Note: Descriptions are shown in the official language in which they were submitted.
CA 02324350 2000-09-19
WO 99/49015 PCT/US99/06356
CARDIAC-DERIVED STEM CELLS
CROSS-REFERENCES TO RELATED APPLICATIONS
This application derives priority from USSN 60/079,123, filed on
March 23, 1998, which is incorporated by reference in its entirety for all
purposes.
TECHNICAL FIELD
The invention resides in the technical fields of cell biology, drug discovery
and medicine.
BACKGROUND OF THE INVENTION
In several tissues, the terminal state of cell differentiation is incompatible
with cell division. For example, in outerlayer skin cells, the cell nucleus is
disintegrated,
in red blood cells, there is no nucleus, and in muscle cells, myofibrils
obstruct mitosis and
cytokinesis. The ability of such cells and tissues to develop, regenerate and
repair is
dependent on the existence of stem cells that on division form additional
differentiated
progeny cells.
Stem cells are not terminally differentiated, can divide without limit, and
give rise to progeny, which can continue to divide or can differentiate. Stem
cells can be
totipotent, pluripotent or unipotent. Totipotent stem cells (e.g., embryonic
stem cells) can
give rise to every cell type in an adult organism. Pluripotent stem cells can
give rise to
more than one differentiated cell type. A unipotent stem cell can give rise to
a single
differentiated cell type. Stem cells are generally characterized by small
size, low
granularity, low cytoplasmic to nuclear ratio and no expression of
osteopontin, collagens
and alkaline phosphatase. The existence of stem cells is well-documented for
epidermis,
intestinal epithelial and hematopoietic systems. Hemopoietic stem cells are
present in
circulation as well as bone marrow. Circulating hemopoietic stem cells can
colonize
organs such as spleen. Stem cells for cells of bone, cartilage, fat and three
types of
muscle (smooth, skeletal and cardiocyte) are thought to be a common
mesenchymal stem
cell precursor, but neither mesenchymal stem cells or the more committed
tissue specific
progenitors have been characterized (Owen et al., Ciba Fdn. Symp. 136, 42-46,
1988);
Owen et al., J. Cell Sci. 87, 731-738 {1987)).
CA 02324350 2000-09-19
WO 99/49015 PCT/US99/06356
Zohar, Blood 90, 3471-3481 (1997) reports attempts to identify
progenitors of osteogenic cells by isolating cells from fetal rat periosteum
and screening
for differential expression of markers, protein content and cell cycle
position. Huss et al.,
PNAS 92, 748-752 (1995) report the identification of a stromal cell precursor
from canine
bone marrow culture. The precursor was reported to differentiate in the
presence of
growth factor and display mature differentiation markers. Robbins et al.,
Trends
Cardiovasc. Med. 2, 44-SO (1992) report that mouse embryonic stem cells can
differentiate into embryoid bodies that simulate some aspects of
cardiogenesis.
DEFIhTITIONS
An isolated cell is a cell that has been at least partially purified from
other
cell types with which it is naturally associated. Often an isolated cell
exists in a
population of cells at least 25, 50, 75, 90, 95 or 99% of which are the
isolated cell type.
Sometimes an isolated cell gives rise to a cell line in which all cells are
essentially
identical except for spontaneous mutations that may arise in propagation of
the cell line.
Sometimes an isolated cell undergoes spontaneous differentiation to generate a
mixed
population of mature cell types.
A set of differentiation markers means one or more phenotypic properties
that can be identified and are specific to a particular cell type.
Differentiation markers are
transiently exhibited at various stages of cell lineage. Pluripotent stem
cells that can
regenerate without commitment to a lineage express a set of differentiation
markers that
may be lost when commitment to a cell lineage is made. Precursor cells express
a set of
differentiation markers that may or may not continue to be expressed as the
cells progress
down the cell lineage pathway toward maturation. Differentiation markers that
are
expressed exclusively by mature cells are usually functional properties such
as cell
products, enzymes to produce cell products and receptors.
Adherence capacity of cells in culture is one marker of progression from
an undifferentiated to differentiated state. Adherent cells form a monolayer
on plastic
substrate.
A cell lineage refers to a differentiated cell type and the ancestor cell
types
from which the differentiated cell type was derived. For example, cardiocytes
and
myoblasts are two cell types in the same lineage.
2
CA 02324350 2000-09-19
WO 99/49015 PCTNS99/06356
SUMMARY OF THE INVENTION
In one aspect, the invention provide isolated nonadherent pluripotent
cardiac-derived human stem cells. On proliferation and differentiation such
cells produce
progeny cells comprising a cell type selected from the group consisting of an
adherent
cardiac-derived stem cell, a fibroblast, a smooth muscle cell, a skeletal
muscle cell, a
cardiocyte, a keratinocyte, an osteoblast and a chondrocyte. Some such stem
cells
generate all of the cell types selected from the group. Nonadherent cardiac-
derived stem
cells of the invention are producible by propagating a population of heart
tissue-derived
cells in a liquid medium on a substrate; and discarding cells from the
population adhering
to the substrate and leaving a suspension of the nonadherent cardiac-derived
stem cells.
The invention further provides an isolated nonadherent pluripotent cardiac-
derived stem cell, which on proliferation and differentiation produces progeny
cells
comprising cardiocytes and either chondrocytes or keratinocytes. Some such
stem cells
on proliferation and differentiation produce progeny cells further comprising
at least one
cell type selected from the group consisting of an adherent cardiac-derived
stem cell, a
fibroblast, a smooth muscle cell, and a skeletal muscle cell. Such stem cells
can be
human or mouse stem cells, for example. Optionally, mouse stem cells can be
obtained
from a p53 deficient mouse.
The invention further provides an isolated adherent human cardiac-derived
stem cell, which proliferates and differentiates to produce progeny cells
comprising a cell
type selected from the group consisting of a fibroblast, a smooth muscle cell,
skeletal
muscle cell, a cardiocyte, a chondrocyte, a keratinocyte and an osteoblast.
The invention further provides an isolated adherent cardiac-derived stem
cell, which proliferates and differentiates to produce progeny cells
comprising a
cardiocyte and either a chondrocyte or a keratinocyte.
In another aspect, the invention provides a method of preparing an isolated
nonadherent cardiac-derived stem cell. Such a method entails centrifuging a
suspension
of cells from heart tissue of a subject on a density gradient; isolating a
band of cells
comprising myocytes; propagating the cells until adherent cardiocytes have
died or been
discarded leaving suspension cells; and culturing the suspension cells until a
population
of nonadherent cardiac cells is detectable.
The invention also provides methods of preparing adherent cardiac derived
stem cells. Such methods start with a nonadherent cardiac-derived stem cell as
described
above. The stem cell is the propagated until adherent progeny cells appears.
An adherent
3
CA 02324350 2000-09-19
WO 99/49015 PCT/US99/06356
cell lacking markers of a cell selected from the group consisting of
myoblasts, smooth
muscle cells, skeletal muscle cells, cardiocytes, osteoblasts, keratinocytes
and
chondroblasts, which on proliferation and differentiation produces progeny
cells
comprising at least one cell type from the group is then identified.
The invention further provides methods of preparing a cardiocyte. Some
such methods entail providing a nonadherent cardiac-derived stem cell as
described
above; propagating the cell under conditions in which the cell proliferates
and
differentiates to produce progeny cells comprising adherent cells; and
identifying an
adherent cell with differentiation markers characteristic of a cardiocyte.
Alternatively,
such methods can start with a nonadherent cardiac-derived stem cell as
described above.
The cell is propagated under conditions in which the cell proliferates and
differentiates to
produce adherent progeny cells. An adherent cell is then identified with
differentiation
markers characteristic of a cardiocyte.
In another aspect the invention provides an isolated population of cells
comprising smooth muscle cells, skeletal muscle cells, cardiocytes,
fibroblasts,
keratinocytes, osteoblasts and chondrocytes.
In another aspect the invention provides methods of treating a patient
suffering from necrotic heart tissue. Such methods entail administering to the
patient an
effective dose of nonadherent or adherent stem cells as described above,
whereby the
stem cells proliferate and differentiate to produce cardiocytes, which replace
the necrotic
tissue. In some such methods, the nonadherent stem cells are administered
directly to the
heart of the patient. In other methods, the nonadherent stem cells are
administered
intravenously. In some methods, fibroblast growth factor is also administered
to the
patient to stimulate proliferation and/or differentiation of the nonadherent
cells. In some
methods, stem cell factor is administered to the patient to stimulate
differentiation of the
nonadherent cells to cardiocytes. In some methods, the patient has a
congestive heart
defect. In some methods, the stem cells are obtained from the blood of the
patient, and
propagated in vitro before readministering to the patient.
In another aspect, the invention provides methods of screening potential
agents for activity in promoting proliferation and/or differentiation of
cardiac-derived
stem cells. Such methods entail propagating nonadherent or adherent cardiac-
derived
stem cell in the presence of a potential agent, and monitoring a change in
differentiation
state of progeny cells relative to the nonadherent or adherent cardiac-derived
stem cells.
4
CA 02324350 2000-09-19
WO 99/49015 PCT/US99/06356
In some such methods, the change of differentiation state is adhesion of
progeny cells. In some methods, the change in differentiation state is
monitored by
detecting the appearance of cardiocytes. In some methods, the appearance of
adherent
cardiac-derived stem cell is monitored.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows the differentiation pathways from cardiac tissue-derived stem
cells to various differentiated cell types.
Fig. 2. shows a density gradient of cells obtained by digesting heart tissue.
The myocyte band is third from top.
DETAILED DESCRIPTION
I. General
The invention provides at least two classes of pluripotent cardiac-derived
1 S stem cells. One class of cells is nonadherent and the other is adherent.
The adherent cells
are partially differentiated progeny of the nonadherent cells. Both classes of
stem cells
can be propagated and differentiated into a variety of differentiated cell
types.
The cardiac-derived stem cells can be isolated from humans and other
vertebrate animals. The cells are isolated from a fraction of a suspension of
cardiac-
derived cells that has typically been discarded as debris by previous workers
in the field.
A band of myocyte cells is typically isolated from a density gradient, and the
cells are
propagated in a culture medium on a substrate. Previous workers have retained
cells
adhering to the substrate and discarded the culture medium in the mistaken
belief that it
contained only debris. The present inventors have kept the culture medium and
discarded
the adherent cells. After propagation of the culture medium a population of
nonadherent
suspension cells becomes apparent.
The cells have several applications in therapy and drug discovery. In
therapeutic applications, the cells are administered to patients suffering
from heart defect,
such as necrotic tissue resulting from a myocardial infarction. The
administered stem
cells colonize the heart of a patient and give rise to myocardial progeny
cells that replace
necrotic tissue and/or supplement preexisting heart tissue.
In drug discovery applications, the stem cells are used to screen
compounds for activity in promoting or inhibiting differentiation of stem
cells to
cardiocytes or other mature differentiated cell types. Compounds that promote
5
CA 02324350 2000-09-19
WO 99/49015 PCT/US99/06356
differentiation of stem cells to cardiocytes can be used for therapy of
patients with heart
defects, optionally in conjunction with the stem cells of the invention. Other
compounds
find uses in other therapeutic application in which promotion or inhibition of
stem cell
differentiation is desired. Another use for stem cells of the present
invention is screening
for compounds which expand the stem cell population in culture. Some such
compounds
also induce differentiation and others do not. Another therapeutic use for
cells of the
present invention is to provide a method to screen for compounds that promote
mobilization of cardiac-derived stem cells from the heart to the circulatory
system.
In vivo assays for evaluating cardiac neogenesis include treating neonatal
and mature animals with the cells of the present invention. The animals'
cardiac function
is measured as heart rate, blood pressure, LV pressure-rate production
(tdp/dt)and cardiac
output to determine left ventricular function. Post-mortem methods for
assessing cardiac
improvement include: increased cardiac weight, nuclei/cytoplasmic volume,
staining of
cardiac histology sections to determine proliferating cell nuclear antigen
(PCNA) vs.
cytoplasmic actin levels (Quaini et al., Circulation Res. 75:1050-1063, 1994
and Reiss et
al., Proc. Natl. Acad. Sci. 93:8630-8635, 1996.)
The cardiac-derived mesenchymal stem cells of the present invention can
be used in treatment of disorders associated with heart disease, i.e.,
myocardial infarction,
coronary artery disease, congestive heart failure, hypertrophic
cardiomyopathy,
myocarditis, congenital heart defects and dilated cardiomyopathy. Cells of the
present
invention are useful for improving cardiac function, either by inducing
cardiac myocyte
neogenesis andlor hyperplasia, by inducing coronary collateral formation, or
by inducing
remodeling of necrotic myocardial area. Cells of the present invention are
also be useful
for promoting angiogenesis and wound healing following angioplasty or
endarterectomy,
to develop coronary collateral circulation, for revascularization in the eye,
for
complications related to poar circulation such as diabetic foot ulcers, for
stroke, following
coronary reperfusion using pharmacologic methods and other indications where
angiogenesis is of benefit.
An ischemic event is the disruption of blood flow to an organ, resulting in
necrosis or infarct of the non-perfused region. Ischemia-reperfusion is the
interruption of
blood flow to an organ, such as the heart or brain, and subsequent restoration
(often
abrupt) of blood flow. While restoration of blood flow is essential to
preserve functional
tissue, the reperfusion itself is known to be deleterious. In fact, there is
evidence that
reperfusion of an ischemic area compromises endothelium-dependent vessel
relaxation
resulting in vasospasms, and in the heart compromised coronary vasodilation,
that is not
seen in an ischemic event without reperfusion (Cuevas et al., Growth Factors
15:29-40,
6
CA 02324350 2000-09-19
WO 99/49015 PCT/US99/06356
1997). Both ischemia and reperfusion are important contributors to tissue
necrosis, such
as a myocardial infarct or stroke. The cells of the present invention have
therapeutic
value to reduce damage to the tissues caused by ischemia or ischemia-
reperfusion events,
particularly in the heart or brain.
Other therapeutic uses for the present invention include induction of
skeletal muscle neogenesis and/or hyperplasia, cartilage regeneration, bone
formation,
tendon regeneration, neural neogenesis, neogenesis in the pancreas and kidney,
and/or for
treatment of systemic and pulmonary hypertension. The various specialized cell
functions
are attained by stimulating the differentiation of cardiac-derived mesenchymal
stem cells
(MSCs) down the appropriate cell pathway, e.g. into cells of the osteoblast,
chondrocyte,
skeletal myocyte or fibroblast lineage. The pathway of differentiation is
determined by
exposure to a growth factor that promotes formation of a particular
differentiated cell.
Several combinations of growth factor and differentiated cell types are
disclosed below
and others are known in the art.
Cardiac-derived MSC-induced coronary collateral development is
measured in rabbits, dogs or pigs using models of chronic coronary occlusion
(Landau et
al., Amer. Heart J. 29:924-931, 1995; Sellke et al., Surgery 120(2):182-188,
1996 and
Lazarous et al., 1996, ibid.) Cardiac-derived MSC induced benefits for
treatment of
stroke are tested in vivo in rats utilizing bilateral carotid artery occlusion
and measuring
histological changes, as well as maze performance (Gage et al., Neurobiol.
Aging 9:645-
655, 1988). Cardiac-derived MSC-induced efficacy in hypertension is tested in
vivo
utilizing spontaneously hypertensive rats (SHR) for systemic hypertension
(Marche et al.,
Clin. Exp. Pharmacol. Physiol. Suppl. 1:5114-116, 1995).
II. Cells of the Invention
1. Nonadherent Pluri~,otent Cardiac-Derived Stem Cells
These cells are characterized by being highly refractory to light, having
small size (typically less than 10 microns and often less than 5 microns),
round shape and
slow growth.
The cells are further characterized by their capacity to proliferate {i.e.,
self
renew) in a medium containing a carbon source, a nitrogen source, insulin and
transferrin.
Addition of stem cell factor (commercially available from Amgen), acid or
basic
fibroblast growth factor, zFGF-5, leukemia inhibitory factor (commercially
available) or
increasing concentration of serum stimulates propagation.
The cells are further characterized by capacity to differentiate into several
cell types. The cell types include cells in various states of differentiation
more advanced
7
CA 02324350 2000-09-19
WO 99/49015 PCT/US99/06356
than the nonadherent cardiac-derived stem cells. These cell types include
adherent
cardiac-derived stem cells (see below), fibroblasts, myoblasts, smooth muscle
cells,
skeletal muscle cells, cardiocytes, chondroblasts, keratinocytes and
chondrocytes.
Probably, adipoblasts, adipocytes, osteoblasts and osteocytes are also present
in progeny
S cells. Tendocytes may also be present. The relationship of the nonadherent
cardiac-
derived stem cells to differentiated progeny cell lineages and types is shown
in Fig. 1.
Cardiac-derived stem cells can be stimulated to differentiate by
propagation in FBS-supplemented media. Differentiation can be stimulated by
treatment
with growth factors, such as stem cell growth factor, and by contact
inhibition. Higher
concentrations of horse serum, although stimulating proliferation, have a
tendency to
inhibit differentiation. The type of growth factor used to induce
differentiation can bias
differentiation toward a selected lineage. Retinoic acid, TGF-~, bone
morphogenic
proteins (BMP), ascorbic acid, and (3-glycerophosphate lead to production of
osteoblasts.
Indomethacin, IBMX (3-isobutyl-1-methylxanthine), insulin, and
triiodithyrocine (T3)
lead to production of adipocytes. aFGF, bFGF, vitamin D3, TNF- (3 and retinoic
acid lead
to production of myocytes. zFGF-5 leads to expansion of cardiocytes
progenitors
although may also be effective later in the pathway from adherent cells to
cardiocytes.
(2) Adherent pluripotent cardiac-derived stem cell
Adherent pluripotent cardiac-derived stem cells result from proliferation
and partial differentiation of the nonadherent stem cells described in the
previous section.
The cells are characterized by amorphous shape and lack of differentiation
markers tested
to-date. Differentiation markers tested include alkaline phosphatase
expression, which is
a marker for pericytes, osteoblast precursors and chondrocyte precursors, and
alpha-actin
expression. The cells can be induced to proliferate and/or differentiate into
the same cell
types as the nonadherent stem cells. The rate of proliferation and the lineage
to which
differentiation can be induced and can be controlled by supplementation of
media with
growth factors as described above.
III. Production of Nonadherent Cardiac-Derived Stem Cells
The starting material is a heart or heart biopsy from a human or animal
subj ect. The subj ect can be embryonic beyond the mesoderm stage, neonatal,
infant or
adult. If immortalized cells are desired, the starting material can be
obtained from the
heart of a transgenic animal that is deficient in one or both copies of a
tumor suppressor
8
CA 02324350 2000-09-19
WO 99149015 PCT/US99/06356
gene. Examples of tumor suppressor genes include p53, p21, and the
retinoblastoma
gene. Transgenic mice with a homozygous mutation in p53 are commercially
available
from Taconic Farms or Jackson Labs.
Methods of processing whole tissue to prepare a suspension of cells are
described by e.g., Methods In Molecular Biology: Animal Cell Culture, 5,
(Pollard et al.
eds., Humana Press, NJ, 1990), incorporated by reference). Typically, heart
tissue is
digested with collagenase, trypsin, other protease and/or DNase to release
cells. Polinger,
Exp. Cell Res. 63:78-82 (1970); Owens et al., J. Natl. Cancer Instit., 53:261-
269, 1974;
Milo et al., In Vitro 16:20-30, 1980; Lasfargues, "Human Mammary Tumors", in
Kruse
et al. (eds) Tissue Culture Methods and Applications (Academic Press, NY,
1973); Paul,
Cell and Tissue Culture, Churchill Livingston, Edinburgh, 1975). Intact cells
are then
separated from debris by low-speed centrifugation. Debris collects in the
supernatant.
The cell pellet is resuspended and cells are fractionated. Fractionation can
be performed
on a density gradient such as a Ficoll or sucrose gradient. The cells form
four bands as
shown in Fig. 2, the myocyte band being the third from the top. The myocyte
band is
removed and the cells are cultured in a complete medium. Such a medium
contains at
least a carbon source, a nitrogen source, essential amino acids, vitamins and
minerals, and
serum. The medium can also be supplemented with growth factors. Cells are
grown at
about 37~C in an atmosphere of 95% 02 and 5% C02 on a plastic substrate.
Initially,
most cells are adherent myocytes. However, most of the adherent myocytes
(e.g., at least
75 or 90%) die within S-30 days and small nonadherent cells appear in
suspension in
increasing concentration. The time period is the shorter end of the above
range for p53
deficient cells and the longer end of the range for normal cells. The small
nonadherent
cells are pluripotent cardiac-derived stem cells.
IV. Differentiation Markers of Different Cell Tyaes
Differentiated cell progeny of stem cells are recognized in part by the
presence of differentiation markers. There are three types of muscle cells,
cardiocytes
(i.e., cardiac muscle cells), striated muscle and smooth muscle cells. The
three types of
cells derive from a common precursor, termed a myoblast. Cardiac myosin
isozyme
expression and the cardiac specific pattern of creatine kinase isozyme
expression when
identified together on the same cell or a clonal population of cells are
markers for cardiac
muscles cells. Cardiocytes can also be recognized by their bifurcated
appearance and
capacity to form gap junctions by light microscopy. Such cells can be
recognized by
9
CA 02324350 2000-09-19
WO 99/49015 PCT/US99/06356
forming an electric potential across confluent cells and detecting transfer of
signal across
the cells. Muscle a-actin mRNA, and smooth muscle cell actin are
differentiation
markers of myocytes. Myosin isozyrne expression and a muscle-specific pattern
of
creatine kinase isozyme expression when identified in a cell or clonal
population are
markers for skeletal muscle cells.
Osteoblast cells secrete bone matrix material. ALP, osteocalcin
expression, PTH-induced cAMP expression, and bone mineralization capacity
identified
together in a cell or clonal population of cells are markers of
differentiation for
osteoblasts.
Chondrocytes secrete type II cartilage. Aggrecan and collagen Type IIB
identified in a cell or clonal population of cells, Alcian Blue staining,
which detects
production of chondroitin sulfate, are markers for chondrocytes.
Keratinocytes secrete keratin and can be recognized using commercially
available stains.
Adipocytes produce lipids and can be recognized by Oil Red O staining.
V. Growth Factors
Basic FGF (also known as FGF-2) is mitogenic in vitro for endothelial
cells, vascular smooth muscle cells, fibroblasts, and generally for cells of
mesoderm or
neuroectoderm origin, including cardiac and skeletal myocytes (Gospodarowicz
et al., J.
Cell. Biol. 70:395-405, 1976; Gospodarowica et al., J. Cell. Biol. 89:568-578,
1981 and
Kardami, J. Mol. Cell. Biochem. 92, 124-134, 1990). Non-proliferative
activities
associated with acidic and/or basic FGF include: increased endothelial release
of tissue
plasminogen activator, stimulation of extracellular matrix synthesis,
chemotaxis for
endothelial cells, induced expression of fetal contractile genes in
cardiomyocytes (Parker
et al., J. Clin. Invest. 85, 507-514, 1990), and enhanced pituitary hormonal
responsiveness
(Baud et al., J. Cellular Physiol. 5, 101-106, 1987.)
zFGF-5 is a type of fibroblast growth factor that is expressed at high levels
in human fetal and adult heart tissue. This growth factor is also expressed at
decreased
levels in fetal lung, skeletal muscle, smooth muscle tissues such as small
intestine, colon
and trachea. The high level of expression of zFGF-5 in fetal and adult heart
and its
effects in cardiac tissue-derived cells suggests that ZFGF-5 has particular
potency in
stimulating proliferation and/or differentiation of cardiac-derived stem cells
to
cardiocytes. The isolation of zFGF-5 and its properties are described in more
detail in
CA 02324350 2000-09-19
WO 99/49015 PCTNS99/06356
commonly owned PCT US97/18635, filed October 16, 1997 (incorporated by
reference).
The nucleotide sequence encoding zFGF-5 is described in SEQ ID NO. 1, and its
deduced
amino acid sequence is described in SEQ ID No. 2. The sequence of zFGF-5 shows
some
sequence similarity with FGF-8.
VI. Immortalization of Cells
Cells that can be continuously cultured and do not die after a limited
number of cell generations are termed immortalized. A cell that survives for
only 20 to
80 population doublings is considered finite (Freshney, Culture of Animal
Cells (Wiley-
Liss, NY, 1994) herein incorporated by reference), and a cell that survives
more than 80,
preferably at least 100, cell generations is considered immortalized.
Some but not all immortalized cells are tumorigenic. As noted above, cells
can be immortalized by using a transgenic animal deficient in a tumor
suppressor gene as
a donor of heart tissue. Cells from other sources can be immortalized by other
means.
These means include transformation and expression by a gene whose product
plays a role
in cell senescence, or overexpression or mutation of one or more oncogenes
that overnde
the action of the senescence genes. Expression of genes that result in
positive signals for
cell proliferation include SV40 large T antigen (Linder et al., Exp. Cell Res.
191, 1-7
(1990), polyoma large T antigen (Ogris et al., Oncogene 8, 1277-1283, 1993),
adenovirus
ElA (Braithwaite et al., J. Virol. 45, 192-199, 1983), myc oncogene
{Khoobyarian et al.,
Virus Res. 30, 113-128, 1993), and the E7 gene of papilloma virus Type 16
(McDougall,
Curr. Top. Microbiol. Immunol. 186:101-119, 1994).
VII. Therapeutic Re igimes
Heart disease is the major cause of death in the United States, accounting
for up to 30% of all deaths. More than 5 million people are diagnosed with
coronary
disease in the US. Coronary disease can be due to congestive heart failure,
hypertrophic
cardiomyopathy, cardiomyopathy, viral infection or myocardial infarction (MI).
Myocardial infarction accounts for 750,000 hospital admissions per year in the
US. In MI
patients, ischemia stimulates growth of fibroblasts and promotes development
of greater
than normal quantities of fibrous tissue to replace necrotic muscular tissue.
Risk factors
for MI include diabetes mellitus, hypertension, truncal obesity, smoking, high
levels of
low density lipoprotein in the plasma or genetic predisposition.
11
CA 02324350 2000-09-19
WO 99/49015 PCT/US99/06356
Although some proliferation of cardiocytes apparently occurs with normal
aging humans and animals (Olivetti et al., J. Am. Coll. Cardiol. 24(1), 140-9
(1994) and
Anversa et al., Circ. Res. 67, 871-885, 1990), this is inadequate to repair
damage due to
coronary disease. Thus, necrosis that occurs in MI and other coronary diseases
is
essentially irreversible.
Such conditions are treated by administration of cardiac-derived stem cells
as described above. Either adherent or nonadherent cells can be used. The
cells can be
administered either intravenously, intracoronary or intraventricularly. A
catheter can be
used for the latter two routes of administration, which are more usual for
adherent cells.
Cells are administered in a therapeutically effective dosage. Such a dosage is
sufficient to
generate significant numbers of new cardiocytes cells in the heart, and/or at
least partially
replace necrotic heart tissue, and/or produce a clinically significant change
in heart
function. A clinically significant improvement in heart performance can be
determined
by measuring the left ventricular ejection fraction, prior to, and after
administration of
cells, and determining at least a 5% increase, preferably 10% or more, in the
total ejection
fraction. Standard procedures are available to determine ejection fraction, as
measured by
blood ejected per beat. Dosages can vary from about 100-10', 1000-106 or 104-
105 cells.
Cells can be administered as pharmaceutical compositions, which can also
include, depending on the formulation desired, pharmaceutically-acceptable,
typically
sterile,. non-toxic Garners or diluents, which are defined as vehicles
commonly used to
formulate pharmaceutical compositions for animal or human administration. The
diluent
is selected so as not to affect the biological activity of the combination.
Examples of such
diluents are distilled water, physiological phosphate-buffered saline,
Ringer's solutions,
dextrose solution, and Hank's solution. In addition, the pharmaceutical
composition or
formulation may also include other carriers, adjuvants, or nontoxic,
nontherapeutic,
nonimmunogenic stabilizers and the like
Administration of stem cells can be preceded, accompanied or followed by
administration of growth factors) that stimulate proliferation and/or
differentiation of the
stem cells into cardiocytes. Growth factors can be administered intravenously
or
intraventricularly. Growth factors are administered in a dosage sufficient to
cooperate
with administered stem cells in generating significant numbers of new
cardiocytes cells in
the heart, and/or at least partially replace necrotic heart tissue, and/or
produce a clinically
significant change in heart function
Suitable growth factors include zFGF-5 and stem cell growth factor.
12
CA 02324350 2000-09-19
WO 99/49015 PCTNS99/06356
In some methods, cardiac-derived stem cells are administered in
combination with anti-inflammatory agents that arrest, reverse or partially
ameliorate
inflammation associated with coronary disease. Suitable antiinflammatory
agents include
antibodies to Mac-1, and L-, E and P-selectin (see Springer, Nature 346, 425-
433 (1990).
Osborn, Cell 62, 3 (1990); Hynes, Cell 69, 11 (1992)). Cardiac-derived stem
cells can
also be administered with diuretics, ACE inhibitors and (3-adrenergic
blockers.
In some methods, the recipient patient of stem cells and the donor from
which the cells are obtained are HLA-matched to reduce allotypic rejections.
In other
methods, cells are administered under cover of an immunosuppressive regime to
reduce
the risk of rejection. Immunosuppressive agents that can be used include
cyclosporin,
corticosteroids, and OKT3. In other methods, immune responses are avoided by
obtaining stem cells from the patient that is to be treated. Stem cells can be
obtained by
biopsy of heart tissue, and expanded in vitro before readministration.
Alternatively, given
the present provision of isolated cardiac-derived stem cells, differentiation
markers can be
identified for these cells, and the cells can be isolated from the blood of
the patient to be
treated.
VIII Use of Cardiac-Derived Stem Cells in Drug Screening
The cardiac-derived stem cells described above can be used to test
compounds for activity in promoting or inhibiting proliferation and/or
differentiation of
the cells. In general, a compound being tested is contacted with a population
of cardiac-
derived stem cells, optionally, in the presence of other agents known to
promote or inhibit
the metabolic pathway or phenotype of interest, and phenotypic and metabolic
changes
are monitored in comparison with a control in which the compound being tested
is absent.
Compounds to be tested include known or suspected growth factors, and analogs
thereof,
libraries of natural compounds not previously known to have activity in
promoting
proliferation or differentiation and combinatorial libraries of compounds.
Large
combinatorial libraries of the compounds can be constructed by the encoded
synthetic
libraries (ESL) method described in Affymax, WO 95/12608, Affymax, WO
93/06121,
Columbia University, WO 94/08051, Pharmacopeia, WO 95/35503 and Scripps, WO
95/30642 (each of which is incorporated by reference for all purposes).
Peptide libraries
can also be generated by phage display methods. See, e.g., Devlin, WO
91/18980.
Compounds that cause cardiac-derived stem cells to proliferate and/or
differentiate into cardiocytes are useful as therapeutic agents in the same
conditions as the
13
CA 02324350 2000-09-19
WO 99/49015 PCT/US99/06356
cardiac-derived stem cells are useful. Such compounds can be administered
alone to
stimulate proliferation and differentiation of endogenous cardiac-derived stem
cells, or
can be administered in conjunction with exogenous cardiac-derived stem cells.
Such
compounds are screened for proliferating activity by contacting them with
cardiac-
derived stem cells in growth media, and monitoring an increase in cell count,
or
incorporation of 3H-thymidine. Compounds are screened for promoting
differentiation to
cardiocytes by monitoring cells with the characteristic morphological
appearance and
differentiation markers of cardiocytes as note above.
Similarly, compounds can be monitored for activity in promoting
differentiation of cardiac-derived stem cells to other differentiated cell
types, such as
smooth muscle cells, skeletal muscle cells, osteoblasts and chondrocytes.
Activity is
detected by detecting the characteristic morphological appearance and
differentiation
markers of one of these cell types. Compounds with activity in promoting
differentiation
to one of the above cell types are useful in treating patients with
degenerative diseases of
bone, muscle or cartilage.
Compounds that inhibit differentiation of stem cells to certain cell types
such as adipocytes can also be useful in some circumstances. For example,
compounds
that inhibit differentiation of stem cells to adipocytes can be used in
treating obesity.
Such compounds are identified by contacting a compound under test with cardiac-
derived
stem cells under conditions that would otherwise lead to differentiation of
the stem cells
to a certain cell type, and monitoring a decreased frequency or extent of
conversion to the
cell type relative to a control in which the compound is omitted.
Compounds identified as therapeutic agents by such screening with the cell
lines of the invention are formulated for therapeutic use as pharmaceutical
compositions.
The compositions can also include, depending on the formulation desired,
pharmaceutically-acceptable, non-toxic carriers or diluents, as described
above.
EXAMPLES
1. Materials
a. Serum-free Media for Myocytes
Dulbecco's Modified Eagle's Medium/Ham's Nutrient Mixture F12 (1:1; Gibco
Laboratories)
Insulin (JRH SC2059), 1 ~,g/ml
Transferrin {JRH 6K2222), 5 p,g/ml
14
CA 02324350 2000-09-19
WO 99/49015 PCT/US99/06356
Selenium (Aldrich Chem. Co. 20010/7), 1 nM
Thyroxine (Sigma T-0397), 1 nM
Ascorbic Acid (Sigma A-4034), 25 wg/ml
LiCI (Sigma L-0505), 1 nM
b. Plating Media (PMl
Dulbecco's Modified Eagle's Medium/Ham's Nutrient Mixture F12
(1:1; Gibco Laboratories), contain 15% HIA FBS
c. Media for Myocyte Isolation
Dulbecco's Modified Eagle's Medium/Ham's Nutrient Mixture F12 (l:l; Gibco
Laboratories)
DF 20, contains 20% HIA FBS
DF 10, contains 10% HIA FBS
DF S, contains 5% HIA FBS
HIA FBS (HyClone lot# AEL4614)
d Enzymes for Digestion of Heart Tissue
Collagenase (Worthington LS004176), 2% solution
DNAse (Worthington LS002007), 0.5% solution
Enzymes are dissolved in PBS (Gibco Laboratories) w/ 1% Glucose
e. Percoll Purification
Percall (Pharmacia #17-0891-O1)
Add buffers (g/L for lx; or g/100m1 for 10x), pH 7
6.8 NaCI
1.0 Dextrose
1.5 NaH2P04
0.4 KCL
0.1 MgS04
4.76 HEPES
0.02 Phenol Red - Sigma
All reagents were passed through a 0.22 ~.M filter before use.
CA 02324350 2000-09-19
WO 99/49015 PCTNS99/06356
A stock solution was made using 9 parts (45 ml) Percoll, plus 1 part (5 ml) l
Ox Ads
without Phenol Red.
Gradient steps were made as follows.
Density (g/ml) Percoll Stock 1 x Ads
(ml) (~ Phenol Red)
(ml)
1.050 18 ml 32 ml without
Phenol Red
1.060 22.2 ml 27.8 ml with
Phenol Red
1.082 31.6 ml 18.4 ml
without Phenol Red
f. Human Fibronectin Plates
Stock solution is 1 mg/ml (1000 pg/ml)
Coating concentration 1 ug/cm2
Dilute stock solution to 10 wg/ml ( 100X: 200 pl in 20 ml SFM)
Plate 3 ml/60 mm tissue culture dishes
Incubate at room temperature for 1 hour
Aspirate remaining material
Rinse plates carefully with dH20 - avoid scraping bottom surface.
Plates are ready for use, store at 4 ~ C
2 Isolation of Nonadherent ardiac-Derived Stem Cells from P53 Deficient Mice
In this example, a code is used to designate months. Thus, A, B and C
represent
successive months starting with A.
On A/19, hearts from 10 P53-/- female mice (about 2 months of age) were
digested as
follows:
16
CA 02324350 2000-09-19
WO 99/49015 PCC/US99/06356
1) The hearts were minced with scissors in a minimal volume of PBS(-). -
2) PBS(-) + 1% glucose was added for a final volume of 18 ml.
3) 1 ml of DNase and 1 ml collagenase were added.
4) The suspension was agitated in a 50 ml Falcon tube on a rotary shaker at
370C
for 30 minutes.
5) This first supernatant was discarded as it contains mostly RBC debris and
fibroblasts.
6) Steps 2-5 were repeated until the hearts were completely digested.
7) After each 30 min agitation period (except for the first digestion, which
was
discarded), the supernatant was removed and added to 20 ml of DF20. The
supernatant with the DF20 was spun at 1650 rpm, 4~C for 10 min. The resulting
pellet was saved and resuspended in 10 ml DF 10 and kept on ice.
8) After complete digestion of the hearts, the pellets were combined (5
pellets per
50 ml Falcon tube) and spun at 1650 rpms, 4~C for 10 min.
9) The final resulting pellet was resuspended in 1.082 gm/ml Percoll (12 ml).
10) A Percoll gradient was prepared in a 50 ml Falcon tube as follows:
12 ml 1.050 gm/ml Percoll loaded, then 12 ml 1.060 gm/ml Percoll loaded
from the bottom, followed by the cell suspension in 1.082 gm/ml Percoll,
loaded from the bottom. The 50 ml Falcon tube was spun in a Beckman
CS-6R centrifuge for 30 min at 3000 rpm, RT.
11) The resulting myocyte fraction in the band between the lower 1.082 gm/ml
Percoll
and the middle 1.06 gm/ml Percoll layers was removed and an equal volume of
DF 10 was added.
12) The myocyte fraction was spun at 1650 rpm for 10 min at RT.
13) The resulting pellet contained 3.75 x 106 cells, which were plated equally
into 5
wells in 5 ml DFS.
14) On A/20 the media was changed to plating media (PM) in 6-well tissue
culture
plates, which was replaced again with fresh PM on A/22 and A/25. Adherent
beating cardiocytes were apparent from A/20 through A/25, although in
diminishing numbers. Additionally, very small suspension cells were present in
the wells.
1 S) On A/26 these suspension cells were split 1:3 into PM, PM plus 1 % horse
serum
(HS), and PM plus 10% HS (HyClone, Logan, Utah).
17
CA 02324350 2000-09-19
WO 99/49015 PCT/US99/06356
16) On B/O1 the suspension cells in PM plus 1% horse serum (HS) and PM plus
10%
HS were split 1:10 into T25 flasks (0.5 ml cell suspension plus 4.5 ml fresh
PM
with either 1% HS or 10% HS). On B/15 the suspension cells in PM plus 1% HS
were split l:l. The flasks were not manipulated again until C/19. The flasks
were
incubated at 37~C in 95% 02 and 5% CO2.
On C/19, the cells maintained in PM plus 10% HS were still viable by light
refraction and trypan blue exclusion. Cells in PM only were not viable. Cells
in PM and
1 % HS had become adherent cells and are described in more detail below. The
suspension cell density was determined, using a hemacytometer, to be 6 x
106/ml.
Also, on C/19, the cells grown in PM + 10% HS were spun at 1500 rpm for
10 min and resuspended at 0.5 x 106/ml in serum free media (SFM), SFM plus 1%
HS,
SFM plus 10% HS, PM, PM plus 1 % HS, or PM plus 10% HS.
On C/26 the suspension cells in PM plus 10% HS were at 8.5 x 105/ml,
while the suspension cells in SFM plus 10% HS were at 8.75 x 105/ml. Half of
these
suspension cells (both the SFM and PM cells) were resuspended in PM plus 10%
HS or
SFM plus 10% HS, and were plated on methylcellulose in the presence of various
cytokines and growth factors. The suspension cells did not form colonies on
methylcellulose, even after 12 days in culture suggesting that the cells could
be primitive
stem cells.
At this time, culturing the suspension cells in 10% HS no longer appeared
to impact the cells significantly, so the cells were passaged in PM only or
SFM only.
zFGF-5 ( 150 pg/ml) enhanced proliferation of the cells 2.5-5.0 fold, and stem
cell factor
(SCF 10 ng/ml) enhanced proliferation about 1.0-2.0 fold determined by short-
pulse
tritiated-thymidine uptake.
A one month treatment of the suspension cells with 10 ng/ml SCF in PM
resulted in the appearance of an adherent layer of cells of mixed lineage
designated
SCF/PM-derived cells. By contrast, treating the suspension cells with SCF in
SFM did
not give rise to adherent cell lineages, implying that some other growth
factor present in
the 1 S% FBS found in PM is essential, in addition to the SCF. Addition of
zFGF-5
(150 pg/ml) to the flasks with SCF in PM prevented the induction of the
adherent cell
lineages, suggesting that zFGF-5 blocked the differentiating activity of the
SCF.
By light microscopy, the morphology of the SCF/PM-derived cells
suggested lineages which include cardiocytes, chondrocytes, fibroblasts,
smooth muscle
myocytes, and skeletal muscle myoblasts. Cardiocyte-like cells had the
appearance of
18
CA 02324350 2000-09-19
WO 99/49015 PCT/US99/06356
cigars or driftwood. Chondrocyte-like cells had the appearance of nests or
cobblestone
streets. Fibroblasts were long and spindly. Cells with a phenotype of smooth
muscle
myocytes were amorphous or star-shaped, skeletal muscle cells had the shape of
a pen.
Myoblasts were intermediate between fibroblasts and skeletal muscle cells.
Other
lineages are probably present, and can be characterized by using
immunohistochemistry,
immunofluorescence and FACS analysis.
3. Adherent Cardiac-Derived Stem Cells
On C/19, the cells maintained in PM plus 1% HS had differentiated into
adherent cells of mixed morphology. These cells were trypsinized, pelleted,
and then
split 1:6 into SFM, SFM plus 1 % HS, SFM plus 10% HS, PM, PM plus 1 % HS or PM
plus 10% HS.
On C/26, the adherent cells were expanded in PM only as the different
media or HS concentrations did not have any noticeable affect on the adherent
cell
morphology. By light microscopy the adherent cells appear to be a mixed
population
including pericytes, fibroblasts, cardiocytes, smooth muscle myocytes,
skeletal muscle
myocytes.
The adherent cells were subcloned on C/08. From the subcloning, 11
clones were obtained and expanded on plating media in 95% Oz/ 5% C02 at 370C.
The
cells were characterized by the following criteria.
(1) Morphology by light microscopy.
(2) Alkaline phosphatase staining (identifies pericytes, osteoblast precursors
and
chondrocyte precursors and mature osteoblasts and chondrocytes)
(3) Acetylated LDL uptake (endothelial cell marker)
(4) MF-20 Ab (Mouse anti-adult chicken pectoralis myosin, which crossreacts
with
mouse myosin and detects both adult skeletal and cardiac myocyte myosin)
(5) M0636 (Dako #5/56 mouse anti-human muscle actin; recognizes alpha actin in
cardiac, skeletal and smooth muscle myocytes, and gamma actin in smooth
muscle myocytes)
{6) Mitogenic response to zFGF-5, acidic FGF and basic FGF. This test tests
for
proliferation capacity. The cells were plated in 96-well plates in PM and
grown to
confluence and then switched to serum-free media for 24 hr after which growth
factor and tritiated thymidine was added.
19
CA 02324350 2000-09-19
WO 99/49015 PCT/US99/06356
(7) Production of zFGF-5 mRNA identified by Northern blotting which is
expressed
at high concentration by cardiocyte lineage cells.
(8) Differentiation induced by contact-inhibition or passage in SFM to
identify
proliferation and/or differentiation capacity.
(9) Differentiation induced by ascorbic acid/-glycerophosphate (AA/0-GP)
to identify osteoblasts.
(al Mixedponulation propagated in PM + 1% HS before isolating clones
Cells in this population showed different morphologies characteristic of
pericytes, fibroblasts, cardiocytes, smooth muscle myocytes, skeletal muscle
myocytes,
and other cells.
About 20-25% of cells were positive for alkaline phosphatase indicating
cells of osteoblast or chondrocyte lineage. All cells were negative for
acetylated LDL
uptake indicating an absence of endothelial cells. All cells were negative for
MF-20
binding indicating an absence of terminally differentiated skeletal cells. All
cells were
positive for M0636 binding indicating the presence of precursors of
cardiocytes,
myocytes, skeletal muscle cells and smooth muscle cells.
The cells showed a mitogenic response to zFGF-5 (cFGF), acidic FGF
(aFGF) and basic FGF. bFGF was most potent in inducing a mitogenic response.
The
response increased in a concentration dependent manner from 1 pg/ml up to 1 0
g/ml.
On treatment with serum free media, the cells showed moderate growth
and differentiated into cells with an appearance akin to frying eggs. This
appearance
suggests early stage myocytes.
On treatment with ascorbic acid/ ~i-GP, cells differentiated into
chondrocyte-like cells by day 6 by morphology. No mineralization occurred even
after
Day 25 indicating absence of osteoblast lineage cells.
Clone 1D9:
Clone 1D9 was isolated from a single cell of the mixed population
described above in 3a. Some cells propagated from 1D9 showed morphology of
cardiocytes, and other cells showed different morphology. A few cells stained
faintly
positive for alkaline phosphatase. The cells did not take up acetylated LDL.
MF-20 did
not bind to the cells. M0636 did bind to cells indicting that some cells were
of smooth
muscle, skeletal muscle, and/or cardiocyte lineage. zFGF-5, acidic FGF and
basic FGF
CA 02324350 2000-09-19
WO 99/49015 PCT/US99/06356
all induced a mitogenic response and bFGF was most potent. Serum-free media
induced
slow growth but not obvious morphological changes. Contact-dependent
differentiation
produced chondrocytes, cardiocytes, and other cell types recognized by
morphology.
Ascorbic acid/ (3-GP-treatment resulted in appearance of chondrocyte-like
cells on Day 6.
No mineralization occurred even after Day 25. These results indicate that
clone 1D9
gives rise to differentiated cells of different lineages.
Clone 2E7:
Cells expanded from 2E7 have the morphology of myoblast (skeletal
myocyte/fibroblast/adipocyte precursor).
The cells were negative for alkaline phosphatase staining, acetylated LDL
uptake and MF-20 binding. The cells were positive for M0636 binding indicating
the
presence of precursors of smooth, skeletal and/or cardiocyte muscle cells. The
cells
proliferated in response to zFGF-5, acidic FGF and basic FGF, with basic
FGFbeing the
1 S most potent. On treatment with serum free media, the cells proliferated
and changed
morphology to appear as large polygonal cells, which may be early stage
myocytes. The
cells were negative for MF-20 binding. On treatment with ascorbic acid/ (3-GP,
cells
differentiated to "ridge-like" formations on Day 11. No mineralization was
observed
even after day 25.
Clone 1611:
Clone 1611 was also isolated as a single cell from the mixed population of
adherent p53 deficient cells described above. Cells expanded from 1611 had the
morphology of skeletal myocytes, smooth muscle myocytes, cardiocytes, and
other cell
types. About 20-25% of cells stained positive for alkaline phosphatase. The
cells showed
a mitogenic response to zFGF-5, acidic FGF and basic FGF, with bFGF the most
potent
and bFGF and aFGF equally active. Addition of serum free media caused a slow
growth
rate and the appearance of star-shaped cells, which are myocyte precursors.
The cells did
not bind MF-20. Treatment with ascorbic acid/ ~i-GP did not result in
mineralization even
after Day 25.
Clone 2B6:
This clone showed the morphology of skeletal myocytes, smooth muscle
myocytes, cardiocytes, and other cell types. Cells were negative for alkaline
phosphatase
21
CA 02324350 2000-09-19
WO 99149015 PCT/US99/06356
staining and acetylated LDL uptake, MF-20 binding. The cells showed a
mitogenic
response to bFGF and aFGF, but not to zFGF-5. SFM-induced differentiation, and
slower
growth rate, producing thicker, rod-shaped cells. Treatment with ascorbic
acid/ (3-GP
induced resulted in cells gathering together in 'ridge-like' formations on Day
11. No
mineralization occurred even after Day 25.
Clone 2A7:
This clone showed the morphology of myoblasts (skeletal
myocyte/fibroblast/adipocyte precursors). The cells were negative for alkaline
phosphatase staining, acetylated LDL uptake, MF-20 binding. The cells were
slightly
positive for M0636 binding. The cells showed a mitogenic response to zFGF-5,
acidic
FGF and basic FGF. aFGF and bFGF were most potent, all 3 FGFs were equally
active.
SFM caused no change in growth rate or morphology. Ascorbic acid/(3-GP
treatment did
not result in mineralization even after Day 25.
Clone 2G7:
This clone had the morphology of multinucleated cardiocytes, and other
cell types. Some cells were positive for alkaline phosphatase staining. The
cells were
negative for acetylated LDL uptake, and MF-20 binding. The cells were positive
for
M0636 binding. The cells showed a mitogenic response to zFGF-5, acidic FGF and
basic
FGF, with bFGF being most potent, and aFGF and bFGF being equally active. SFM
induced moderate growth rate, and no distinct morphology change. Cells were
negative
for MF-20 binding. Ascorbic acid/ ~-GP treatment did not result in
mineralization even
after Day 25.
Clone 2F11:
This clone had the morphology of myoblasts (skeletal
myocyte/fibroblast/adipocyte precursors). 5% of cells stained with alkaline
phosphatase.
The cells were negative for acetylated LDL uptake, MF-20 binding, and M0636
binding.
SFM induced moderate growth rate resulting in star-shaped cells.
Ascorbic acid/ ~i-GP-induced differentiation resulted in cells gathering in a
'ridge-like'
formation on Day 11.
No mineralization occurred even after Day 25.
22
CA 02324350 2000-09-19
WO 99/49015 PCT/US99/06356
Clone 2A6:
These cells had a stellate appearance characteristic of myoblasts. The cells
were negative for alkaline phosphatase staining, negative for acetylated LDL,
negative for
MF-20 binding and negative for M0636 binding. The cells showed a mitogenic
response
to zFGF-5, acidic FGF and basic FGF, with
bFGF being the most potent, and all 3 FGFs being equally active. SFM
treatment caused moderate growth and the appearance of some large star-shaped
cells.
Treatment with ascorbic acid/ (3-GP induced differentiation with cells
gathering together
in 'ridge-like' formations on Day 11 (many formations were evident). No
mineralization
occurred even after Day 25.
Clone 2612:
These cells showed the morphology of cardiocytes, skeletal myocytes,
smooth muscle myocytes, and others. The cells were negative for alkaline
phosphatase
staining, acetylated LDL uptake, MP-20 binding, M0636 binding.
The cells showed a mitogenic response to zFGF-5, acidic FGF and basic
FGF. bFGF and cFGF were equally potent and active from 10 fg/ml up to 1 ~g/ml.
FGF
stimulation regenerated the original suspension cell progenitor population and
produced
zFGF-5 mRNA. zFGF-5 150 pg/ml (1 day) stimulation produced clear adult
cardiocytes
by morphology. Treatment for five days resulted in formation of haystack
structure,
ridges and troughs. SFM treatment resulted in strong growth, loss of
multinucleation, and
appearance of large polygonal cells. Contact-dependent differentiation
produced
cardiocytes, chondrocytes, and other cell types. Ascorbic acid/ (3-GP-
treatment resulted
in differentiation and larger cells by Day 6. No mineralization occurred even
after Day
25.
Clone 2D6:
The cells had a stellate or haystack appearance when sub-confluent
suggesting myoblasts (skeletal myocyte/fibroblast/adipocyte precursor). The
cells were
negative for acetylated LDL uptake, negative for alkaline phosphatase
staining, negative
for MF-20 binding and positive for M0636 binding. SFM treatment caused rapid
growth,
and loss of haystack or stellate appearance. Ascorbic acid/~i-GP treatment
resulted in
appearance of chondrocyte-like cells on Day 6. No mineralization occurred even
after
Day 25.
23
CA 02324350 2000-09-19
WO 99/49015 PCT/US99/06356
Clone 2H6:
The cells showed the morphology of cardiocytes and other cells. The cells
were negative for alkaline phosphatase staining, acetylated LDL uptake, MF-20
binding
and positive for M0636 binding. The cells showed a mitogenic response to zFGF-
5,
acidic FGF and basic FGF. bFGF was the most potent, and all 3 FGFs were
equally
active. SFM treatment caused reduced growth rate, reduced adherence to
plastic, and
appearance of large polygonal cells. Treatment with ascorbic acid/ (i-GM
resulted in cells
gathering together in 'ridge-like' formations on Day 11. No mineralization
occurred even
after Day 25.
5. Isolation of Cardiac-Derived Stem Cells from Neonatal Mice
1) 1-4 day old neonates (mice) are sacrificed by cervical dislocation and
placed in a
70% ETOH bath. When 10-15 neonates have been collected, one is placed it on
I S its back and a midline sternotomy is performed. Pressure is applied to the
chest
cavity with forceps, the heart popped out and the ventricle easily excised.
The
ventricles are placed in ice cold PBS (50 ml Falcon Tube, with PBS, on ice),
with
approximately 100 ventricles in each tube.
2) When all the hearts have been collected, rinse in PBS several times. Then
heart
tissue is minced with scissors in a minimal volume of PBS, and rinsed again in
PBS several times. (Rinse until all red blood cells and debris is removed).
From step 3) PBS is supplemented with 1% Glucose.
3) a) To begin dissociation, hearts are brought up in 18 ml PBS.
b) 1 ml of each stock solution of DNase/Collagenase is added.
4) Agitate on a rotary shaker at 37°C for 30 minutes. (1. DIGESTION)
5) Discard supernatant from the 1. Digestion, which contains mainly
fibroblasts, red
blood cells and debris.
6) a) To begin dissociation, hearts are brought up in 18 ml PBS.
b) 1 ml of each stock solution of DNase/Collagenase is removed.
7) Agitate on a rotary shaker at 37°C for 20 minutes.
8) Transfer supernatant (20 ml) from tube, to a 50 ml Falcon Tube containing
20 ml
DF20.
Mix carefully (turn tube upside-down).
9) Spin tubes) at 1650 rpm, 4°C for 10 min.
24
CA 02324350 2000-09-19
WO 99/49015 PCT/US99/06356
10) Keep pelleted cells on the bottom, and discard supernatant (~40 ml).
11 ) Add 10 ml of DF10 to pellet, mix well up and down (using the sterile
pipette),
keeping cell suspensions) on ice.
S Repeat steps 6)-11) until all tissue is digested.
12) When dissociation is complete, combine all steps 11).
13) Pellet cells (spin tubes at 1650 rpms 4~C for 10 minutes, discard
supernatant).
14) Resuspend myocytes in 1.082 g/ml Percoll (12 ml Percoll/100 neonates).
1 S) Prepare Percoll Gradient using 10 ml pipette to load from bottom 12 ml
each of
1.050 g/ml, 1.060 g/ml, then 1.082 g/ml (containing cells) Percoll in as many
SO
mI Falcon tubes as needed. (1 tube Percoll Gradient/100 neonates).
16) Centrifuge for 30 minutes at 3000 rpms, room temperature. A Beckman CS-6R
centrifuge is suitable.
18) After the centrifugation step in the Percoll Gradient, 4 bands appear (see
Fig. 2).
19) Aspirate off the upper band, collect (fibroblast layer first, if want to
keep, transfer
into a SO ml Falcon Tube, add same amount of DF10 volume cell suspension
collected), then collect myocyte layer, transfer into a 50 ml Falcon Tube, add
same amount of DF10 as volume cell suspension collected.
20) Mix well but carefully, and spin cells at 1650 rpm, 10 min, room
temperature.
21) Discard supernatant. Add 5 ml DFS/"100 neonates".
22) Incubate myocytes on human fibronectin (HFn) for 1 hr at 37°C. The
remaining
fibroblasts will attach to coated plate within an hour.
23) Collect non-adherent cells, wash the dish several times with DFS to make
sure that
all nonadherent cells are collected.
24) Spin cells 10 min at 1650 rpm, remove supernatant, and resuspend cells in
DFS.
6 Expansion of MSCs from Bone Marrow
Assays were performed to measure the frequency of fibroblast colony
forming units from monkey low density, non-adherent cells isolated from bone
marrow.
This assay is indicative of mesenchymal stem cell frequency.
One half of a 96 well microtiter plate is inoculated with cells at a density
of 10,000 cells/well and the other half of the plate is inoculated with cells
at a density of
CA 02324350 2000-09-19
WO 99/49015 PCTNS99/06356
1,000 cells/well. The culture medium is aMEM (GIBCO-BRL, Gaithersburg, MD), 2%
bovine serum albumin, 10 :~g/ml insulin, 200 ug/ml transferrin, antibiotic and
50 uM 13-
Mercaptoethanol. The cells are incubated at 37°C in 5% C02 for 14 days
and then stained
with toluidine blue to improve cell visibility and examined microscopically.
Positive
wells have at least 50 cells exhibiting a "stromal" morphology, i.e., large,
spread out cells.
The positive control is medium containing 20% fetal bovine serum. Results
demonstrated
that zFGFS, at a concentration of 100 ng/ml increased the frequency of CFU-F
to levels
equivalent to the positive control of 20% FBS.
7. Identification of Cardiac-Derived MSCs for zFGFS
Identification of a putative mesenchymal stem cell as a target (i.e. having a
receptor) for zFGFS was made using FITC-labeled protein and either neonatal
mouse or
fetal lamb (third trimester) heart tissue.
zFGFS, purified as described above, was dialyzed into 0.1 M sodium
1 S bicarbonate pH 9Ø Fluorescein isothiocyanate (FITC; Molecular Probes,
Eugene, OR)
was dissolved at 1 mg/ml in the same buffer without exposure to strong light.
The
mixture was prepared containing 1 mg FITC/l mg zFGFS, and reacted for 1-2
hours in
the dark at room temperature. The reaction was stopped by adding 1 M glycine
to a final
concentration of 0.1 M, then reacted for 1 hour at room temperature. The
mixture was
then dialyzed against 0.1 M sodium bicarbonate to make a 1:500-1:1000 dilution
for 3
hours. The dialysis solution was changed and the process repeated for 3-l8
hours to
remove unlabeled FITC.
Neonatal mouse or fetal iamb heart ventricles were isolated, minced, and
repeatedly washed in phosphate buffered solution (PBS) until all red blood
cells and
debris were removed. The minced ventricles were placed in a solution
containing 18 ml
PBS and 1% glucose and 1 ml of 2% DNAse/Collagenase solution was added. The
mixture was incubated on a shaker for 30 minutes at 37°C. The
supernatant was
discarded and the process was repeated once more. After incubation, the
supernatant
(~20 ml) was transferred to a tube containing 20 ml DF 20 (Dulbecco's Modified
Eagle's
Medium/Ham's Nutrient Mixture F12, 1:1 (GIBCO-BRL, Gaithersburg, MD) and 20%
fetal bovine serum). After mixing, the tubes were centrifuged at 1650 rpms in
a Beckman
CS-6R centrifuge (Beckman, Fullerton, CA) at 4°C for 10 minutes. The
supernatant was
. discarded and the pellet was resuspended in DF 10 (10% FBS). The cells were
kept cold
and spun again and resuspended in 40 ml of DF 10. The cell mixture was passed
over a
40 :m filter (Becton Dickinson, Detroit, MI) and counted using a
hemacytometer.
The cells were incubated in FITC-labeled zFGFS at 4°C for 30
minutes at
a concentration of 2 x 106 cells/1 ug zFGFS. After incubation, the cells were
spun at
26
CA 02324350 2000-09-19
WO 99/49015 PCT/US99/06356
1650 rpms in a Beckman CS-6R centrifuge (Beckman) for 5 minutes. The
supernatant
was discarded and the pellet washed once in 10 ml of DF 10 and resuspended in
4 ml DF
10.
~1 of MACS anti-FITC microbeads (Miltenyi Biotech, Auburn, CA)
5 were mixed with 10' cells in 4 ml of DF 10 and incubated at 4°C for
30 minutes.
MACS positive selection type LS+ separation columns (Miltenyi Biotech)
were washed with 3 ml of MAC buffer (PBS, 0.5% BSA, 2 mM EDTA) and the
celUbead
mixture was washed in 10 ml MAC buffer and then resuspended in 6 ml MAC
buffer.
The cell/bead mixture was divided between the two columns and the first
negative
10 fraction was discarded. 1.5 ml of 0.6 M NaCI was added to each column and
eluted but
not collected. The columns were then washed with 1.5 ml MAC buffer. The cells
bound
with FITC-labeled zFGFS were collected by adding 3 ml MAC buffer, removing the
column from the magnet and flushing out the positive cells using the plunger.
The
positive cell fraction was plated in a T75 flask and 50 ml of plating medium
was added
(DF with 15% FBS and antibiotics). The cells were incubated at 37°C for
1 week and
counted. The yield of positive cells was approximately 0.1 % of original total
cells
counted.
Cells binding FITC-labeled zFGFS were examined by transmission
electronmicroscopy (TEM). The cells were between 3-5 microns in diameter. The
cell
nuclei occupied the majority of the cell volume, and few cytoplasmic
organelles were
apparent. The phenotype identified by TEM identifies the zFGFS-isolated cells
as
primitive mesenchymal stem cells.
8 In vivo Study, of Cardiomvopathic Rats
Rats infused subcutaneously with epinephrine for 2 weeks develop a
cardiomyopathy quite similar to human idiopathic dilated cardiomyopathy
(Deisher et al.,
Am. J. Cardiovasc. Pathol. 5(1):79-88, 1994 and Deisher et al., J. Pharmacol.
Exp. Ther.
266(1) 262-269, 1993.)
The effect of cardiac-derived MSCs on the initiation and progression of
the catecholamine-induced cardiomyopathy is evaluated by administering cardiac-
derived
MSCs isolated from healthy rats with a similar genetic background by infra-
pericardial,
infra-coronary, infra-arterial, infra-ventricular or infra-venous injection to
rats receiving
subcutaneous infusions of epinephrine or saline.
In one protocol, rats (300 gm) are implanted with epinephrine in an
osmotic pump and are injected with cardiac-derived MSCs or control cells and
mortality
is monitored for 2 weeks, at the end of which the rats are sacrificed, the
hearts are
27
CA 02324350 2000-09-19
WO 99!49015 PCT/US99l06356
weighed wet, and fixed in 10% neutral buffered formalin for histology. Prior
to
sacrificing, cardiac function is measured.
Measurement include body weight, heart weight, cardiac fibrosis and
cardiohistomorphometry in epinephrine-infused rats. Cardiac fibrosis is
determined by
scoring Masson's Trichrome stained heart sections. Three sections are scored
for each
heart, and the average score taken. Positive results indicate that infusion of
cardiac-
derived MSCs can be beneficial in the setting of heart failure of varying
etiologies, of
which can include myocardial infarct (MI), idiopathic dilated cardiomyopathy
(IDCM),
hypertrophic cardiomyopathy, viral myocarditis, congenital abnormalities, and
i 0 obstructive diseases.
It is apparent from the forgoing that the invention includes a number of
uses, some of which can be expressed concisely as follows. The invention
provides for
the use of nonadherent cardiac-derived stem or adherent cardiac-derived stem
cells in the
treatment of disease and or in the discovery of drugs for use in the same. The
invention
further provides for the use of nonadherent or adherent cardiacderived stem
cells in the
manufacture of a medicament for treatment of disease.
While the foregoing invention has been described in some detail for
purposes of clarity and understanding, it will be clear to one skilled in the
art from a
reading of this disclosure that various changes in form and detail can be made
without
departing from the true scope of the invention. All publications and patent
documents
cited in this application are incorporated by reference in their entirety for
all purposes to
the same extent as if each individual publication or patent document were so
individually
denoted.
28
