD placement.
Dosages, number of injections an injection sites will vary in other
embodiments.
[0377] In one embodiment, patients treated with allogeneic cells
(or autologous cells) according to several embodiments described
herein, will show one or more of the following improvements: (i)
weaning success during the study period such that LVAD removal is
feasible, (ii) improved LVAD RPM at which aortic valve opening in
every cardiac cycle is seen by transthoracic echo, the
corresponding LV ejection fraction, wall thickness, and mass, (iii)
improved perfusion and coronary blood flow reserve, and (iv)
improved walk distance/times and other exercise test
parameters.
[0378] In some embodiments, patients will be weaned of LVAD (or
other therapy) through the use of regenerative cell therapy. In one
embodiment, weaning will take place over the course of 1-3 months.
In one embodiment, on a weekly basis, patients will undergo
transthoracic echo to assess LV function and aortic valve opening
and to assure aortic valve closure and adequate volumetric pump
flow rate during RPM adjustment. LVAD flow will be adjusted
downward 100-500 RPM per week until the target level of 9000 RPM is
reached (baseline settings are typically 9500-10000 RPM). Pump flow
rate and RPM will be recorded before and after pump adjustment.
During this time, INR will be maintained between 1.5 and 2.5 to
minimize the risk of thrombus formation. During these weekly
visits, the 6-minute walk test and cardiopulmonary exercise test
(as patients are able) will be performed. LVAD explantation will be
considered if patients are able to perform cardiopulmonary exercise
tests.
[0379] In one embodiment, once the LVAD flow has been adjusted to
9000 RPM, patients will be admitted to the hospital to undergo a
rapid wean. An echocardiogram will be performed at 9000 RPM to
measure baseline EF, LV dimensions and Systolic velocity (Sm) of
the basal segments (septal and lateral wall) by Tissue Doppler. The
device will then be gradually adjusted to 6000 RPM with reductions
of 1000 RPM occurring every 4-6 hours. A BNP value will also be
obtained every 4-6 hours. If the patient reports symptoms or there
is an increase in BNP to a value >1000 pg/mL, the rapid weaning
attempt will be terminated, and the LVAD speed will be adjusted
back to 9000 RPM. 6000 RPM has been determined to be a safe speed
for device function, without increased risk for thrombosis or
backflow from the aorta to the LV. The device will remain at this
speed for 16-24 hours. If the patient successfully completes the
16-24 hour period at 6000 RPM (asymptomatic, BNP <1000 pg/mL),
an echocardiogram to measure changes in EF, LV dimensions and Sm
will follow. If EF>45%, right heart catheterization will follow,
with the device at 6000 RPM, in order to measure PCWP. Finally, a
symptom limited CPX will follow, with the device set at 6000 RPM.
If the patient satisfies the explantation criteria (see below) the
device will be set back to the optimal RPM level specified before
the initiation of the weaning protocol, until explantation occurs.
In some embodiments, LVAD explantation will be performed if the
following criteria are met: (i) a LVEF>45%, (ii) LVEDD<55 mm,
(iii) Sm>8 cm/sec, (iv) a change in these three parameters of
<10% at 6000 RPM compared to 9000 RPM, (v) LVEDP<12 mm (vi)
Hg or PCWP<12 mm Hg, (vii) resting cardiac index>2.8
L/min/m.sup.2, (viii) exercise VO2 max>16 mL/kg/min at 6000 RPM,
and (ix) adequate right ventricular function (under minimal LVAD
support at 6000 rpm), assessed by echocardiography and right heart
catheterization. In one embodiment, at least one of the criteria is
met before explanation. Thus, according to several embodiments of
the invention, administration of regenerative cells advantageously
permits LVAD explantation.
Example 16
Efficacy of CDCs for Myocardial Repair Versus Alternative Stem Cell
Types
[0380] As discussed above, several embodiments of the present
invention comprise use of CDCs for regeneration and/or repair of
damaged cardiac tissue. Use of heart-derived cells e.g.,
cardiospheres, CDCs) for regenerative cardiology is but one of
several approaches presently being employed in the pursuit of
cardiac cell therapy. Multiple extra-cardiac cell types, including
bone marrow mononuclear cells (BM-MNCs), bone marrow-derived
mesenchymal stem cells (BM-MSCs), adipose tissue-derived
mesenchymal stem cells (AD-MSCs), endothelial progenitor cells, and
myoblasts are under investigation for use in regeneration of the
damaged heart. Moreover, even within heart-derived cells, there are
multiple approaches being investigated. For example, the CDCs
disclosed herein (mixture of stromal, mesenchymal and progenitor
cells) are used in several embodiments to effect cardiac repair
and/or regeneration. While in some embodiments, selection is
performed, in several embodiments, the CDCs are not selected for,
or enriched based on expression of any particular markers. One
alternative approach is to purify the c-kit.sup.+ subpopulation
from mixed heart-derived cells. Thus, the present study was
performed to compare the efficacies of several of these various
cell types in repairing and/or regenerating cardiac tissue by
direct and/or indirect mechanisms.
Methods
Cell Sources
[0381] Human CDCs were obtained and expanded as described above.
Human BM-MSCs and BM-MNCs were purchased from Lonza (Walkersville,
Md.). Human AD-MSCs were purchased from Invitrogen (Carlsbad,
Calif.). These cells were freshly-isolated from healthy donors. The
c-kit.sup.+ stem cell subpopulation was purified from the expanded
CDC population using a CELLection Pan Mouse IgG Kit and a Dynal
Magnetic Particle Concentrator-15 (Invitrogen).
[0382] For confirmatory rat studies, four-month-old Wistar Kyoto
rats were used to expand CDCs, BM-MSCs, and AD-MSCs. BM-MNCs were
also collected from the same rats by gradient centrifugation.
Freshly-collected BM-MNCs and twice-passaged CDCs, BM-MSCs, and
AD-MSCs were used for the rat experiments discussed below.
[0383] Unless otherwise noted, IMDM basic medium (Gibco)
supplemented with 10% FBS (Hyclone) and 20 mg/ml gentamycin was
used to culture all cell lines.
Flow cytometry
[0384] Characterization of CDCs, BM-MSCs, AD-MSCs, and BM-MNCs was
evaluated by flow cytometry using established methods. Briefly,
cells were incubated with FITC or PE-conjugated antibodies against
CD29, CD31, CD34, CD45, CD90, CD105, c-kit, and CD133 (eBioscience)
for 30 minutes. Isotype-identical antibodies served as negative
control. Quantitative analysis was performed using a FACSCalibur
flow cytometer with CellQuest software (BD Biosciences).
ELISA
[0385] For evaluation of growth factor production, cells were
seeded in 24-well culture plates at densities of
1.times.10.sup.6/ml (BM-MNCs) or 1.times.10.sup.5/ml (all other
cell types) in FBS-free IMDM media (all cell types) for 3 days.
Supernatants were collected and the concentrations of
angiopoietin-2, bFGF, HGF, IGF-1, PDGF, SDF-1, and VEGF were
measured with human ELISA kits (R&D Systems Inc.), according to
the manufacturer's instructions. For evaluation of cytokine
production by cultured rat cells, concentrations of HGF (B-Bridge
International, Inc.), IGF-1, and VEGF (R&D Systems Inc.) were
measured in the supernatants after 3 days of culture.
[0386] To compare the production of growth factors from the
purified c-kit.sup.+ subpopulation and unsorted CDCs, cells
(5.times.10.sup.4/ml) were seeded in 24-well culture plates and
cultured for 2 days under 20% O.sub.2. Growth factors in
conditioned media were measured by ELISA as described above.
Immunostaining
[0387] To determine myogenic differentiation in vitro, cells were
seeded on fibronectin-coated 4-chamber culture slides. After 7 days
of culture, cells were fixed, blocked with goat serum for 30
minutes, and then incubated with mouse anti-human troponin T
antibody (R&D Systems Inc.) for human cells or with goat
anti-rat troponin T antibody for rat cells. After 1 hour incubation
at room temperature, culture slides were washed and then incubated
with a PE-conjugated secondary antibody. Cell nuclei were stained
with DAPI. Cardiomyogenic differentiation was quantified by
counting positively-stained cells.
In Vitro Angiogenesis Assay
[0388] Angiogenic potency was assayed by tube formation using a kit
(Chemicon Int.), according to the manufacturer's instructions.
Briefly, cells were seeded on ECMatrix.TM.-coated 96-well plates at
a density of 2.times.10.sup.5 cells (BM-MNCs) or 2.times.10.sup.4
cells (all other cell types) per well. HUVEC cells were included as
positive controls. After 6 hours, tube formation was imaged. The
total tube length was then measured with Image-Pro Plus software
(version 5.1.2, Media Cybernetics Inc., Carlsbad, Calif.).
TUNEL Assay
[0389] To quantify the resistance to oxidative stress in vitro,
cells were seeded on fibronectin-coated 4-chamber culture slides.
After 24 hours of culture, cells were cultured with or without the
addition of 100 .mu.M H.sub.2O.sub.2 to the medium for another 24
hours. Cells were fixed, and apoptotic cells were detected by TUNEL
assay using the In Situ Cell Death Detection Kit (Roche
Diagnostics, Mannheim, Germany), according to the manufacturer's
instructions. Cell nuclei were stained with DAPI; apoptotic cells
were counted by TUNEL-positive nuclei.
Myocardial Infarction Model and Cell Implantation
[0390] Acute myocardial infarction was created in male SCID-beige
mice (10-12 weeks old), as described above. Cells were injected at
four points in the infarct border zone with a total of 40 .mu.l of
one of the following: phosphate-buffered saline (Control, n=8),
1.times.10.sup.5 CDCs (CDCs, n=20), 1.times.10.sup.5 BM-MSCs
(n=20), 1.times.10.sup.5 AD-MSCs (n=20), 1.times.10.sup.6 BM-MNCs
(high BM-MNCs, n=11), or 1.times.10.sup.5 BM-MNCs (low BM-MNCs,
n=9). Two dosages were studied with the BM-MNCs, including one with
10-fold more cells than in the comparator groups, because MNCs are
smaller than the other cell types, thus the higher dose avoids
experimental bias against this cell type in terms of total
transplanted cell mass.
[0391] C-kit-selected and non-selected CDCs were compared in a
separate study by injecting 1.times.10.sup.5 purified c-kit.sup.+
cells (c-kit.sup.+, n=16) and 1.times.10.sup.5 unsorted CDCs
(unsorted, n=11) into the infarcted hearts of SCID mice, using the
methods described above.
Echocardiography
[0392] Mice underwent echocardiography 3 hours (baseline) and 3
weeks after surgery using Vevo 770.TM. Imaging System
(VISUALSONICS.TM., Toronto, Canada). After the induction of light
general anesthesia, the hearts were imaged two-dimensionally in
long-axis views at the level of the greatest left ventricular (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.
Blinded reading of echos was conducted independently by two
experienced echocardiographers (K. M. and J. T.). The results
correlated well (FIGS. 39A-39B), so the averages of the two
readings for LVEF in each mouse were used for statistical analysis.
Histology
[0393] Mice were sacrificed 3 weeks after treatment. Hearts were
sectioned in 5 .mu.m sections and fixed with 4% paraformaldehyde.
The engraftment of implanted human cells was identified by
immunostaining for human nuclear antigen (HNA; Chemicon Int.). To
measure cell engraftment, 10 images of the infarct and border zones
were selected randomly from each animal. To quantify the apoptotic
cells in the heart, slides were fixed and apoptotic cells were
detected by TUNEL assay as described above. The differentiation of
implanted human cells into cardiomyocytes in the infarcted hearts
of SCID mice was identified by immunostaining with monoclonal
antibodies against human specific .alpha.-sarcomeric actin (Sigma),
as described above. For morphometric analysis, animals in each
group were euthanized at 3 weeks (after cardiac function
assessment) and the hearts were harvested and frozen in OCT
compound. Sections every 100 .mu.m (5 .mu.m thick) were prepared.
Masson's trichrome staining was performed as per manufacturer's
instructions (HT15 Trichrome Staining (Masson) Kit; Sigma). Images
were acquired with a PathScan Enabler IV slide scanner (Advanced
Imaging Concepts, Princeton, N.J.). From the Masson's
trichrome-stained images, morphometric parameters including infarct
wall thickness and infarct perimeter were measured in each section
with NIH ImageJ software.
Statistical Analysis
[0394] All results are presented as mean.+-.standard deviation (SD)
except as noted. Statistical significance was determined by one-way
ANOVA followed by LSD post hoc test (Dr. SPSS II, Chicago, Ill.).
Three outliers (2 from CDC group and 1 from AD-MSC group), which
differed from the means of each group by >2 SDs, were omitted,
and differences were considered statistically significant when
p<0.05.
Results
Characterization of Cell Phenotypes
[0395] Unlike BM-MNCs, which grow in suspension as small round
cells, all other cell types studied (CDCs, BM-MSCs, and AD-MSCs)
typically grow as adherent monolayers (compare FIGS. 40A-40C to
40D). Flow cytometry distinguished BM-MNCs from other cell types by
the predominant expression of pan-hematopoietic marker CD45
(74.7%), as compared to <1% in CDCs, BM-MSCs, and AD-MSCs (FIG.
40E).
[0396] Conversely, >99% of CDCs, BM-MSCs, and AD-MSCs expressed
CD105, a TGF-.beta. receptor subunit commonly associated with MSCs.
However, these three cell types can be distinguished by CD90:
>99% of BM-MSCs and 85% of AD-MSCs expressed CD90, but only 18%
of CDCs expressed this marker. CD90 (well-known as Thy-1) was
originally discovered as a thymocyte antigen. In humans, Thy-1 is
also expressed by endothelial cells, smooth muscle cells, a subset
of CD34.sup.+ bone marrow cells, and umbilical cord blood,
fibroblasts, and fetal liver-derived hematopoietic cells. CD90 is
widely used as a marker of a variety of stem cells, e.g. MSCs,
hepatic stem cells, keratinocyte stem cells, putative endometrial
progenitor/stem cells, and hematopoietic stem cells. Thus, in some
embodiments, CDCs contain a minority of fibroblast and/or
weakly-committed hematopoietic cells, which is in contrast to the
dominance of such populations in the cells of bone marrow and
adipose origins. In some embodiments, CD90 expression in CDCs marks
the cardiac mesenchymal subpopulation. In still additional
embodiments, at least a portion of CDCs do not express CD90.
In Vitro Secretion of Growth Factors
[0397] As discussed above, paracrine mechanisms are at least
partially responsible, in some embodiments, for the repair and or
regeneration of cardiac tissue. Production of six growth factors
(angiopoietin-2, bFGF, HGF, IGF-1, SDF-1, and VEGF) by the various
cell types was compared. Compared to the other cell types, CDCs
were unique in their ability to secrete large amounts of all growth
factors studied (see FIGS. 41A-41F). In contrast to CDCs, the other
cell types failed to express comparable levels of one or more
growth factors: BM-MNCs produced little VEGF and SDF-1; BM-MSCs
secreted little IGF-1 and bFGF; and AD-MSCs were not rich sources
of HGF and SDF-1. FIGS. 41G-41J depicts schematically the secretion
of each of the six studied cytokines in each given cell type, as
wheel-and-spoke diagrams in which the length of each spoke is
proportional to the growth factor concentration in conditioned
media. The symmetrical starburst pattern highlights the uniquely
well-balanced paracrine profile of CDCs.
[0398] In order to ensure that these findings did not reflect
donor-specific idiosyncrasies (commercially purchased cell types
were from different donors), growth factor secretion by the various
cell types derived from individual rats were also compared.
Correlating with the results above, higher levels of VEGF, IGF-1,
and HGF (FIGS. 42A, 42B, and 42C, respectively) were detected in
media conditioned by rat CDCs as compared to rat BM-MSCs, AD-MSCs,
and BM-MNCs, all collected from the same animals. In several
embodiments, it is this unexpectedly balanced secretion of various
paracrine factors that provides, at least in part, the therapeutic
benefits of the CDCs. In some embodiments, the well-balanced
release of growth factors by CDCs, which are acting as localized
production factories post-administration, favors enhanced
myocardial repair through paracrine mechanisms after implantation
into the heart. However, in some embodiments, the generation of one
or more of these factors that is greater than the amount generated
by the other cell types is responsible, at least in part, for the
enhanced efficacy of CDCs. For example, in some embodiments, the
amount of VEGF secreted by CDCs is greater than that of the other
cell types, and lead (at least in part) to the enhanced therapeutic
effect seen post-CDC administration. In still additional
embodiments, generation of one or more of these factors (or other
factors disclosed herein) functions in concert with direct
mechanisms (e.g., engraftment of the CDCs themselves) to effect
cardiac repair and or regeneration.
Tube Formation
[0399] The angiogenic ability of the various cell types was
quantified using an in vitro tube-forming assay. All cell types
showed the ability to form capillary-like networks on matrigel
within 6 hours (FIGS. 43C, upper left, upper right, and lower left
panels), with the exception of BM-MNCs (FIG. 43C, lower right
panel). Quantitative analysis showed that the mean tube length of
the capillary-like networks was greater in CDCs than in the other
cell types (p<0.05, FIG. 43D). Thus, in several embodiments,
administration of CDCs yields a greater angiogenic effect in the
target tissue. In some embodiments, this effect yields formation of
longer vessels, thereby maintaining or improving blood supply to
cardiac tissue. In some embodiments, the longer vessels formed
allow blood supply to bypass a damaged region. In some embodiments,
the increased angiogenic effect yields a more dense or more
branched network of vessels (e.g., capillaries), which thereby
maintains or improves regionalized blood supply to cardiac tissue
(e.g., a certain region of cardiac tissue is more thoroughly
perfused). Combinations of increased length and increased density
result in some embodiments. This increase in the vascular
infrastructure results in greater capacity for distribution of
blood to the cardiac tissue. As a result, in several embodiments,
the increased angiogenic effect of CDCs mediates the functional
recovery and or anatomical repair and/or regeneration of cardiac
tissue through one or more of increased blood flow, increased
ability to distribute paracine factors, increased oxygen delivery
to the myocardium, and combinations thereof.
Resistance to Oxidative Stress
[0400] According to several embodiments disclosed herein, the
short-term survival and/or engraftment of administered cells plays
a role in the repair or regeneration of damaged cardiac tissue.
Survival of the administered cells despite the rigors of
transplantation procedures enables CDCs, in some embodiments, to
provide greater therapeutic benefits. For example, enhanced cell
resilience to oxidative stress favors both transplanted cell
engraftment and resultant functional benefit. Sensitivity to
oxidative stress was assessed by exposing cells to H.sub.2O.sub.2,
a powerful oxidant. After 24 hours of exposure to 100 .mu.M
H.sub.2O.sub.2, the number of apoptotic cells tended to be lower in
human CDCs as compared to human BM-MNCs (p=0.067, FIG. 44B), but
there was no significant difference among CDCs, BM-MSCs, and
AD-MSCs. In rat cells, higher apoptosis was observed in BM-MNCs
compared to any of the other three cell types after H.sub.2O.sub.2
(p<0.05, FIG. 45B). Taken together, these data highlight a
relative deficiency of BM-MNCs in terms of resistance to oxidative
stress. These data also suggest, that, in some embodiments, CDCs
show resistance to oxidative stressors. As a result, the increased
resistance may enable CDCs (or cardiospheres) to survive longer,
function more robustly, and/or engraft to a greater degree than
other stem cell types. In combination with other characteristics
(e.g., paracrine profile) of the CDCs generated according the
methods disclosed herein, a greater therapeutic efficacy is
realized, in several embodiments.
Cardiomyogenic Differentiation
[0401] As discussed above, in several embodiments CDCs are suitable
for differentiation into cardiomyocytes. In some embodiments, this
accounts for (at least a portion of) the repair or regeneration of
damaged or diseased cardiac tissue via a direct mechanism. The
ability of the various cell types to undergo spontaneous
cardiomyogenic differentiation in vitro was assessed by
immunostaining for cardiac-specific troponin T. Many human CDCs
expressed troponin T (see, e.g., FIG. 43A and 43B), in contrast to
human BM-MSCs, AD-MSCs, or BM-MNCs, few of which were positive for
troponin T. Quantitative analysis showed that .about.9% of CDCs
expressed troponin T, while <1% did so in the other cell types
(FIG. 43B). Similar findings were observed using rat CDCs, BM-MSCs,
AD-MSCs, and BM-MNCs, all collected from the same animals (FIGS.
46A-46B). Thus, in some embodiments, the direct differentiation of
CDCs (or other cardiac stem cells such as cardiospheres) into
cardiac cells serves as a primary mechanism for the repair and/or
regeneration of functional cardiac tissue. In some embodiments, the
direct differentiation is a as a complementary mechanism, working
in concert with the paracrine effects discussed herein.
Cell Engraftment and In Vivo Differentiation
[0402] As disclosed above, the methods and compositions disclosed
herein yield a positive correlation between long-term cell
engraftment and functional benefit (e.g., functional and/or
anatomical cardiac repair and/or recovery). Engraftment and
differentiation of human cells 3 weeks after direct intramyocardial
injection into the infarcted hearts of SCID mice was evaluated.
Histology revealed expression of .alpha.-sarcomeric actin (aSA) in
some of the surviving progeny of human CDCs (positive for human
nuclear antigen [HNA]; FIG. 47A), confirming the cardiomyogenic
differentiation in vivo. In contrast, human cells positive for
a-sarcomeric actin were observed rarely and inconsistently in mice
injected with BM-MSCs, AD-MSCs, and BM-MNCs (data not shown).
Quantitative image analysis confirmed that the engraftment (e.g.,
the numbers of HNA.sup.+ cells) was greater in mice implanted with
human CDCs than with comparator cells (p<0.05, FIG. 47B). In
addition, the numbers of cardiomyocytes derived from the
transplanted cells (HNA.sup.-/.alpha.SA.sup.+) were greater in mice
implanted with human CDCs than with any of the other cell types
(p<0.01, FIG. 47C). In several embodiments, the increased
cardiomyogenic differentiation attributable to CDCs is responsible
for the greater therapeutic efficacy of CDCs. In some embodiments,
the increased engraftment alone is responsible for improved
efficacy, not only because a greater number of administered CDCs
are retained in the target cardiac tissue, but because there is an
associated greater increase of paracrine factor production. In some
embodiments, the greater degreed of cardiomyogenic differentiation
is responsible for the greater efficacy (e.g., direct mechanisms of
repair are dominant). In several embodiments, these effects of CDCs
(increased engraftment, increased cardiac differentiation,
paracrine effects) function in concert to for a multi-pronged
mechanism of cardiac tissue repair and/or regeneration.
Cell Apoptosis
[0403] In addition to tissue regeneration, tissue preservation may
be a salutary component of cell therapy for acute myocardial
infarction. To evaluate this possibility, apoptotic nuclei in the
infarcted region of control mice and mice injected with each of the
comparator cell types were evaluated. TUNEL staining revealed
apoptotic nuclei in the infarcted hearts 3 weeks after treatment
(FIGS. 48A-48B). Given the time-point, it is likely that the acute
phase of cell death due to ischemia may have already resolved.
Thus, the apoptotic nuclei may be a reflection of long-term
remodeling and heart failure. The total number of apoptotic cells
in the infarct and peri-infarct area was counted. The hearts of
mice implanted with CDCs exhibited fewer TUNEL-positive cells,
compared to all other cell-treated groups (p<0.05, FIG. 48C). As
discussed above, the greater production of pro-angiogenic and
anti-apoptotic factors by CDCs may be responsible, at least in
part, for the reduced apoptosis of CDCs. Moreover, in some
embodiments, the reduced apoptosis may be responsible for the
increased engraftment/survival of CDCs. In some embodiments,
however, the increased engraftment may be responsible for the
reduced apoptosis (e.g., the engrafted cells are in a preferred
environment for survival as compared to loosely attached or
non-engrafted cells). Regardless of the temporal order of these
mechanisms, the reduced amount of apoptosis allows for one or more
of increased CDC survival, increased cardiomyogenic
differentiation, and increased paracrine factor production, which
in turn result in one or more of improved cardiac anatomy (e.g.,
reduced infarct size) or improved cardiac function (e.g., increased
LVEF). Moreover, in several embodiments the reduction in apoptosis
is realized not only in the acute phase of cell death (e.g., at
short time periods after an ischemic event), but also in the
long-term (e.g., ameliorating long-term remodeling and/or heart
failure).
Cardiac Function
[0404] Clinically, one of the most meaningful endpoints of cardiac
cell therapy is the ability to produce functional benefit after
transplantation into the injured heart. Echocardiography was used
to measure cardiac function, and all images were interpreted
blindly and independently by two experienced sonographers (see
FIGS. 39A-39B). FIG. 49 summarizes the results. The LVEF at
baseline (i.e., two hours post-infarction) was comparable among all
groups. This indicates similar ischemic injury among the groups.
Among the various treatments, the implantation of CDCs resulted in
the greatest LEVF at 3 weeks (p=0.038 vs. BM-MSC; p=0.002 vs.
AD-MSC; p=0.002 vs. high BM-MNC; p=0.001 vs. low BM-MNC group; and
p<0.001 vs. Control). BM-MSCs also improved cardiac function
(p=0.009 vs. Control) and AD-MSCs tended to improve function
(p=0.073 vs. Control), while the other cell types, although higher
on average than controls, had no statistically significant
functional benefit. Thus, in some embodiments, administration of
stem cells prevents the decline in cardiac function that results
from cardiac injury absent any therapeutic intervention.
Advantageously, the administration of cardiac stem cells (e.g.,
CDCs) not only prevents this decline in function, but yields
improved function over time. In several embodiments function is
improved at least about 5% over a baseline function (e.g., function
after injury). In several embodiments function is improved at least
about 10% over a baseline function. In still additional
embodiments, greater improvements in function are realized.
Ventricular Remodeling
[0405] Potentiating the functional benefits of cell therapy is
attenuation of adverse ventricular remodeling. To evaluate this
effect, the morphological consequences of transplantation of the
various cell types on myocardial infarct size and wall thinning
were evaluated. Heart morphometry at 3 weeks showed severe LV
chamber dilatation and infarct wall thinning in the control hearts
(FIGS. 50A-50F). In contrast, all the cell-treated groups exhibited
attenuated LV remodeling. Compared to control, the implantation of
any type of human cells decreases fractional infarct perimeter and,
conversely, increases the minimal infarct wall thickness, 3 weeks
after treatment (p<0.05 vs. Control group, FIGS. 50G-50H).
Despite the positive benefits due to administration of any cell
type, the protective effect was greatest in the CDC-treated hearts.
CDC-treated hearts had thicker infarcted walls (FIG. 50G;
p<0.01), but a smaller fractional infarct perimeter (FIG. 50H;
p<0.05) as compared to any of the other cell-treated groups.
Thus, in addition to the above-discussed improved function, the
administration of CDCs, in several embodiments, also improves or
mitigates the remodeling that occurs after an injury to the heart
(e.g., an ischemic event). In some embodiments, infarct size is
reduced by 10% to about 50% as compared to infarct size in an
untreated subject. In some embodiments, infarct size is reduced
2-fold, 3-fold, 5-fold or greater. In some embodiments, functional
cardiac tissue mass is increased (e.g., increased wall thickness).
In several embodiments, the increases are about 2-fold, 3-fold,
5-fold or greater than an untreated control. In some embodiments,
the combination of reduced infarct (or other damage) size and
increased functional mass provide a synergistic increase in cardiac
function.
Unsorted CDCs Versus the c-kit.sup.+-Purified Cell
Subpopulation
[0406] Having established that CDC populations that are unselected
for any particular marker were the most efficacious cell type among
those studied, a comparison of such CDCs was made against purified
c-kit.sup.+ cells. Unsorted CDCs were compared to equal numbers of
c-kit.sup.+ stem cells purified from CDCs by magnetic cell sorting.
Purified c-kit.sup.+ cells were determined to be inferior to
unsorted CDCs in terms of functional benefit after transplantation
into the infarcted heart, although they did outperform
vehicle-injected controls (FIG. 51A). The sorting procedure did not
itself compromise cell functional efficacy, as CDCs sorted for
CD105 (expressed by >99% of CDCs) exhibited an LVEF comparable
to that of unsorted CDCs (data not shown). The c-kit antibody used
for purification is known to interfere only minimally with ligand
binding, receptor phosphorylation, and internalization in
c-kit-expressing cell lines. Also, magnetic-activated cell sorting
for mast cells using this c-kit antibody neither induced histamine
release nor did it impair the ability of cells to release histamine
when stimulated. The therapeutic superiority of the CDCs versus the
purified c-kit.sup.+ subpopulation suggests that, in some
embodiments, the mixed CDC population (e.g., stromal, mesenchymal,
and c-kit.sup.+ cells) function in concert to enhance overall
paracrine potency, direct repair mechanisms, and in turn functional
and anatomical benefits.
[0407] To investigate one potential mechanism for the functional
superiority of CDCs, production of a variety of paracrine factors
in conditioned media from sorted and unsorted cells was evaluated.
Indeed, unsorted CDCs produced higher amounts of paracrine factors
in vitro as compared to purified c-kit.sup.+ cells (FIGS. 51B-51E).
As discussed above, in some embodiments, the well-balanced release
of growth factors by CDCs promotes enhanced myocardial repair
through paracrine mechanisms after implantation into the heart. In
some embodiments, the combination of the balanced profile and the
overall greater amount of production is responsible for the
increased efficacy of cell therapy with unsorted CDCs. In some
embodiments, it is the overall greater amount of production which
is responsible for the increased efficacy of cell therapy with
unsorted CDCs. In still additional embodiments, generation of one
or more of these factors (or other factors disclosed herein)
functions in concert with direct mechanisms (e.g., engraftment of
the CDCs themselves) to effect cardiac repair and or
regeneration.
* * * * *