Note: Descriptions are shown in the official language in which they were submitted.
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EMBRYONIC-LIKE STEM CELLS DERIVED FROM
POST-PARTUM MAMMALIAN PLACENTA, AND USES
AND METHODS OF TREATMENT USING SAID CELLS
1. INTRODUCTION
The present invention relates to the use of embryonic-like stem cells that
originate
from a post-partum placenta with conventional cord blood compositions or other
stem or
progenitor cells. The embryonic-like stem cells can be used alone or in a
mixture with other
stem cell populations. In accordance with the present invention, the embryonic-
like stem
cells may be mixed with other stem cell populations, including but not limited
to, umbilical
cord blood, fetal and neonatal hematopoietic stem cells and progenitor cells,
human stem
cells and progenitor cells derived from bone marrow. The embryonic-like stem
cells and
the mixed populations of embryonic-like stem cells and stem cells have a
multitude of uses
and applications, including but not limited to, therapeutic uses for
transplantation,
diagnostic and research uses. The embryonic-like stem cells and the mixed
populations are
also useful in the treatment of diseases or disorders, including vascular
disease, neurological
diseases or disorders, autoimmune diseases or disorders, diseases or disorders
involving
inflammation, and cancer or the disorders associated therewith. In particular,
the
embryonic-like stem cells or mixtures including them are administered in high
doses and
without HLA typing.
2. BACKGROUND OF THE INVENTION
There is considerable interest in the identification, isolation and generation
of human
stem cells. Human stem cells are totipotential or pluripotential precursor
cells capable of
generating a variety of mature human cell lineages. This ability serves as the
basis for the
cellular differentiation and specialization necessary for organ and tissue
development.
Recent success at transplanting such stem cells have provided new clinical
tools to
reconstitute and/or supplement bone marrow after myeloablation due to disease,
exposure to
toxic chemical and/or radiation. Further evidence exists that demonstrates
that stem cells
can be employed to repopulate many, if not all, tissues and restore
physiologic and anatomic
functionality. The application of stem cells in tissue engineering, gene
therapy delivery and
cell therapeutics is also advancing rapidly.
Many different types of mammalian stem cells have been characterized. For
example, embryonic stem cells, embryonic germ cells, adult stem cells or other
committed
stem cells or progenitor cells are known. Certain stem cells have not only
been isolated and
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characterized but have also been cultured under conditions to allow
differentiation to a
limited extent. A basic problem remains, however, in that obtaining sufficient
quantities
and populations of human stem cells which are capable of differentiating into
all cell types
is near impossible. Stem cells are in critically short supply. These are
important for the
treatment of a wide variety of disorders, including malignancies, inborn
errors of
metabolism, hemoglobinopathies, and immunodeficiencies. It would be highly
advantageous to have a source of more embryonic stem cells.
Obtaining sufficient numbers of human stem cells has been problematic for
several
reasons. First, isolation of normally occurnng populations of stem cells in
adult tissues has
been technically difficult and costly due, in part, to very limited quantity
found in blood or
tissue. Secondly, procurement of these cells from embryos or fetal tissue,
including
abortuses, has raised religious and ethical concerns. The widely held belief
that the human
embryo and fetus constitute independent life has prompted governmental
restrictions on the
use of such sources for all purposes, including medical research. Alternative
sources that do
not require the use of cells procured from embryonic or fetal tissue are
therefore essential
for further progress in the use of stem cells clinically. There are, however,
few viable
alternative sources of stem cells, particularly human stem cells, and thus
supply is limited.
Furthermore, harvesting of stem cells from alternative sources in adequate
amounts for
therapeutic and research purposes is generally laborious, involving, e.g.,
harvesting of cells
or tissues from a donor subject or patient, culturing and/or propagation of
cells iya vitro,
dissection, etc.
For example, Caplan et al. (U.S. Patent No. 5,46,359 entitled "Human
mesenchymal stem cells," issued January 23, 1996), discloses human mesenchymal
stem
cell (hMSC) compositions derived from the bone maxrow that serve as the
progenitors for
mesenchymal cell lineages. Caplan et al. discloses that hMSCs are identified
by specific
cell surface markers that are identified with monoclonal antibodies.
Homogeneous hMSC
compositions are obtained by positive selection of adherent marrow or
periosteal cells that
are free of markers associated with either hematopoietic cell or
differentiated mesenchymal
cells. These isolated mesenchymal cell populations display epitopic
characteristics
associated with mesenchymal stem cells, have the ability to regenerate in
culture without
differentiating, and have the ability to differentiate into specific
mesenchymal lineages
when either induced ih. vitro or placed in vivo at the site of damaged tissue.
The drawback
of such methods, however, is that they require harvesting of marrow or
periosteal cells from
a donor, from which the MSCs must be subsequently isolated.
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Hu et al. (WO 00/73421 entitled "Methods of isolation, cryopreservation, and
therapeutic use of human amniotic epithelial cells," published December 7,
2000) discloses
human amniotic epithelial cells derived from placenta at delivery that are
isolated, cultured,
cryopreserved for future use, or induced to differentiate. According to Hu et
al. a placenta
is harvested immediately after delivery and the amniotic membrane separated
from the
chorion, e.g., by dissection. Amniotic epithelial cells are isolated from the
amniotic
membrane according to standard cell isolation techniques. The disclosed cells
can be
cultured in various media, expanded in culture, cryopreserved, or induced to
differentiate.
Hu et al. discloses that amniotic epithelial cells are multipotential (and
possibly
pluripotential), and can differentiate into epithelial tissues such as corneal
surface
epithelium or vaginal epithelium. The drawback of such methods, however, is
that they are
labor-intensive and the yield of stem cells is very low. For example, to
obtain sufficient
numbers of stem cells for typical therapeutic or research purposes, amniotic
epithelial cells
must be first isolated from the amnion by dissection and cell separation
techniques, then
cultured and expanded in vitro.
Umbilical cord blood ("cord blood") is a known alternative source of
hematopoietic
progenitor stem cells. Stem cells from cord blood are routinely cryopreserved
for use in
hematopoietic reconstitution, a widely used therapeutic procedure used in bone
marrow and
other related transplantations (see e.g., Boyse et al., U.S. 5,004,681,
"Preservation of Fetal
and Neonatal Hematopoietin Stem and Progenitor Cells of the Blood", Boyse et
al., U.S.
Patent No. 5,192,553, entitled "Isolation and preservation of fetal and
neonatal
hematopoietic stem and progenitor cells of the blood and methods of
therapeutic use",
issued March 9, 1993). Conventional techniques for the collection of cord
blood are based
on the use of a needle or cannula, which is used with the aid of gravity to
drain cord blood
from (i.e., exsanguinate) the placenta (Boyse et al., U.S. Patent No.
5,192,553, issued
March 9, 1993; Boyse et al., U.S. Patent No. 5,004, 681, issued April 2, 1991;
Anderson,
U.S. Patent No. 5,372,581, entitled Method and apparatus for placental blood
collection,
issued December 13, 1994; Hessel et al., U.S. Patent No. 5,415,665, entitled
Umbilical cord
clamping, cutting, and blood collecting device and method, issued May 16,
1995). The
needle or cannula is usually placed in the umbilical vein and the placenta is
gently massaged
to aid in draining cord blood from the placenta. Thereafter, however, the
drained placenta
has been regarded as having no further use and has typically been discarded. A
major
limitation of stem cell procurement from cord blood, moreover, has been the
frequently
inadequate volume of cord blood obtained, resulting in insufficient cell
numbers to
effectively reconstitute bone marrow after transplantation.
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Naughton et al. (U.S. Patent No. 5,962,325 entitled "Three-dimensional stromal
tissue cultures" issued October 5, 1999) discloses that fetal cells, including
fibroblast-like
cells and chondrocyte-progenitors, may be obtained from umbilical cord or
placenta tissue
or umbilical cord blood.
I~raus et al. (U.S. Patent No. 6,338,942, entitled "Selective expansion of
target cell
populations", issued January 15, 2002) discloses that a predetermined target
population of
cells may be selectively expanded by introducing a starting sample of cells
from cord blood
or peripheral blood into a growth medium, causing cells of the target cell
population to
divide, and contacting the cells in the growth medium with a selection element
comprising
binding molecules with specific affinity (such as a monoclonal antibody for
CD34) for a
predetermined population of cells (such as CD34 cells), so as to select cells
of the
predetermined target population from other cells in the growth medium.
Rodgers et al. (U.S. Patent No. 6,335,195 entitled "Method for promoting
hematopoietic and mesenchylnal cell proliferation and differentiation," issued
January 1,
2002) discloses methods for ex vivo culture of hematopoietic and mesenchymal
stem cells
and the induction of lineage-specific cell proliferation and differentiation
by growth in the
presence of angiotensinogen, angiotensin I (AI), AI analogues, AI fragments
and analogues
thereof, angiotensin II (AII), All analogues, All fragments or analogues
thereof or All ATZ
type 2 receptor agonists, either alone or in combination with other growth
factors and
cytokines. The stem cells are derived from bone marrow, peripheral blood or
umbilical cord
blood. The drawback of such methods, however, is that such ex vivo methods for
inducing
proliferation and differentiation of stem cells are time-consuming, as
discussed above, and
also result in low yields of stem cells.
Because of restrictions on the collection and use of stem cells, and the
inadequate
numbers of cells typically collected from cord blood, stem cells are in
critically short
supply. Stem cells have the potential to be used in the treatment of a wide
variety of
disorders, including malignancies, inborn errors of metabolism,
hemoglobinopathies, and
immunodeficiencies. There is a critical need for a readily accessible source
of large
numbers of human stem cells for a variety of therapeutic and other medically
related
purposes. The present invention addresses that need and others.
Additionally, there remains a need for the treatment of neurological
conditions such
as amylotrophic lateral sclerosis (ALS). Although recent studies using
irradiated mouse
models of familial ALS, a less-common form of ALS, have suggested that cord
blood may
be useful in the treatment of this disease, the source issue discussed above
makes this option
less than ideal. See Ende et al., Life Sci. 67:53059 (2000). Thus, there
remains a need for
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stem or progenitor cell populations that can be used to treat diseases,
particularly larger
amounts of these populations when diseases such as ALS are being treated.
3. SUMMARY OF THE INVENTION
The present invention relates to cord blood compositions or stem or progenitor
cells
therefrom in which said compositions are supplemented with or contacted with
embryonic-
like stem cells that originate from a post-partum placenta. The embryonic-like
stem cells
which are the subject of other applications can be used herein as a
composition or a mixture
With other stem or progenitor cell populations. In accordance with the present
invention,
the embryonic-like stem cells may contacted with other stem or progenitor cell
populations,
including but not limited to, umbilical cord blood, fetal and neonatal
hematopoietic stem
cells and progenitor cells, human stem cells and progenitor cells derived from
bone marrow.
The embryonic-like stem cells and the mixed populations of embryonic-like stem
cells and
stem or progenitor cells have a multitude of uses and applications, including
but not limited
to, therapeutic uses for transplantation and treatment and prevention of
disease, and
diagnostic and research uses.
In accordance with the present invention, populations of stem cells are mixed
with
populations of embryonic-like stem cells in order to supplement, augment or
enhance the
concentrations of pluripotent and multipotent stem cells in the stem cell
populations. for
example, in one embodiment, umbilical cord blood, or stem or progenitor cells
therefrom, is
augmented or contacted with the embryonic-like stem cells of the invention
prior to
administration to the patient. It is recognized that the embryonic-like stem
cells may also be
administered simultaneously or sequentially with the umbilical cord blood, or
cells
therefrom, However, contacting the cells of each before administration is
preferred.
The embryonic-like stem cells of the invention may be characterized by the
presence
of the following cell surface markers: CD10, CD29, CD44, CD54, CD90, SH2, SH3,
SH4,
OCT-4 and ABC-p, and the absence of the following cell surface markers: CD34,
CD38,
CD45, SSEA3 and SSEA4. In a preferred embodiment, such embryonic-like stem
cells may
be characterized by the presence of cell surface markers OCT-4 and APC-p.
Embryonic-
like stem cells originating from placenta have characteristics of embryonic
stem cells but
are not derived from the embryo. In other words, the invention encompasses
mixtures of
cord blood and embryonic-like stem cells isolated from a placenta that are OCT-
4+ and/or
ABC-p+. Such embryonic-like stem cells are as versatile (e.g., pluripotent) as
human
embryonic stem cells.
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Tn accordance with the present invention, populations of stem cells are mixed
with
embryonic-like stem cells that are pluripotent or multipotent. Such embryonic-
like stem
cells can be isolated from the perfused placenta at different time points
e.g., CD34+
/CD38+, CD34+/CD38-, and CD34-/CD38- hematopoietic cells. In one embodiment,
such
cells may be used to supplement populations of hematopoietic stem cells, such
as those
found in umbilical cord blood, according to the methods of the invention.
The invention also provides a composition in which a mixture of stem cells
with
embryonic-like stem cells is contained within one bag or container. In a
preferred
embodiment, the composition is a pharmaceutically acceptable unit dose
composition. In
another embodiment, the invention provides a composition in which a population
of stem
cells and a population of embryonic-like stem cells are contained within two
separate bags
or containers. In certain embodiments, such a "two bag" kit may be mixed
prior, in
particular immediately prior to, or at the time of administration to a patient
in need thereof.
In other embodiments, the contents of each bag may be administered separately
to a patient,
wherein the mixing of the two cell populations occurs ifz vivo. In other
embodiments, the
container is sealed, air tight, and sterile.
The present invention relates to populations of stem cells are mixed with
embryonic-
like stem cells. 111 accordance with the present invention, stems cells that
may be mixed
with embryonic-like stem cells include, but are not limited to, umbilical cord
blood, fetal
and neonatal hematopoietic stem cells and progenitor cells, human stem cells
and progenitor
cells derived from bone marrow. In a preferred embodiment of the present
invention, the
embryonic-like stem cells of the invention are mixed with umbilical cord
blood.
The present invention also provides methods of treating a patient in need
thereof by
administration of a population of stem cells supplemented with embryonic-like
stem cells.
In one embodiment, the supplementation of the population of cord blood cells
with
embryonic-like stem cells occurs by mixing the stem cells and embryonic-like
stem cells
prior to administration of the combined or "spiked" population to the patient.
In another
embodiment, the supplementation of the population of stem cells with embryonic-
like stem
cells occurs upon administration of the supplemented population to the
patient, e.g., by
simultaneous administration of the cord blood cells and the embryonic-Iike
stem cells. In
another embodiment, the supplementation of the population of stem cells with
embryonic-
Iike stem cells occurs after administration of the cord blood cells to the
patient, e.g., by
administering the embryonic-stern cells separately from, and before or after,
administration
of the stem cells.
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According to the invention, populations of stem cells, e.g., umbilical cord
blood,
supplemented with embryonic-like stem cells from the placenta have a multitude
of uses,
including prophylactic, therapeutic and diagnostic uses. The supplemented
populations of
stem cells can be used for transplantation and/or to treat or prevent disease.
In one
embodiment of the invention, the supplemented populations of cells are used to
renovate
and repopulate tissues and organs, thereby replacing or repairing diseased
tissues, organs or
portions thereof. In another embodiment, the supplemented populations of stem
cells can
be used as a diagnostic to screen for genetic disorders or a predisposition
for a particular
disease or disorder.
In another embodiment, the invention provides a method for isolating other
embryonic-like and/or multipotent or pluripotent stem cells from an extract or
perfusate of a
exsanguinated placenta and using them to supplement populations of cord blood
cells
according to the methods of the invention.
The present invention also provides pharmaceutical compositions that comprise
populations of stem cells, e.g., umbilical cord blood cells, that have been
supplemented with
one or more populations of embryonic-like stem cells of the invention.
The present invention provides an isolated homogenous population of human
placental stem cells that has the potential to differentiate into all cell
types. In another
embodiment, the population of human placental stem cells has the potential to
differentiate
into one cell type. In yet another embodiment, the population of human
placental stem cells
has the potential to differentiate into several different cell types. Such
cells may be used to
supplement populations of stem cells, e.g., umbilical cord blood, according to
the methods
of the invention.
The invention also encompasses pharmaceutical compositions that comprise
populations of hematopoietic stem cells supplemented with one or more
populations of cells
that have high concentrations (or larger populations) of homogenous
hematopoietic stem
cells including, but not limited to, CD34+ /CD38- cells; CD34-/CD38- cells,
and CD133+
cells. One or more of these cell populations can be used with, or mixed with,
hematopoietic
stem cells i.e., CD34+/CD38+ hematopoietic cells, obtained from umbilical cord
blood or
other sources, for transplantation and other uses.
The present invention also provides methods of mixing a population of stem,
progenitor or cord blood cells, including banked or cryopreserved cord blood
cells, with a
population of embryonic-like stem cells. In one embodiment, the two
populations are
physically mixed. In another aspect of this embodiment, the two populations
are physically
mixed and then treated with a growth factor, e.g., a cytokine andlor an
interleukin, to induce
cell differentiation. In another aspect of this embodiment, the stem cells
and/or the
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embryonic-like stem cells are treated with a growth factor, e.g., a cytokine
and/or an
interleukin, to induce cell differentiation and then physically mixed. In one
embodiment,
the mixed populations are treated with a growth factor to induce
differentiation into a
variety of cell types. In another embodiment, the mixed populations are
treated with a
growth factor to induce differentiation into a particular cell type. In
another embodiment,
the mixed populations are treated with a growth factor to prevent or suppress
differentiation
into a particular cell type. In certain embodiments, the culture conditions
can be controlled,
e.g., the mixed population of cells can be treated with a specific cocktail of
cytokines or
interleukins to direct or induce differentiation to a specific cell type.
In another embodiment, the invention provides a method of treating a patient
in need
thereof comprising administration of a plurality of umbilical cord blood cells
and a plurality
of embryonic-like stem cells.
In another embodiment, the invention provides a method of treating
myelodysplasia
which comprises administering umbilical cord blood cells (or stem cells
isolated therefrom)
and embryonic-like stem cells to a patient in need thereof.
The invention also relates to new uses of human placental stem cells
(embryonic-like stem
cells). Methods of treating or preventing disease with the compositions
containing
embryonic-like stem cells and other stem or progenitor cells or sources
thereof are also
encompassed herein. Similarly, methods of dosing such compositions are
encompassed.
finally, it should be noted that the compositions of the invention can contain
stem or
progenitor cell populations from multiple donors. The invention includes the
use of non-
HLA matched compositions in patients as well as HLA-matched compositions.
blood type
matching with the patient is preferred but not required when the compositions
containing
both embryonic-like stem cells and stem or progenitor cells are used.
3.1. DEFINITIONS
As used herein, the term "bioreactor" refers to an ex vivo system for
propagatilig
cells, producing or expressing biological materials and growing or culturing
cells tissues,
organoids, viruses, proteins, polynucleotides and microorganisms.
As used herein, the terms "cord blood" and "umbilical cord blood" are
interchangeable.
As used herein, the term "embryoni c stem cell" refers to a cell that is
derived from
the inner cell mass of a blastocyst (e.g., a 4- to 5-day-oId human embryo) and
that is
pluripotent.
As used herein, the term "embryonic-like stem cell" refers to a cell that is
not
derived from the inner cell mass of a blastocyst. As used herein, an
"embryonic-Iike stem
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cell" may also be referred to as a "placental stem cell," preferably a human
placental stem
cell derived from a post-partum perfused placenta. An embryonic-like stem cell
is
preferably pluripotent. However, the stem cells which may be obtained from the
placenta
include embryonic-Iike stem cells, multipotent cells, and committed progenitor
cells.
According to the methods of the invention, embryonic-like stem cells derived
from the
placenta may be collected from the isolated placenta once it has been
exsanguinated and
perfused for a period of time sufficient to remove residual cells.
As used herein, the term "exsanguinated" or "exsanguination," when used with
respect to the placenta, refers to the removal and/or draining of
substantially all cord blood
from the placenta. In accordance with the present invention, exsMguination of
the placenta
can be achieved by, for example, but not by way of limitation, draining,
gravity induced
efflux, massaging, squeezing, pumping, etc. In a preferred embodiment,
exsMguination of
the placenta may fiu ther be achieved by perfusing, rinsing or flushing the
placenta with a
fluid that may or may not contain agents, such as anticoagulants, to aid in
the
exsanguination of the placenta.
As used herein, the term to "mix" means to combine or blend into one mass or
mixture; to put together into one mass so that the constituent parts are more
or less
homogeneous; to create or form by combining ingredients; to form by admixture,
augmentation, supplementation, or commingling; or to add an ingredient or
element to
Mother ingredient or element, and vice-versa.
As used herein, the term "perfuse" or "perfusion" refers to the act of pouring
or
passaging a fluid over or through an organ or tissue, preferably the passage
of fluid through
an organ or tissue with sufficient force or pressure to remove any residual
cells, e.g., non-
attached cells from the organ or tissue. As used herein, the term "perfusate"
refers to the
fluid collected following its passage through an organ or tissue. In a
preferred embodiment,
the perfusate contains one or more anticoagulants.
As used herein, the term "exogenous cell" refers to a "foreign" cell, i.e., a
heterologous cell (i. e., a "non-self ' cell derived from a source other than
the placental
donor) or autologous cell (i.e., a "self' cell derived from the placental
donor) that is-derived
from M organ or tissue other than the placenta.
As used herein, the term "organoid" refers to an aggregation of one or more
cell
types assembled in superficial appearance or in actual structure as any organ
or gland of a
mammalian body, preferably the human body.
As used herein, the term "multipotent cell" refers to a cell that has the
capacity to
grow into any of subset of the mammalian body's approximately 260 cell types.
Unlike a
pluripotent cell, a multipotent cell does not have the capacity to form all of
the cell types.
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As used herein, the term "pluripotent cell" refers to a cell that has complete
differentiation versatility, i. e., the capacity to grow into any of the
mammalian body's
approximately 260 cell types. A pluripotent cell can be self renewing, and can
remain
dormant or quiescent within a tissue. Unlike a totipotent cell (e.g., a
fertilized, diploid egg
cell), an embryonic stem cell cannot usually form a new blastocyst.
As used herein, the term "progenitor cell" refers to a cell that is committed
to
differentiate into a specific type of cell or to form a specific type of
tissue.
As used herein, the term "stem cell" refers to a master cell that can
reproduce
indefinitely to form the specialized cells of tissues and organs. A stem cell
is a
l 0 developmentally pluripotent or multipotent cell. A stem cell can divide to
produce two
daughter stem cells, or one daughter stem cell and one progenitor ("transit")
cell, which
then proliferates into the tissue's mature, fully formed cells. The "stem
cell" used herein
includes "progenitor cells" unless otherwise noted.
As used herein, the term "totipotent cell" refers to a cell that is able to
form a
15 complete embryo (e.g., a blastocyst).
4. DETAILED DESCRIPTION OF THE INVENTION
The present invention is based in part on the unexpected discovery that
embryonic-
like stem cells produced by the exsanguinated, perfused and/or cultured
placenta are
20 pluripotent stem cells that can be readily differentiated into any desired
cell type. These
embryonic-like stem cells can be used to supplement, augment or enhance
populations of
stem cells, including, but not limited to umbilical cord blood, fetal and
neonatal
hematopoietic stem cells and progenitor cells, human stem cells and progenitor
cells derived
from bone marrow. In accordance with the present invention, populations of
stem cells are
2$ mixed with populations of embryonic-Like stem cells in order to supplement,
augment or
enhance the concentrations of pluripotent and multipotent stem cells in the
stem cell
populations. In accordance with the present invention, the populations of stem
cells mixed
with populations of embryonic-like stem cells have a multitude of uses and
applications,
including but not limited to, therapeutic uses for transplantation and
treatment and
30 prevention of disease, and diagnostic and research uses.
The invention also provides a composition in which a mixture of stem cells and
embryonic-like stem cells is contained within one bag or container. In another
embodiment,
the invention provides a composition in which a population of stem cells and a
population
of embryonic-like stem cells are contained within two sepaxate bags or
containers. In
35 certain embodiments, such a "two bag" composition may be mixed prior, in
particular
immediately prior, to or at the time of administration to a patient in need
thereof. In other
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embodiments, the contents of each bag may be administered separately to a
patient, wherein
two cell populations are used adjunctively ih vivo.
The present invention also provides methods of mixing a population of stem or
progenitor cells or cord blood includiilg banked or cryopreserved cord blood
with a
population of embryonic-like stem cells. In one embodiment, the two
populations are
physically mixed. In another aspect of this embodiment, the two populations
are physically
mixed and then treated with a growth factor, e.g., a cytokine and/or an
interleukin, to induce
cell differentiation. In another aspect of this embodiment, the stem cells
and/or the
embryonic-like stem cells are treated with a growth factor, e.g., a cytokine
and/or an
interleukin, to induce cell differentiation and then physically mixed.
The present invention also provides methods of mixing a population of
committed
cells, e.g., a population of cells committed to differentiate into neurons,
muscle cells,
hematopoietic, vascular cells, adipocytes, chondrocytes, osteocytes,
hepatocytes, pancreatic,
or cardiac cells, with a population of embryonic-like stem cells. In one
embodiment, the
~o populations are physically mixed. In another aspect of this embodiment, the
two
populations are physically mixed and then treated with a growth factor, e.g.,
a cytokine
and/or an interleukin, to induce cell differentiation. In another aspect of
this embodiment,
the committed cells andlor the embryonic-like stem cells are treated with a
growth factor,
e.g., a cytokine and/or an interleukin, to induce cell differentiation and
then physically
mixed.
According to the methods of the invention, embryonic-like stem cells are
extracted
from a drained placenta by means of a perfusion technique that utilizes either
or both of the
umbilical artery and the umbilical vein. The placenta is preferably drained by
exsanguination and collection of residual blood (e.g., residual umbilical cord
blood). The
drained placenta is then processed in such a manner as to establish the ex
vivo, natural
bioreactor environment in which the resident embryonic-like stem cells within
the
parenchyma and extravascular space are recruited. The embryonic-like stem
cells migrate
into the drained, empty microcirculation where, according to the methods of
the invention,
they are collected, preferable by washing into a collecting vessel by
perfusion.
As disclosed above, a number of different pluripotent or multipotent stem
cells can
be isolated from the perfused placenta at different time points during the
perfusion, e.g.,
CD34+ /CD38+, CD34+ /CD38-, and CD34-/CD38- hematopoietic cells. In one
embodiment, such cells may be used to supplement populations of stem cells,
e.g., cord
blood cells, according to the methods of the invention.
The present invention further provides an isolated homogenous population of
human
placental stem cells that has the potential to differentiate into all cell
types. In another
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embodiment, the population of human placental stem cells has the potential to
differentiate
into one cell type. In yet another embodiment, the population of human
placental stem cells
has the potential to differentiate into several different cell types. Such
cells may be used to
supplement populations of stem cells, e.g., cord blood cells, according to the
methods of the
invention.
The present invention also provides methods of mixing a population of stem
cells
with a population of embryonic-like stem cells. W one embodiment, the two
populations
are physically mixed. In another aspect of this embodiment, the two
populations are
physically mixed and then treated with a growth factor, e.g., a cytokine
and/or an
interleukin, to induce cell differentiation. In another aspect of this
embodiment, the stem
cells and/or the embryonic-like stem cells are treated with a growth factor,
e.g., a cytokine
and/or an interleukin, to induce cell differentiation and then physically
mixed. In one
embodiment, the mixed populations are treated with a growth factor to induce
differentiation into a variety of cell types. In another embodiment, the mixed
populations
~'e heated with a growth factor to induce differentiation into a particular
cell type. In
another embodiment, the mixed populations are treated with a growth factor to
prevent or
suppress differentiation into a particular cell type. Tn certain embodiments,
the culture
conditions can be controlled, e.g., the mixed population of cells can be
treated with a
specific cocktail of cytokines or interleukins to direct or induce
differentiation to a specific
cell type.
The present invention provides pharmaceutical compositions that comprise
populations of stem cells, e.g., cord blood cells, that have been supplemented
with one or
more populations of embryonic-like stem cells of the invention.
The invention also encompasses pharmaceutical compositions that comprise
populations of stem cells, e.g., cord blood cells, supplemented with one or
more populations
of cells that have high concentrations (or larger populations) of homogenous
hematopoietic
stem cells including, but not limited to, CD34+ /CD38- cells; and CD34-/ CD38-
cells. One
or more of these cell populations can be used with, or mixed with, cord blood
hematopoietic
cells, i. e., CD34+/CD38+ hematopoietic cells for transplantation and other
uses.
According to the invention, populations of stem cells, e.g., umbilical cord
blood,
supplemented with embryonic-like stem cells from the placenta have a multitude
of uses,
including therapeutic and diagnostic uses. The supplemented populations of
stem cells can
be used for transplantation or to treat or prevent disease. In one embodiment
of the
invention, the supplemented populations of cells are used to renovate and
repopulate tissues
and organs, thereby replacing or repairing diseased tissues, organs or
portions thereof. In
another embodiment, the supplemented populations of stem cells can be used as
a
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diagnostic to screen for genetic disorders or a predisposition for a
particular disease or
disorder.
The present invention also provides methods of treating a patient in need
thereof by
administration of a population of stem cells supplemented with embryonic-like
stem cells.
In one embodiment, the supplementation of the population of cord blood cells
with
embryonic-like stem cells occurs by mixing the stem cells and embryonic-like
stem cells
prior to administration of the supplemented population to the patient. In
another
embodiment, the supplementation of the population of stem cells with embryouc-
like stem
cells occurs upon administration of the supplemented population to the
patient, e.g., by
simultaneous administration of the cord blood cells and the embryonic-like
stem cells. In
another embodiment, the supplementation of the population of stem cells with
embryonic-
like stem cells occurs after administration of the cord blood cells to the
patient, e.g., by
administering the embryonic-stem cells separately from, and before or after,
administration
of the stem cells.
4.1. METHODS OF ISOLATING AND CULTURING PLACENTA
4.I.1. Pretreatment of Placenta
According to the methods of the invention, a human placenta is recovered
shortly
after its expulsion after birth and, in certain embodiments, the cord blood in
the placenta is
recovered. In certain embodiments, the placenta is subjected to a conventional
cord blood
recovery process. Such cord blood recovery may be obtained commercially, e.g.,
LifeBank
Inc., Cedar Knolls, N.J., ViaCord, Cord Blood Registry and Cryocell. The cord
blood can
be drained shortly after expulsion of the placenta.
In other embodiments, the placenta is pretreated according to the methods
disclosed
in co-pending application No. 10/076,180, filed February 13, 2002, which is
incorporated
herein by reference in its entirety.
4.1.2. Exsanguination of Placenta and Removal of Residual Cells
As disclosed in PCT publication WO 02/064755, published August 22, 2002, which
is incorporated herein by reference in its entirety, the placenta after birth
contains quiescent
cells that can be activated if the placenta is properly processed after birth.
For example,
after expulsion from the womb, the placenta is exsanguinated as quickly as
possible to
prevent or minimize apoptosis. Subsequently, as soon as possible after
exsanguination the
placenta is perfused to remove blood, residual cells, proteins, factors and
any other
materials present in the organ. Materials debris may also be removed from the
placenta.
Perfusion is normally continued with an appropriate perfusate for at Ieast two
to more than
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twenty-four hours. The placenta can therefore readily be used as a rich and
abundant source
of embryonic-like stem cells, which cells can be used for research, including
drug
discovery, treatment and prevention of diseases, in particular transplantation
surgeries or
therapies, and the generation of committed cells, tissues and organoids.
Further, surprisingly and unexpectedly, the human placental stem cells
produced by
the exsanguinated, perfused and/or cultured placenta are pluripotent stem
cells that can
readily be differentiated into any desired cell type.
According to the methods of the invention, stem or progenitor cells,
including, but
not limited to embryonic-like stem cells, may be recovered from a placenta
that is
exsanguinated, i.e., completely drained of the cord blood remaining after
birth and/or a
conventional cord blood recovery procedure. According to the methods of the
invention,
the methods for exsanguination of the placenta and removal of residual cells
may be
accomplished using any method known in the art, e.g., the methods disclosed in
PCT
publication WO 02/064755, published August 22, 2002, which is incorporated
herein by
reference in its entirety.
4.1.3. Culturing Placenta
After exsanguination and a sufficient time of perfusion of the placenta, the
embryonic-like stem cells are observed to migrate into the exsanguinated and
perfused
microcirculation of the placenta where, according to the methods of the
invention, they are
collected, preferably by washing into a collecting vessel by perfusion. In
other
embodiments, the placenta is cultured, and the cells propagated are monitored,
sorted and/or
characterized according to the methods described in PCT publication WO
02/064755,
published August 22, 2002, which is incorporated herein by reference in its
entirety.
4.2. COLLECTION OF CELLS FROM THE PLACENTA
After exsanguination and perfusion of the placenta, embryonic-like stem cells
migrate into the drained, empty microcirculation of the placenta where,
according to the
invention, they axe collected, preferably by collecting the effluent perfusate
in a collecting
vessel.
In preferred embodiments, cells cultured in the placenta are isolated from the
effluent perfusate using techniques known by those skilled in the art, such
as, for example,
density gradient centrifugation, magnet cell separation, flow cytometry, or
other cell
separation or sorting methods well known in the art, and sorted.
In a specific embodiment, the embryonic-like stem cells are collected from the
placenta and, in certain embodiments, preserved, according to the methods
described in
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PCT publication WO 02/064755, published August 22, 2002, which is incorporated
herein
by reference in its entirety.
4.3. EMBRYONIC-LIKE STEM CELLS
Embryonic-like stem cells obtained in accordance with the methods of the
invention
may include pluripotent cells, i.e., cells that have complete differentiation
versatility, that
are self renewing, and can remain dormant or quiescent within tissue. The stem
cells which
may be obtained from the placenta include embryonic-like stem cells,
multipotent cells,
committed progenitor cells, and fibroblastoid cells.
The first collection of blood from the placenta is referred to as cord blood
which
contains predominantly CD34+ and CD38+ hematopoietic progenitor cells. Within
the frst
twenty-four hours of post-partum perfusion, high concentrations of CD34+ and
CD38-
hernatopoietic progenitor cells may be isolated from the placenta, along with
high
concentrations of CD34- and CD38+ hematopoietic progenitor cells. After about
twenty-
four hours of perfusion, high concentrations of CD34- and CD38- cells can be
isolated from
the placenta along with the aforementioned cells. The isolated perfused
placenta of the
invention provides a source of large quantities of stem cells enriched for
CD34+ and CD38-
stem cells and CD34- and CD38+ stem cells. The isolated placenta which has
been
perfused for twenty-four hours or more provides a source of large quantities
of stem cells
einiched for CD34- and CD38- stem cells.
In a preferred embodiment, embryonic-like stem cells obtained by the methods
of
the invention are viable, quiescent, pluripotent stem cells that exist within
a full-teen
human placenta and that can be recovered following successful birth and
placental
expulsion, resulting in the recovery of as many as one billion nucleated
cells, which yield
50-100 million multipotent and pluripotent stem cells.
The human placental stem cells provided by the placenta are surprisingly
embryonic-like, for example, the presence of the following cell surface
markers have been
identified for these cells: SSEA3-, SSEA4-, OCT-4+ and ABC-p+. Preferably, the
embryonic-like stem cells of the invention are characterized by the presence
of OCT-4+ and
~C_p+ cell surface markers. Thus, the invention encompasses stem cells which
have not
been isolated or otherwise obtained from an embryonic source but which can be
identified
by the following markers: SSAE3-, SSAE4-, OCT-4+ and ABC-p+. In one
embodiment,
the human placental stem cells do not express MHC Class 2 antigens.
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The stem cells isolated from the placenta are homogenous, and sterile.
Further, the
stem cells are readily obtained in a form suitable for administration to
humans, i.e., they are
of pharmaceutical grade.
Preferred embryonic-like stem cells obtained by the methods of the invention
may
be identified by the presence of the following cell surface markers: OCT-4+
and ABC-pt.
Further, the invention encompasses embryonic stem cells having the following
markers:
CD10+, CD38-, CD29+, CD34-, CD44+, CD45-, CD54+, CD90+, SH2+, SH3+, SH4+,
SSEA3-, SSEA4-, OCT-4+, and ABC-p+. Such cell surface markers are routinely
determined according to methods well known in the art, e.g. by flow cytometry,
followed by
washing and staining with an anti-cell surface marker antibody. For example,
to determine
the presence of CD-34 or CD-38, cells may be washed in PBS and then double-
stained with
anti-CD34 phycoerythrin and anti-CD38 fluorescein isothiocyanate (Becton
Dickinson,
Mountain View, CA).
In another embodiment, cells cultured in the placenta bioreactor are
identified and
characterized by a colony forming unit assay, which is commonly known in the
art, such as
Mesen CuItTM medium (stem cell Technologies, Inc., Vancouver British Columbia)
The embryonic-like stem cells obtained by the methods of the invention may be
induced to differentiate along specific cell lineages, including adipogenic,
chondrogenic,
osteogenic, hematopoietic, myogenic, vasogenic, neurogenic, and hepatogenic.
In certain
embodiments, embryonic-Iike stem cells obtained according to the methods of
the invention
are induced to differentiate for use in transplantation and ex vivo treatment
protocols. In
certain embodiments, embryouc-like stem cells obtained by the methods of the
invention
are induced to differentiate into a particular cell type and genetically
engineered to provide a
therapeutic gene product. In a specific embodiment, embryonic-like stern cells
obtained by
the methods of the invention are incubated with a compound iya vitf°o
that induces it to
differentiate, followed by direct transplantation of the differentiated cells
to a subj ect. Thus,
the invention encompasses methods of differentiating the human placental stem
cells using
standard culturing media. Further, the invention encompasses hematopoietic
cells, neuron
cells, fibroblast cells, strand cells, mesenchymal cells and hepatic cells.
Embryonic-like stem cells may also be further cultured after collection from
the
placenta using methods well known in the art, for example, by culturing on
feeder cells,
such as irradiated fibroblasts, obtained from the same placenta as the
embryonic-like stem
cells or from other human or nonhuman sources, or in conditioned media
obtained from
cultures of such feeder cells, in order to obtain continued long-term cultures
of embryonic-
like stem cells. The embryonic-like stem cells may also be expanded, either
within the
placenta before collection from the placental bioreactor or ih vitro after
recovery from the
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placenta. In certain embodiments, the embryonic-like stem cells to be expanded
are
exposed to, or cultured in the presence of, an agent that suppresses cellular
differentiation.
Such agents are well-known in the art and include, but are not limited to,
human Delta-1
and human Serrate-1 polypeptides (see, Sakano et al., U.S. Patent No.
6,337,387 entitled
"Differentiation-suppressive polypeptide", issued January 8, 2002), leukemia
inhibitory
factor (LIF) and stem cell factor. Methods for the expansion of cell
populations are also
known in the art (see e.g., Emerson et al., U.S. Patent No. 6,326,198 entitled
"Methods and
compositions for the ex vivo replication of stem cells, for the optimization
of hematopoietic
progenitor cell cultures, and for increasing the metabolism, GM-CSF secretion
and/or IL-6
secretion of human stromal cells", issued December 4, 2001; Kraus et al., U.S.
Patent No.
6,338,942, entitled "Selective expansion of target cell populations", issued
January 1 S,
2002).
The embryonic-like stern cells may be assessed for viability, proliferation
potential,
and longevity using standard techniques known in the art, such as trypan blue
exclusion
1S assay, fluorescein diacetate uptake assay, propidium iodide uptake assay
(to assess
viability); and thymidine uptake assay, MTT cell proliferation assay (to
assess
proliferation). Longevity may be determined by methods well known in the art,
such as by
determining the maximum number of population doubling in an extended culture.
In certain embodiments, the differentiation of stem cells or progenitor cells
that are
cultivated in the exsanguinated, perfused and/or cultured placenta is
modulated using an
agent or pharmaceutical compositions comprising a dose and/or doses effective
upon single
or multiple administration, to exert an effect sufficient to inhibit, modulate
and/or regulate
the differentiation of a cell collected from the placenta.
Agents that can induce stem or progenitor cell differentiation are well known
in the
2S art and include, but are not limited to, Ca2~, EGF, a-FGF, (3-FGF, PDGF,
keratinocyte
growth factor (KGF), TGF-j3, cytokines (e.g., IL-la, IL-1(3, IFN-'y, TFN),
retinoic acid,
transfernn, hormones (e.g., androgen, estrogen, insulin, prolactin,
triiodothyronine,
hydrocortisone, dexamethasone), sodium butyrate, TPA, DMSO, NMF, DMF, matrix
elements (e.g., collagen, laminin, heparan sulfate, MatrigelTM), or
combinations thereof.
Agents that suppress cellular differentiation are also well-known in the art
and
include, but are not limited to, human Delta-1 and human Serrate-1
polypeptides (see,
Sakano et al., U.S. Patent No. 6,337,387 entitled "Differentiation-suppressive
polypeptide",
issued January 8, 2002), leukemia inhibitory factor (LIF), and stem cell
factor.
The agent used to modulate differentiation can be introduced into the
placental
3S bioreactor to induce differentiation of the cells being cultured in the
placenta. Alternatively,
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the agent can be used to modulate differentiation iya vitro after the cells
have been collected
or removed from the placenta.
Determination that a stem cell has differentiated into a particular cell type
may be
accomplished by methods well-known in the art, e.g., measuring changes in
morphology
and cell surface markers using techniques such as flow cytometry or
immunocytochemistry
(e.g., staining cells with tissue-specific or cell-marker specific
antibodies), by examination
of the morphology of cells using light or confocal microscopy, or by measuring
changes in
gene expression using techniques well known in the art, such as PCR and gene-
expression
profiling.
4.4. SUPPLEMENTING POPULATIONS OF STEM CELLS
WITH EMBRYONIC-LIKE STEM CELLS
The present invention relates to populations of stem cells are mixed with
embryonic-
like stem cells. In accordance with the present invention, stems cells that
may be mixed
with embryonic-like stem cells include, but are not limited to, umbilical cord
blood, fetal
and neonatal hematopoietic stem cells and progenitor cells, human stem cells
and progenitor
cells derived from bone marrow. In a preferred embodiment of the present
invention, the
embryonic-like stem cells of the invention are mixed with umbilical cord
blood.
The present invention provides an isolated homogenous population of human
placental stem cells (embryonic-like stem cells) which has the potential to
differentiate into
all cell types. Such cells may be used to supplement populations of stem
cells, e.g., cord
blood cells, according to the methods of the invention.
The invention also provides populations of cord blood cells that have been
supplemented (i.e., mixed, combined or augmented) with populations of
embryonic-like
stem cells that originate from a placenta.
The supplemented populations are very versatile, in that they contain
populations of
cells that are pluripotent or multipotent stem cells, e.g., cells displaying a
CD34+ /CD38+,
CD34+ /CD38- or CD34-/CD38- phenotype.
In accordance with the present invention the supplemented populations of stem
cells
°f the invention contain embryonic-like stem cells and other stem or
progenitor cells at a
ratio of 100,000,000:1, 50,000,000:1, 20,000,000:1, 10,000,000:1, 5,000,000:1,
2,000,000:1, 1,000,000:1, 500,000:1, 200,000:1, 100,000:1, 50,000:1, 20,000:1,
10,000:1,
5,000:1, 2,000:1, 1,000:1, 500:1, 200:1, 100:1, 50:1, 20:1, 10:1, 5:1, 2:1,
1:1; 1:2; 1:5; 1:10;
1:100; I:200; 1:500; 1:1,000; 1:2,000; 1:5,000; 1:10,000; 1:20,000; 1:50,000;
1:100,000;
1:500,000; 1:1,000,000; 1:2,000,000; 1:5,000,000; 1:10,000,000; 1:20,000,000;
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I :50,000,000; or 1:100,000,000, comparing numbers of total nucleated cells in
each
population.
In another embodiment, the invention provides methods for supplementing,
mixing,
combining or augmenting stem cells, e.g., umbilical cord blood, with a
composition of the
invention, e.g., a population of pure embryonic-like placental stem cells or a
population of
cells enriched for embryonic-like placental stem cells. In one embodiment, an
aliquot (or
population) of embryonic-like placental stem cells is added to an aliquot of
umbilical cord
blood. before delivery to a patient in need thereof.
The present invention also provides methods of supplementing a population of
cord
blood cells with a population of embryonic-like stem cells. In one embodiment,
the two
populations are physically mixed. In another aspect of this embodiment, the
two
populations are physically mixed and then treated with a growth factor, e.g.,
a cytokine
and/or an interleukin, to induce cell differentiation. In another aspect of
this embodiment,
the cord blood cells and/or the embryonic-like stem cells are treated with a
growth factor,
e.g., a cytokine and/or an interleukin, to induce cell differentiation and
then physically
mixed.
The present invention also provides methods of treating a patient in need
thereof by
administration of a population of cord blood cells supplemented with embryonic-
Iike stem
cells. In one embodiment, the supplementing of the population of cord blood
cells with
embryonic-like stem cells occurs by mixing the cord blood cells and embryonic-
like stem
cells prior to administration of the supplemented population to the patient.
In another
embodiment, supplementing the population of cord blood cells with embryonic-
like stem
cells occurs upon administration of the supplemented population to the
patient, e.g., by
simultaneous administration of the cord blood cells and the embryonic-like
stem cells. In
another embodiment, supplementing of the population of cord blood cells with
embryonic-
like stem cells occurs after administration of the cord blood cells to the
patient, e.g., by
administering the embryonic-stem cells separately from, and before or after,
administration
of the cord blood cells.
In one embodiment, the invention provides methods for supplementing cord blood
cells with embryonic-like stem cells, wherein the mixture is contained within
one bag. In
another embodiment, the invention provides methods for supplementing cord
blood cells
with embryonic-like stem cells, wherein the cord blood cells and the embryonic-
like stem
cells are each contained in a separate bags. Such a "two bag" composition may
be mixed
prior to or at the time of administration to a patient in need thereof.
In another embodiment, an aliquot (or population) of embryonic-like placental
stem
cells are conditioned before being added to, and mixed into, an aliquot of
umbilical cord
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blood before delivery to a patient in need thereof. For example, in one aspect
of this
embodiment, a population of embryonic-like placental stem cells is induced to
differentiate
into a particular cell lineage, e.g., a hematopoietic, neuronal, adipogenic,
chondrogenic,
osteogenic, hepatogenic, pancreatic, or myogenic lineage, as disclosed above
in Section 4.3,
by exposure to, e.g., cytokines (e.g., IL-la, IL-1~, IFN-y, TFN), retinoic
acid, transferrin,
hormones (e.g., androgen, estrogen, insulin, prolactin, triiodothyronine,
hydrocortisone,
dexamethasone), sodium butyrate, TPA, DMSO, NMF, DMF, matrix elements (e.g.,
collagen, laminin, heparan sulfate, MatrigelTM), or combinations thereof,
before being added
to, and mixed into, an aliquot of umbilical cord blood.
In another aspect of this embodiment, a population of embryonic-like placental
stem
cells is conditioned by being exposed to an agent that suppresses
differentiation, e.g., human
Delta-1 and human Serrate-1 polypeptides, or combinations thereof, before
being added to,
and mixed into, an aliquot of umbilical cord blood.
In another embodiment, an aliquot (or population) of non-conditioned embryonic-
like placental stem cells and an aliquot of umbilical cord blood are mixed,
and the mixed
population of cells is conditioned before being delivery to a patient in need
thereof. In
specific embodiments, the mixed population of embryonic-like placental stem
cells and
umbilical cord blood cells are conditioned with an agent that induces or
suppresses cell
differentiation as disclosed above.
In a specific embodiment, a population of embryonic-like stem cells of the
invention
is added to, or mixed into, a population of umbilical cord blood cells prior
to administration
to a patient in need thereof. In another specific embodiment, a population of
embryonic-
like stem cells of the invention is added to, or mixed into, a population of
umbilical cord
blood cells during, or simultaneous with, administration to a patient in need
thereof. In
another specific embodiment, a population of embryonic-like stern cells of the
invention
and a population of umbilical cord blood cells are administered sequentially
to a patient in
need thereof. In one embodiment, the population of embryonic-like stem cells
is
administered first and the population of umbilical cord blood cells is
administered second.
In another embodiment, the population of umbilical cord blood cells is
administered first
~d the population of embryonic-like placental stem cells is administered
second.
The populations of cord blood cells spiked with embryonic-like stem cells may
be
cultured, induced to propagate, and/or induced to differentiate under a
variety of conditions,
including but not limited to treating the spiked populations by introduction
of nutrients,
hormones, vitamins, growth factors, or any combination thereof, into the
culture medium.
Serum and other growth factors maybe added to the culture medium. Growth
factors are
usually proteins and include, but are not limited to: cytokines, lymphokines,
interferons,
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colony stimulating factors (CSF's), interferons, chemokines, and interleukins.
Other growth
factors that may be used include recombinant human hematopoietic growth
factors
including ligands, stem cell factors, thrombopoeitin (Tpo), granulocyte colony-
stimulating
factor (G-CSF), leukemia inhibitory factor, basic fibroblast growth factor,
placenta derived
growth factor and epidermal growth factor. In one embodiment, the supplemented
populations are treated with a growth factor to induce differentiation into a
variety of cell
types. In another embodiment, the spiked populations are treated with a growth
factor to
induce differentiation into a particular cell type. In another embodiment, the
supplemented
populations are treated with a growth factor to prevent or suppress
differentiation into a
p~icular cell type.
In certain embodiments of the invention, the methods of supplementing a
population
of cord blood comprise (a) induction of differentiation of embryonic-like stem
cells, (b)
mixing the embryonic-like stem cells with a population of cord blood cells and
(c)
administration of the mixture to a patient in need thereof.
In other embodiments of the invention, the methods of supplementing a
population
of cord blood comprise (a) mixing the embryonic-like stem cells with a
population of cord
blood cells; (b) induction of differentiation of the mixture of the spiked
population of cord
blood cells and embryonic-like stem cells and (c) administration of the
mixture to a patient
in need thereof.
hl other embodiments of the invention, the methods of supplementing a
population
of cord blood comprise (a) administration of a mixture of cord blood cells
supplemented
with embryonic-like stem cells to a patient in need thereof and (b) induction
of
differentiation of the mixture and (c) administration of the mixture to a
patient in need
thereof.
In certain embodiments, stem or progenitor cells are induced to differentiate
into a
particular cell type, by exposure to a growth factor, according to methods
well known in the
art. In specific embodiments, the growth factor is: GM-CSF, IL-4, Flt3L,
CD40L, IFN-
alpha, TNF-alpha, IFN-gamma, IL-2, IL-6, retinoic acid, basic fibroblast
growth factor,
TGF-beta-1, TGF-beta-3, hepatocyte growth factor, epidermal growth factor,
cardiotropin-1,
~giotensinogen, angiotensin I (AI), angiotensin II (AID, All ATZ type 2
receptor agonists,
or analogs or fragments thereof.
In one embodiment, stem or progenitor cells are induced to differentiate into
neurons, according to methods well known in the art, e.g., by exposure to (3-
mercaptoethanol or to DMSO/butylated hydroxyanisole, according to the methods
disclosed
in Section 5.4.1.
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In another embodiment, stem or progenitor cells are induced to differentiate
into
adipocytes, according to methods well known in the art, e.g., by exposure to
dexamethasone, indomethacin, insulin and IBMX, according to the methods
disclosed in
Section 5.4.2.
S In another embodiment, stem or progenitor cells are induced to differentiate
into
chondrocytes, according to methods well known in the art, e.g., by exposure to
TGF-.beta-3,
according to the methods disclosed in Section 5.4.3.
In another embodiment, stem or progenitor cells are induced to differentiate
into
osteocytes, according to methods well known in the art, e.g., by exposure to
dexamethasone,
ascorbic acid-2-phosphate and beta-glycerophosphate, according to the methods
disclosed in
Section 5.4.4.
fit another embodiment, stem or progenitor cells are induced to differentiate
into
hepatocytes, according to methods well known in the art, e.g., by exposure to
IL-6 +/- IL-IS,
according to the methods disclosed in Section S.4.S.
IS In another embodiment, stem or progenitor cells are induced to
differentiate into
pancreatic cells, according to methods well known in the art, e.g., by
exposure to basic
fibroblast growth factor, and transforming growth factor beta-I, according to
the methods
disclosed in Section 5.4.6.
Tn another embodiment, stem or progenitor cells are induced to differentiate
into
cardiac cells, according to methods well known in the art, e.g., by exposure
to retinoic acid ,
basic fibroblast growth factor, TGF-beta-1 and epidermal growth factor, by
exposure to
cardiotropin-1 or by exposure to human myocardium extract, according to the
methods
disclosed in Section 5.4.7.
In another embodiment, the embryonic-like stem cells are stimulated to produce
2S bioactive molecules, such as immunoglobulins, hormones, enzymes.
In another embodiment, the embryonic-like stem cells are stimulated to
proliferate,
for example, by administration of erythropoietin, cytokines, lyrnphokines,
interferons,
colony stimulating factors (CSF's), interferons, chernokines, interleukins,
recombinant
human hematopoietic growth factors including ligands, stem cell factors,
thrombopoeitin
(Tpo), interleukins, and granulocyte colony-stimulating factor (G-CSF) or
other growth
factors.
In another embodiment, the embryonic-like stem cells are genetically
engineered
either prior to, or after collection from, the placenta, using, for example, a
viral vector such
as an adenoviral or retroviral vector, or by using mechanical means such as
liposomal or
3S chemical mediated uptake of the DNA.
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A vector containing a transgene can be introduced into a cell of interest by
methods
well known in the art, e.g., transfection, transformation, transduction,
electroporation,
infection, microinjection, cell fusion, DEAF dextran, calcium phosphate
precipitation,
liposomes, LIPOFECTINTM, lysosome fusion, synthetic cationic lipids, use of a
gene gun or
a DNA vector transporter, such that the transgene is transmitted to daughter
cells, e.g., the
daughter embryonic-like stem cells or progenitor cells produced by the
division of an
embryonic-like stem cell. For various techniques for transformation or
transfection of
mammalian cells, see Keown et al., 1990, Methods Enzymol. 185: 527-37;
Sambrook et al.,
2001, Molecular Cloning, A Laboratory Manual, Third Edition, Gold Spring
Harbor
Laboratory Press, N.Y.
Preferably, the transgene is introduced using any technique, so long as it is
not
destructive to the cell's nuclear membrane or other existing cellular or
genetic structures. In
certain embodiments, the transgene is inserted into the nucleic genetic
material by
microinjection. Microinjection of cells and cellular structures is commonly
known and
practiced in the art.
For stable transfection of cultured mammalian cells, such as the embryonic-
like stem
cells, only a small fraction of cells may integrate the foreign DNA into their
genome. The
efficiency of integration depends upon the vector and transfection technique
used. In order
to identify and select integrants, a gene that encodes a selectable marker
(e.g., for resistance
to antibiotics) is generally introduced into the host embryonic-like stem cell
along with the
gene sequence of interest. Preferred selectable markers include those that
confer resistance
to drugs, such as 6418, hygromycin and methotrexate. Cells stably transfected
with the
introduced nucleic acid can be identified by drug selection (e.g., cells that
have incorporated
the selectable marker gene will survive, while the other cells die). Such
methods are
particularly useful in methods involving homologous recombination in mammalian
cells
(e.g., in embryonic-like stem cells) prior to introduction or transplantation
of the
recombinant cells into a subject or patient.
A number of selection systems may be used to select transformed host embryonic-
like cells. In particular, the vector may contain certain detectable or
selectable markers.
Other methods of selection include but are not limited to selecting for
another marker such
as: the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:
223),
hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalski, 1962,
Proc.
Natl. Acad. Sci. USA 48: 2026), and adenine phosphoribosyltransferase (Lowy et
al., 1980,
Gell 22: 817) genes can be employed in tk-, hgprt- or aprt- cells,
respectively. Also,
antimetabolite resistance can be used as the basis of selection for the
following genes: dhfr,
which confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl.
Acad. Sci. USA
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77: 3567; O'Hare et al., 1981, Proe. Natl. Acad. Sci. USA 78: 1527); gpt,
which confers
resistance to mycophenolic acid (Mulligan and Berg, 1981, Proc. Natl. Acad.
Sci. USA 78:
2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-
Garapin et al.,
1981, J. MoI. Biol. 150: 1); and hygro, which confers resistance to hygromycin
(Santerre et
al., 1984, Gene 30: 147).
The transgene may integrate into the genome of the cell of interest,
preferably by
random integration. In other embodiments the transgene may integrate by a
directed
method, e.g., by directed homologous recombination (i.e., "knock-in" or "knock-
out" of a
gene of interest in the genome of cell of interest), Chappel, U.S. Patent No.
5,272,071; and
PCT publication No. WO 91/06667, published May 16, 1991; U.S. Patent
5,464,764;
Capecchi et al., issued November 7, 1995; U.S. Patent 5,627,059, Capecchi et
al. issued,
May 6, 1997; U.S. Patent 5,487,992, Capecchi et al., issued January 30, 1996).
Methods for generating cells having targeted gene modifications through
homologous recombination are known in the art. The construct will comprise at
least a
portion of a gene of interest with a desired genetic modification, and will
include regions of
homology to the target locus, i. e., the endogenous copy of the targeted gene
in the host's
genome. DNA constructs for random integration, in contrast to those used for
homologous
recombination, need not include regions of homology to mediate recombination.
Markers
can be included in the targeting construct or random construct for performing
positive and
negative selection for insertion of the transgene.
To create a homologous recombinant cell, e.g., a homologous recombinant
embryonic-like stem cell, endogenous placental cell or exogenous cell cultured
in the
placenta, a homologous recombination vector is prepared in which a gene of
interest is
flanked at its 5' and 3' ends by gene sequences that are endogenous to the
genome of the
targeted cell, to allow for homologous recombination to occur between the gene
of interest
carried by the vector and the endogenous gene in the genome of the targeted
cell. The
additional flanking nucleic acid sequences are of sufficient length fox
successful
homologous recombination with the endogenous gene in the genome of the
targeted cell.
Typically, several kilobases of flanking DNA (both at the 5' and 3' ends) axe
included in the
vector. Methods for constructing homologous recombination vectors and
homologous
recombinant animals from recombinant stem cells are commonly known in the art
(see, e.g.,
Thomas and Capecchi, 1987, Cell 51: 503; Bradley, 1991, Curr. Opin.
Bio/Technol. 2: 823-
29; and PCT Publication Nos. WO 90/11354, WO 91/01140, and WO 93/04169.
In one embodiment, the genome of an exogenous cell cultured in the placenta
according to the methods of the invention is a target of gene targeting via
homologous
recombination or via random integration.
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In a specific embodiment, the methods of Bonadio et al. (U.S. Patent No.
5,942,496,
entitled Methods and compositions for multiple gene transfer into bone cells,
issued August
24, 1999; and PCT W095/22611, entitled Methods and compositions for
stimulating bone
cells, published August 24, 1995 ) are used to introduce nucleic acids into a
cell of interest,
such as a stem cell, progenitor cell or exogenous cell cultured in the
placenta, e.g., bone
progenitor cells.
4.5. USES OF EMBRYONIC-LIKE STEM CELLS AND
SUPPLEMENTED POPULATIONS OF STEM CELLS
Embryonic-like stem cells may be obtained from perfused placentas according to
the
methods described in copending United States Application No. 01/076,180, filed
February
13, 2002.
The placental stem cell (embryonic-like stem cell) may be induced to
differentiate
into a particular cell type, either ex vivo or iya vivo. For example,
pluripotent embryonic-like
stem cells may be injected into a damaged organ, and for organ neogenesis and
repair of
injury in vivo. Such injury may be due to such conditions and disorders
including, but not
limited to, myocardial infarction, seizure disorder, multiple sclerosis,
stroke, hypotension,
cardiac arrest, ischemia, inflammation, age-related loss of cognitive
function, radiation
damage, cerebral palsy, neurodegenerative disease, Alzheimer's disease,
Parkinson's
disease, Leigh disease, AmS dementia, memory loss, amyotrophic lateral
sclerosis,
ischemic renal disease, brain or spinal cord trauma, heart-lung bypass,
glaucoma, retinal
ischemia, or retinal trauma.
The embryonic-like stem cells isolated from the placenta, alone or in
combination
with stem or progenitor cell populations (i. e., the cell compositions of the
invention) may be
used, in specific embodiments, in autologous or heterologous enzyme
replacement therapy
to treat specific diseases or conditions, including, but not limited to
lysosomal storage
diseases, such as Tay-Sachs, Niemann-Pick, Fabry's, Gaucher's, Hunter's, and
Hurler's
syndromes, as well as other gangliosidoses, mucopolysaccharidoses, and
glycogenoses.
In other embodiments, the embryonic-like stem cells, alone or in combination
with
stem or progenitor cell populations, may be used as autologous or heterologous
transgene
carriers in gene therapy to correct inborn errors of metabolism,
adrenoleukodystrophy,
cystic fibrosis, glycogen storage disease, hypothyroidism, sickle cell anemia,
Pearson
syndrome, Pompe's disease, phenylketonuria (PKU), porphyrias, maple syrup
urine disease,
homocystinuria, mucoplysaccharide nosis, chronic granulomatous disease and
tyrosinemia
and Tay-Sachs disease or to treat cancer, tumors or other pathological
conditions.
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In other embodiments, the cell compositions may be used in autologous or
heterologous tissue regeneration or replacement therapies or protocols,
including, but not
limited to treatment of corneal epithelial defects, cartilage repair, facial
dermabrasion,
mucosal membranes, tympanic membranes, intestinal linings, neurological
structures (e.g.,
retina, auditory neurons in basilar membrane, olfactory neurons in olfactory
epithelium),
bum and wound repair for traumatic injuries of the skin, or for reconstruction
of other
damaged or diseased organs or tissues.
The large numbers of embryonic-like stem cells and/or progenitor obtained
using the
methods of the invention would, in certain embodiments, reduce the need for
large bone
marrow donations. Approximately 1 x 10$ to 2 x 108 bone marrow mononuclear
cells per
kilogram of patient weight must be infused for engraftment in a bone marrow
transplantation (i.e., about 70 ml ofmarrow for a 70 kg donor). To obtain 70
ml requires an
intensive donation and significant loss of blood in the donation process. W a
specific
embodiment, cells from a small bone marrow donation (e.g., 7-10 ml) could be
expanded by
propagation in a placental bioreactor before infusion into a recipient.
Furthermore, a small number of stem cells and progenitor cells normally
circulate in
the blood stream. In another embodiment, such exogenous stem cells or
exogenous
progenitor cells are collected by apheresis, a procedure in which blood is
withdrawn, one or
more components are selectively removed, and the remainder of the blood is
reinfused into
the donor. The exogenous cells recovered by apheresis are expanded by
propagation in a
placental bioreactor, thus eliminating the need for bone marrow donation
entirely.
While the blood cells regenerate between chemotherapy treatments, however, the
cancer has time to grow and possibly become more resistant to the chemotherapy
drugs due
to natural selection. Therefore, the longer chemotherapy is given and the
shorter the
d~ation between treatments, the greater the odds of successfully killing the
cancer. To
shorten the time between chemotherapy treatments, embryonic-like stem cells or
progenitor
cells collected according to the methods of the invention, alone or in
combination with other
stern cell or progenitor cell populations, could be introduced into the
patient. Such
treatment would reduce the time the patient would exhibit a low blood cell
count, and
would therefore permit earlier resumption of the chemotherapy treatment.
The embryonic-like stem cells, progenitor cells, foreign cells, or engineered
cells
obtained from a placenta according to the methods of the invention, alone or
in combination
with other stem cell or progenitor cell populations, can be used in the
manufacture of a
tissue or organ ifa vivo. The methods of the invention encompass using cells
obtained from
the placenta, e.g., embryonic-like stem cells, progenitor cells, or foreign
stem or progenitor
cells, to seed a matrix and to be cultured under the appropriate conditions to
allow the cells
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to differentiate and populate the matrix. The tissues and organs obtained by
the methods of
the invention may be used for a variety of purposes, including research and
therapeutic
purposes.
The embryonic-like stem cells and the supplemented populations of stem cells
of the
invention can also be used for a wide variety of prophylactic or therapeutic
protocols in
which a tissue or organ of the body is augmented, repaired or replaced by the
engraftment,
transplantation or infusion of a desired cell population, such as a stem cell
or progenitor cell
population. The embryonic-like stem cells and the supplemented populations of
stem cells
of the invention can be used to replace or augment existing tissues, to
introduce new or
altered tissues, or to join together biological tissues or structures. The
embryonic-like stem
and supplemented stem cell populations of the invention can also be
substituted for
embryonic stern cells in therapeutic protocols described herein in which
embryonic stem
cells would be typically be used.
In a preferred embodiment of the invention, embryonic-like stem cells and
supplemented stem cell populations may be used as autologous and allogenic,
including
matched and mismatched HLA type hematopoietic traalsplants. In accordance with
the use
of embryonic-like stem cells as allogenic hematopoietic transplants it may be
necessary to
treat the host to reduce immunological rejection of the donor cells, such as
those described
in U.S. Patent No. 5,800,539, issued September 1, 1998; and U.S. Patent No.
5,806,529,
issued September 15, 1998, both of which are incorporated herein by reference.
For example, embryonic-like stem cells and supplemented stem cell populations
of
the invention can be used in therapeutic transplantation protocols, e.g., to
augment or
replace stem or progenitor cells of the liver, pancreas, kidney, lung, nervous
system,
muscular system, bone, bone marrow, thymus, spleen, mucosal tissue, gonads, or
hair.
Embryonic-like stem cells and supplemented stem cell populations may be used
instead of specific classes of progenitor cells (e.g., chondrocytes,
hepatocytes,
hematopoietic cells, pancreatic parenchyma) cells, neuroblasts, muscle
progentor cells,
etc.) in therapeutic or research protocols in which progenitor cells would
typically be used.
Embryonic-like stem cells and supplemented stem cell populations of the
invention
can be used for augmentation, repair or replacement of cartilage, tendon, or
ligaments. For
example, in certain embodiments, prostheses (e.g., hip prostheses) are coated
with
replacement cartilage tissue constructs grown from embryonic-like stem cells
of the
invention. In other embodiments, joints (e.g., knee) are reconstructed with
cartilage tissue
constructs grown from embryonic-like stem cells. Cartilage tissue constructs
can also be
employed in major reconstructive surgery for different types of joints (for
protocols, see
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e.g., Resnick, D., and Niwayama, G., eds., 1988, Diagnosis of Bone and Joint
Disorders, 2d
ed., W. B. Saunders Co.).
The embryonic-like stem cells and supplemented stem cell populations of the
invention can be used to repair damage of tissues and organs resulting from
trauma,
metabolic disorders, or disease. In such an embodiment, a patient can be
administered
embryonic-like stem cells, alone or combined with other stem or progenitor
cell
populations, to regenerate or restore tissues or organs which have been
damaged as a
consequence of disease, e.g., enhance immune system following chemotherapy or
radiation,
repair heart tissue following myocardial infarction.
The embryonic-like stem cells and supplemented stern cell populations of the
invention can be used to augment or replace bone marrow cells in bone marrow
transplantation. Human autologous and allogenic bone marrow transplantation
are currently
used as therapies for diseases such as leukemia, lymphoma and other life-
threatening
disorders. The drawback of these procedures, however, is that a large amount
of donor bone
marrow must be removed to insure that there is enough cells for engraftment.
The embryonic-like stem cells and supplemented stem cell populations of the
invention can provide stem cells and progenitor cells that would reduce the
need for large
bone marrow donation. It would also be, according to the methods of the
invention, to
obtain a small marrow donation and then expand the number of stem cells and
progenitor
cells culturing and expanding in the placenta before infusion or
transplantation into a
recipient.
The embryonic-like stem cells and supplemented stem cell populations of the
invention may be used, in specific embodiments, in autologous or heterologous
enzyme
replacement therapy to treat specific diseases or conditions, including, but
not limited to
lysosomal storage diseases, such as Tay-Sachs, Niemann-Pick, Fabry's,
Gaucher's,
Hunter's, Hurler's syndromes, as well as other gangliosidoses,
mucopolysaccharidoses, and
glycogenoses.
In other embodiments, the cells may be used as autologous or heterolagous
transgene carriers in gene therapy to correct inborn errors of metabolism such
as
a~'enoleukodystrophy, cystic fibrosis, glycogen storage disease,
hypothyroidism, sickle cell
anemia, Pearson syndrome, Pompe's disease, phenylketonuria (PKIJ), and Tay-
Sachs
disease, porphyrias, maple syrup urine disease, homocystinuria,
mucopolypsaccharide nosis,
chronic granulomatous disease, and tyrosinemia. or to treat cancer, tumors or
other
pathological or neoplastic conditions.
In other embodiments, the cells may be used in autologous or heterologous
tissue
regeneration or replacement therapies or protocols, including, but not limited
to treatment of
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corneal epithelial defects, cartilage repair, facial dermabrasion, mucosal
membranes,
tympanic membranes, intestinal linings, neurological structures (e.g., retina,
auditory
neurons in basilar membrane, olfactory neurons in olfactory epithelium), burn
and wound
repair for traumatic injuries of the skin, scalp (hair) transplantation, or
for reconstruction of
other damaged or diseased organs or tissues.
The large numbers of embryonic-like stem cells and/or progenitor obtained
using the
methods of the invention would, in certain embodiments, reduce the need for
large bone
marrow donations. Approximately 1 x 10$ to 2 x 108 bone marrow mononuclear
cells per
kilogram of patient weight must be infused for engraftment in a bone marrow
transplantation (i.e., about 70 ml of marrow for a 70 kg donor). To obtain 70
ml requires an
intensive donation and significant loss of blood in the donation process. In a
specific
embodiment, cells from a small bone marrow donation (e.g., 7-10 ml) could be
expanded by
propagation in a placental bioreactor before infusion into a recipient.
In another embodiment, the embryonic-like stem cells and supplemented stem
cell
populations of the invention can be used in a supplemental treatment in
addition to
chemotherapy. Most chemotherapy agents used to target and destroy cancer cells
act by
killing all proliferating cells, i. e., cells going through cell division.
Since bone marrow is
one of the most actively proliferating tissues in the body, hematopoietic stem
cells are
frequently damaged or destroyed by chemotherapy agents and in consequence,
blood cell
production is diminishes or ceases. Chemotherapy must be terminated at
intervals to allow
the patient's hematopoietic system to replenish the blood cell supply before
resuming
chemotherapy. It may take a month or more for the formerly quiescent stem
cells to
proliferate and increase the white blood cell count to acceptable levels so
that chemotherapy
may resume (when again, the bone marrow stem cells are destroyed).
While the blood cells regenerate between chemotherapy treatments, however, the
cancer has time to grow and possibly become more resistant to the chemotherapy
drugs due
to natural selection. Therefore, the longer chemotherapy is given and the
shorter the
duration between treatments, the greater the odds of successfully killing the
cancer. To
shorten the time between chemotherapy treatments, embryonic-like stem cells or
progenitor
cells collected according to the methods of the invention could be introduced
into the
patient. Such treatment would reduce the time the patient would exhibit a low
blood cell
count, and would therefore permit earlier resumption of the chemotherapy
treatment.
In another embodiment, the human placental stem cells can be used to treat or
prevent genetic diseases such as chronic granulomatous disease.
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4.6. PHARMACEUTICAL COMPOSITIONS
The present invention encompasses pharmaceutical compositions which comprise
the embryonic-like stem cells and supplemented stem cell populations of the
invention. The
present invention encompasses pharmaceutical compositions comprising a dose
and/or
S doses effective upon single or multiple administration, prior to or
following transplantation
of conditioned or unconditioned human progenitor stem cells, that are able to
exert an effect
sufficient to inhibit, modulate and/or regulate the differentiation of human
pluripotent and
multipotent progenitor stem cells of placental origin into one or more cell
lineages, for
example, mesodermal, adipose, chondrocytic, osteocytic, myocytic, vascular,
neural,
endothelial, hepatic, kidney, pancreatic, and/or hematopoietic lineage cells.
In accordance with this embodiment, the embryonic-like stem cells and
supplemented stem cell populations of the invention may be formulated as an
injectable
(e.g., PCT WO 96/39101, incorporated herein by reference in its entirety). In
an alternative
embodiment, the cells and tissues of the present invention may be formulated
using
1S polymerizable or cross linking hydrogels as described in U.S. Patent Nos.
5,709,854;
S,S 16,532; S,6S4,381; each of which is incorporated by reference in their
entirety. The
embryonic-like stem cells may be administered as obtained from the placenta,
or may be
spiked into umbilical cord blood and administered as a mixed cell composition,
or may be
placed into any physiologically-acceptable buffer or fluid for administration
to an
individual.
The invention also encompasses pharmaceutical compositions that have high
concentrations (or larger populations) of homogenous embryonic-like stem
cells, wherein
one or more of these cell populations can be used with, or as a mixture with,
other stem or
progenitor cells, for use in transplantation and other uses. Other stem or
progenitor cells
2S may include but are not limited to: adipogenic, chondrogenic, osteogenic,
hematopoietic,
myogenic, vasogenic, neurogenic, and hepatogeuc stem cells; mesenchymal stem
cells,
stromal cells, endothelial cells, hepatocytes, keratinocytes, and stem or
progenitor cells for a
particular cell type, tissue or organ, including but not limited to neurons,
myelin, muscle,
blood, bone marrow, skin, heart, connective tissue, lung, kidney, liver, and
pancreas (e.g.,
p~creatic islet cells).
In one embodiment, the invention provides pharmaceutical compositions that
have
high concentrations (or larger populations) of homogenous hematopoietic stem
cells
including but not limited to CD34+ /CD38- cells; and CD34-/ CD38- cells. One
or more of
these cell populations can be used with, or as a mixture with, other stem
cells, for use in
3S ~'~splantation and other uses. In a specific embodiment, the pharmaceutical
composition
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comprises embryonic-like placental stem cells of the invention and cord blood
hematopoietic cells i. e., CD34+/CD38+ hematopoietic cells.
One or more of these cell populations can be used with or as a mixture with
cord
blood hematopoietic cells i.e., CD34+/CD38+ hematopoietic cells for
transplantation and
other uses.
In one embodiment, the invention provides heterogeneous population of
nucleated
cells that comprises embryonic-like placental stem cells. In certain
embodiments, a
heterogeneous population of nucleated cells (rather than a pure population
CD34+ cells
embryonic-like placental stem cells) is preferred.
In another embodiment, the invention provides a mixed population of cells
(e.g.,
cord blood cells and embryonic-like placental stem cells). The population of
mixed cells
may be frozen or unfrozen. Such a mixed population may be stored and/or used
in one
container, e.g., one bag or one syringe.
In another embodiment, the invention provides two or more separate or distinct
populations of different cell types (e.g., cord blood cells and embryonic-like
placental stem
cells). Each separate population may be stored and/or used in a separate
container, e.g., one
bag (e.g., blood storage bag from Baxter, Becton-Dickinson, Medcep, National
Hospital
Products or Terumo) or one syringe, which contains a single type of cell or
cell population.
In certain aspects of this embodiment, the invention provides separate
containers of
different cell types to be mixed before administration. Such cells may be
unfrozen or frozen.
In a specific embodiment, cord blood cells are contained in one bag and
embryonic-
like placental stem cells are contained in a second bag.
In another embodiment, the invention provides embryonic-like placental stem
cells
that are "conditioned" before freezing.
In another embodiment, a population of cells including, but not limited to,
embryonic-like placental stem cells may be conditioned by the removal of red
blood cells
and/or granulocytes according to standard methods, so that a population of
nucleated cells
remains that is enriched for embryonic-like placental stem cells. Such an
enriched
population of embryonic-like placental stem cells may be used unfrozen, or
frozen for later
use. If the population of cells is to be frozen, a standard cryopreservative
(e.g., DMSO,
glycerol, EpilifeTM Cell Freezing Medium (Cascade Biologics)) is added to the
enriched
population of cells before it is frozen.
In another embodiment, a population of cells including, but not limited to,
embryonic-like placental stem cells may be conditioned by the removal of red
blood cells
~~or granulocytes after it has been frozen and thawed.
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According to the invention, agents that induce cell differentiation may be
used to
condition a population of embryonic-like stem cells. Tn certain embodiments,
an agent that
induces differentiation can be added to a population of cells within a
container, including,
but not limited to, Ca2~, EGF, a-FGF, (3-FGF, PDGF, keratinocyte growth factor
(KGF),
TGF-J3, cytokines (e.g., IL-la, IL-1[3, IFN-y, TFN), retinoic acid,
transferrin, hormones
(e.g., androgen, estrogen, insulin, prolactin, triiodothyronine,
hydrocortisone,
dexamethasone), sodium butyrate, TPA, DMSO, NMF, DMF, matrix elements (e.g.,
collagen, laminin, heparan sulfate, MatrigelTM), or combinations thereof.
In another embodiment, agents that suppress cellular differentiation can be
added to
1 p a population of embryonic-like stem cells. In certain embodiments, an
agent that suppresses
differentiation can be added to a population of cells within a container,
including, but not
limited to, human Delta-l and human Serrate-1 polypeptides (see, Sakano et
al., IJ.S. Patent
No. 6,337,387 entitled "Differentiation-suppressive polypeptide", issued
January 8, 2002),
leukemia inhibitory factor (LIF), stem cell factor, or combinations thereof.
15 W certain embodiments, one or more populations of embryonic-like stem cells
are
delivered to a patient in need thereof. In certain embodiments, two or more
populations of
fresh (never frozen) cells are delivered from a single container or single
delivery system.
In another embodiment, two or more populations of frozen and thawed cells are
delivered from a single container or single delivery system.
20 In another embodiment, each of two or more populations of fresh (never
frozen)
cells are transferred to, and delivered from, a single container or single
delivery system. In
another embodiment, each of two or more populations of frozen and thawed cells
are
transferred to, and delivered from, a single container or single delivery
system.. In another
aspect of these embodiments, each population is delivered from a different IV
infusion bag
25 (e.g., from Baxter, Becton-Dickinson, Medcep, National Hospital Products or
Terurno).
The contents of each container (e.g.,1V infusion bag) may be delivered via a
separate
delivery system, or each container may be "piggybacked" so that their contents
are
combined or mixed before delivery from a single delivery system.. For example,
the two or
more populations of cells may be fed into and/or mixed within a common flow
line (e.g.,
30 tubing), or they may be fed into and/or mixed within a common container
(e.g., chamber or
bag).
According to the invention, the two or more populations of cells may be
combined
before administration, during or at administration or delivered
simultaneously.
In one embodiment, a minimum of 1.7 x 10' nucleated cells/kg is delivered to a
35 patient in need thereof. Preferably, at least 2.5 x 10' nucleated cells/kg
is delivered to a
patient in need thereof.
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In one embodiment, the invention provides a method of treating or preventing a
disease or disorder in a subject comprising administering to a subject in
which such
treatment or prevention is desired a therapeutically effective amount of the
embryonic-like
stem cells, or supplemented cell populations, of the invention.
In another embodiment, the invention provides a method of treating or
preventing a
disease or disorder in a subject comprising administering to a subject in
which such
treatment or prevention is desired a therapeutically effective amount of the
embryonic-like
stem cells of the invention.
The embryonic-like stem cells of the invention are expected to have an anti-
inflammatory effect when administered to an individual experiencing
inflammation. In a
prefexred embodiment, the embryonic-like stem cells or supplemental cell
populations of
the invention may be used to treat any disease, condition or disorder
resulting from, or
associated with, inflammation. The inflammation may be present in any organ or
tissue, for
example, muscle; nervous system, including the brain, spinal cord and
peripheral nervous
system; vascular tissues, including cardiac tissue; pancreas; intestine or
other organs of the
digestive tract; lung; kidney; liver; reproductive organs; endothelial tissue,
or endodermal
tissue.
The embryonic-like stem cells or supplemented cell populations of the
invention
may also be used to treat autoimmune or irmnune system-related disorders,
including those
associated with inflammation. Thus, in cextain embodiments, the invention
provides a
method of treating an individual having an autoimmune disease or condition,
comprising
administering to such individual a therapeutically effective amount of the
cells or
supplemented cell populations of the invention, wherein said disease or
disorder can be, but
is not limited to, diabetes, amylotrophic lateral sclerosis, myasthenia
gravis, diabetic
neuropathy or lupus. In related embodiments, the embryonic-like stem cells or
supplemented cell populations of the invention may be used to treat immune-
related
disorders, such as chronic or acute allergies.
In certain embodiments, the disease or disordex includes, but is not limited
to, any of
the diseases ox disorders disclosed herein, including, but not limited to
aplastic anemia,
myelodysplasia, myocardial infarction, seizure disorder, multiple sclerosis,
stroke,
hypotension, cardiac arrest, ischemia, inflammation, age-related loss of
cognitive function,
radiation damage, cerebral palsy, neurodegenerative disease, Alzheimer's
disease,
Parkinson's disease, Leigh disease, AIRS dementia, memory loss, amyotrophic
lateral
sclerosis (ALS), ischemic renal disease, brain or spinal cord trauma, heart-
lung bypass,
glaucoma, retinal ischemia, retinal trauma, lysosomal storage diseases, such
as Tay-Sachs,
Niemann-Pick, Fabry's, Gaucher's, Hunter's, and Hurler's syndromes, as well as
other
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gangliosidoses, mucopolysaccharidoses, glycogenoses, inborn errors of
metabolism,
adrenoleukodystrophy, cystic fibrosis, glycogen storage disease,
hypothyroidism, sickle cell
anemia, Pearson syndrome, Pompe's disease, phenylketonuria (PK~, porphyrias,
maple
syrup urine disease, homocystinuria, mucoplysaccharide nosis, chronic
granulomatous
disease and tyrosinemia, Tay-Sachs disease, cancer, tumors or other
pathological or
neoplastic conditions.
In other embodiments, the cells may be used in the treatment of any kind of
injury
due to trauma, particularly trauma involving inflammation. Examples of such
trauma-
related conditions include central nervous system (CNS) injuries, including
injuries to the
brain, spinal cord, or tissue surrounding the CNS injuries to the peripheral
nervous system
(PNS); or injuries to any other part of the body. Such trauma may be caused by
accident, or
may be a normal or abnormal outcome of a medical procedure such as surgery or
angioplasty. The trauma may be related to a rupture or occlusion of a blood
vessel, for
example, in stroke or phlebitis. In specific embodiments, the cells may be
used in
autologous or heterologous tissue regeneration or replacement therapies or
protocols,
including, but not limited to treatment of corneal epithelial defects,
cartilage repair, facial
dermabrasion, mucosal membranes, tympa~lic membranes, intestinal linings,
neurological
structures (e.g., retina, auditory neurons in basilar membrane, olfactory
neurons in olfactory
epithelium), burn and wound repair for traumatic injuries of the skin, or for
reconstruction
of other damaged or diseased organs or tissues.
In a specific embodiment, the disease or disorder is aplastic anemia,
myelodysplasia,
leukemia, a bone marrow disorder or a hematopoietic disease or disorder. In
another
specific embodiment, the subject is a human.
In another embodiment, the invention provides a method of treating an
individual
having a disease, disorder or condition associated with or resulting from
inflammation. In
other embodiments, the invention provides a method of treating an individual
having a
neurological disease, disorder or condition. In a more specific embodiment,
said
neurological disease is ALS. W another more specific embodiment, said
neurological
disease is Parkinson's disease. In another specific embodiment, said disease
is a vascular or
cardiovascular disease. In a more specific embodiment, said disease is
atherosclerosis. In
another specific embodiment, said disease is diabetes.
In a specific embodiment, the pharmaceutical compositions of the invention
comprise an aliquot of umbilical cord blood to which embryonic-like placental
stem cells
have been added as disclosed above in Section 4.4.
A number of the embryonic-like stem cells, or of the supplemented cell
populations,
once administered, are able to engraft into the host, forming long-term
"colonies." This
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results in a host that is essentially chimeric. Because chimeras in other
genetic contexts are
generally more vigorous and resilient, such chimerism is expected enhance the
host's health
and well-being. As such, the embryonic-like stem cells may be administered not
simply to
an individual suffering from a specific disease, disorder or condition, but
may be
administered to an individual in order to increase the individual's overall
health and well-
being.
The embryonic-like stem cells or supplemented cell populations of the
invention
may be treated prior to administration to an individual with compounds that
modulate the
activity of TNF-a. Such compounds are disclosed in detail in copending United
States
Provisional Application No. 60/372,348, filed April I2, 2002, which disclosure
is
incorporated herein in its entirety. Preferred compounds are referred to as
IMiDs and
SelCids, and particularly preferred compounds are available under the trade
names
ActimidTM and RevimidTM.
A particularly useful aspect of the embryonic-like stem cells of the invention
is that,
in certain embodiments, there is no need to HLA-type the cells prior to
administration. In
other words, embryonic-like stem cells may be taken from a heterologous donor,
or a
plurality of heterologous donors, and transplanted to an individual in need of
such cells, and
the transplanted cells will remain within the host indefinitely. This
elimination of the need
for HLA typing greatly facilitates both the transplantation procedure itself
and the
identification of donors for transplantation. However, the embryonic-like stem
cells or
supplemented cell populations containing them may be HLA matched (donor to
recipient)
prior to administration.
The inventors have discovered that the efficacy of treating an individual with
the
embryonic-like stem cells or supplemented cell populations is enhanced if
these cells are
preconditioned. Preconditioning comprises storing the cells in a gas-permeable
container of
a period of time at approximately -5 to23°C, 0-10°C, or
preferably 4-5°C. The period of
time may be between I8 hours and 21 days, between 48 hours and 10 days, and is
preferably
between 3-5 days. The cells may be cryopreserved prior to preconditioning or,
preferably,
are preconditioned immediately prior to administration.
Thus, in one embodiment, the invention provides a method of treating an
individual
comprising administering to said individual embryonic-Iike stem cells
collected from at
least one donor. "Donor" as used herein means an adult, child, infant, or,
preferably, a
placenta. In another, preferred, embodiment, the method comprises
administering to said
individual embryonic-like stem cells that are collected from a plurality of
donors and
pooled. In a specific embodiment, said embryonic-like stem cells are stem
cells taken from
a plurality of donors. When collected form multiple donors, the dosage units,
where a
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"dosage unit" is a collection from a single donor, may be pooled prior to
administration,
may be administered sequentially, or may be administered alternatively. In
another
embodiment of the method, said embryonic-like stem cells are mixed with, or
"spiked" into
umbilical cord blood, and the mixture adminstered to an individual. In more
specific
embodiments of the method, the ratio of embryonic-like stem cells to cord
blood may be at
least 20:80, 30:70, 40:60, 50:50, 60:40, 70:30 or 80:20, by number of total
nucleated cells.
4.7 ADMINISTRATION OF STEM CELLS: DOSAGES
A particularly useful aspect of the invention is the administration of high
doses of
stem cells to an individual; such numbers of cells are significantly more
effective than the
material (for example, bone marrow or cord blood) from which they were
derived. In this
context, "high dose" indicates 5, 10, 15 or 20 or more times the number of
total nucleated
cells, including stem cells, particularly embryonic-like stem cells, than
would be
administered, for example, in a bone marrow transplant. Typically, a patient
receiving a
stem cell infusion, for example for a bone marrow transplantation, receives
one unit of cells,
where a unit is approximately 1 x 10~ nucleated cells (corresponding to 1-2 X
10g stem
cells). For high-dose therapies, therefore, a patient would be administered 3
billion, 5
billion, 10 billion, 15 billion, 20 billion, 30 billion, 40 billion, 50
billion or more, or,
alternatively, 3, 5, 10, 20, 30, 40, or 50 units or more, of total nucleated
cells, either
embryonic-like stem cells alone, or embryonic-like stern cells spiked into
another stem or
progenitor cell population (e.g., embryonic-like stem cells spiked into
umbilical cord
blood). In one preferred embodiment, for example, an individual is given 15
units of spiked
cord blood, where the unit contains approximately 750 million cord blood cells
and 500
million embryonic-like stem cells. Thus, in one embodiment, the number of
nucleated cells
administered to an individual is at least five times the number of cells
normally
administered in a bone marrow replacement. In another specific embodiment of
the
method, the number of nucleated cells administered to an individual is at
least ten times the
number of cells normally administered in a bone marrow replacement. In another
specific
embodiment of the method, the number of nucleated cells administered to an
individual is at
least fifteen times the number of cells normally administered in a bone marrow
replacement.
In another embodiment of the method, the total number of nucleated cells,
which includes
stem cells, administered to an individual is between 1-100 x 10$ per kilogram
of body
weight. In another embodiment, the number of total nucleated cells
administered is at least
5 billion cells. In another embodiment, the total number of nucleated cells
administered is
at least 15 billion cells.
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In another embodiment of the method, said embryonic-like stem cells and said
cord
blood are mixed immediately prior to (i. e., within five minutes ofJ
administration to said
individual. In another embodiment, said embryonic-like stern cells and said
cord blood are
mixed at a point in time more than five minutes prior to administration to
said individual.
In another embodiment of the method, the embryonic-like stem cells are
cryopreserved and
thawed prior to administration to said individual. In another embodiment, said
embryonic-
like stem cells and said cord blood are mixed to form a supplemented cell
population at a
point in time more than twenty-four hours prior to administration to said
individual,
wherein said supplemented cell population has been cryopreserved and thawed
prior to said
administration. In another embodiment, said embryonic-like stem cells and/or
supplemented cell populations may be administered more than once. In another
embodiment, said embryonic-like stem cells and/or supplemented cell
populations are
preconditioned by storage from between 18 hours and 21 days prior to
administration. In a
more specific embodiment, the cells are preconditioned for 48 hours to 10 days
prior to
administration. In a preferred specific embodiment, said cells are
preconditioned for 3-5
days prior to transplantation. In a preferred embodiment of any of the methods
herein, said
embryonic-Iike stem cells are not HLA typed prior to administration to an
individual.
W another specific embodiment of the method, said embryonic-like stem cells
are
primarily (i.e., > 50%) CD34+ cells. In a more specific embodiment of the
method, said
embryonic-like stem cells are primari.Iy CD34t33+ stem cells.
Therapeutic or prophylactic treatment of an individual with embryonic-like
stem
cells or supplemented cell populations containing them may be considered
efficacious if the
disease, disorder or condition is measurably improved in any way. Such
improvement may
be shown by a number of indicators. Measurable indicators include, for
example, detectable
changes in a physiological condition or set of physiological conditions
associated with a
particular disease, disorder or condition (including, but not limited to,
blood pressure, heart
rate, respiratory rate, counts of various blood cell types, levels in the
blood of certain
proteins, carbohydrates, lipids or cytokines or modulation expression of
genetic markers
associated with the disease, disorder or condition). Treatment of an
individual with the
stem cells or supplemented cell populations of the invention would be
considered effective
if any one of such indicators responds to such treatment by changing to a
value that is
within, or closer to, the normal value. The normal value may be established by
normal
ranges that are known in the art fox various indicators, or by comparison to
such values in a
control. In medical science, the efficacy of a treatment is also often
characterized in terms
of an individual's impressions and subjective feeling of the individual's
state of health.
Improvement therefore may also be characterized by subjective indicators, such
as the
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individual's subjective feeling of improvement, increased well-being,
increased state of
health, improved level of energy, or the like, after administration of the
stem cells or
supplemented cell populations of the invention.
The embryonic-like stem cells and supplemented cell populations of the
invention
may be administered to a patient in any pharmaceutically or medically
acceptable manner,
including by injection or transfusion. The cells or supplemented cell
populations may be
contain, or be contained in any pharmaceutically-acceptable carrier (See
Section 4.8). The
embryonic-like stem cells or supplemented cell populations may be carried,
stored, or
transported in any pharmaceutically or medically acceptable container, for
example, a blood
bag, transfer bag, plastic tube or vial.
4.8 HITS
The invention also provides a pharmaceutical pack or kit comprising one or
more
containers filled with one or more of the ingredients of the pharmaceutical
compositions of
the invention. Optionally associated with such containers) can be: an
apparatus for cell
culture, one or more containers filled with a cell culture medium or one or
more
components of a cell culture medium, an apparatus for use in delivery of the
compositions
of the invention, e.g., an apparatus for the intravenous injection of the
compositions of the
invention, and/or a notice in the form prescribed by a governmental agency
regulating the
m~ufacture, use or sale of pharmaceuticals or biological products, which
notice reflects
approval by the agency of manufacture, use or sale for human administration.
In a specific
embodiment, the kit comprises one or more containers filled with embryonic-
like stem cells
of the invention and one or more different containers filled with stem cells,
e.g., umbilical
cord blood, as disclosed above.
In one embodiment, the kit comprises a mixture of stem cells, e.g., cord blood
cells,
supplemented with embryonic-like stem cells contained within one bag or
container. In
another embodiment, the kit comprises a population of cord blood cells and a
population of
embryonic-Iike stem cells that are contained within two separate bags or
containers. In
certain embodiments, the kit comprises a "two bag" composition wherein the bag
containing
the cord blood cells and the bag containing the embryonic-like stem cells is
mixed prior to,
or at the time of, administration to a patient in need thereof. In other
embodiments, the kit
comprises a population of cord blood cells and a population of embryonic-like
stem cells
that are contained within two separate bags or containers and that are
administered
separately (e.g., simultaneously or sequentially) to a patient, wherein the
mixing of the two
cell populations occurs iya vivo.
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W another embodiment, the kit provides a population of cord blood cells and a
population of embryonic-like stem cells that are physically mixed prior to
administration.
In another aspect of this embodiment, the kit comprises a container containing
a growth
factor, e.g.,GM-CSF, IL-4, Flt3L, CD40L, IFN-alpha, TNF-alpha, IFN-gamma, IL-
2, IL-6,
retinoic acid, basic fibroblast growth factor, TGF-beta-l, TGF-beta-3,
hepatocyte growth
factor, epidermal growth factor, cardiotropin-1, angiotensinogen, angiotensin
I (AI),
angiotensin II (AII), All ATZ type 2 receptor agonists, or analogs or
fragments thereof. In
another aspect of this embodiment, the two populations are physically mixed
and then
treated with the growth factor comprised in the kit, to induce cell
differentiation, prior to
administration to the patient. In another aspect of this embodiment, the cord
blood cells
and/or the embryonic-like stem cells are treated with the growth factor
comprised in the kit,
to induce cell differentiation and then physically mixed prior to
administration to the
patient.
The following experimental examples are offered by way of illustration and not
by
way of limitation.
S. EXAMPLES
5.1. EXAMPLE 1: ANALYSIS OF CELL TYPES RECOVERED FROM
PERFUSATE OF DRAINED PLACENTA
This example describes the analysis of the cell types recovered from the
effluent
perfusate of a placenta cultured according to the methods of the invention.
Twenty ml of phosphate buffered saline solution (PBS) was added to the
perfusion
liquid and a 10 ml portion was collected and centrifuged for 25 minutes at
3000 rpm
(revolutions per minute). The effluent was divided into four tubes and placed
in an ice bath.
2.5 ml of a 1% fetal calf serum (FCS) solution in PBS was added and the tubes
were
centrifuged (140 minutes x 10 g (acceleration due to gravity)). The pellet was
resuspended
in 5 ml of 1% FCS and two tubes were combined. The total mononucleocytes were
calculated by adding the total lymphocytes and the total monocytes, and then
multiplying
the result by the total cell suspension volume.
The following table discloses the types of cells obtained by perfusion of a
cultured
placenta according to the methods described hereinabove.
WBC Lym% MID% GRA% Total # of
10001m1 Volume Cells
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CB 10.5 43.2 8 48.8 60 ml 6.3 X 10$
(Cord
Blood)
PP 12.0 62.9 18.2 18.9 1 S ml 1. 8 X 1 O8
(Placenta
perfusate,
room
temperature)
PPz 11.7 56.0 19.2 24.8 30 ml 3.5 X 10$
(Placenta
perfusate,
37° C)
Samples of PP were after Ficoll.
Total cell number of PP after Ficoll was 5.3 X 108 and number of CB before
processing is 6.3 X 10g. Lym% indicates percent of lymphocytes; M)D% indicates
percent of midrange white blood cells; and GRA% indicates percent of
granulocytes.
5.2. EXAMPLE 2: ANALYSIS OF CELLS OBTAINED BY PERFUSION
AND INCUBATION OF PLACENTA
The following example describes an analysis of cells obtained by perfusion and
incubation of placenta according to the methods of the invention.
5.2.1. MATERIALS AND METHODS
Placenta donors were recruited from expectant mothers that enrolled in private
umbilical cord blood banking programs and provided informed consent permitting
the use
of the exsanguinated placenta following recovery of cord blood for research
purposes.
Donor data may be confidential. These donors also permitted use of blinded
data generated
from the normal processing of their umbilical cord blood specimens for
cryopreservation.
This allowed comparison between the composition of the collected cord blood
and the
effluent perfusate recovered using the experimental method described below.
Following exsanguination of cord blood from the umbilical cord and placenta is
stored at room temperature and delivered to the laboratory within four to
twenty-four hour,
according to the methods described hereinabove, the placenta was placed in a
sterile,
insulated container at room temperature and delivered to the laboratory within
4 hours of
birth. Placentas were discarded if, on inspection, they had evidence of
physical damage such
as fragmentation of the organ or avulsion of umbilical vessels. Placentas were
maintained
at room temperature (232 °C) or refrigerated (4 °C) in sterile
containers for 2 to 20 hours.
Periodically, the placentas were immersed and washed in sterile saline at 253
°C to remove
any visible surface blood or debris.
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The wnbilical cord was transected approximately 5 cm from its insertion into
the
placenta and the umbilical vessels were cannulated with TEFLON~ or
polypropylene
catheters connected to a sterile fluid path allowing bi-directional perfusion
of the placenta
and recovery of the effluent fluid. The methods described hereinabove enabled
all aspects of
placental conditionng, perfusion and effluent collection to be performed under
controlled
ambient atmospheric conditions as well as real-time monitoring of
intravascular pressure
and flow rates, core and perfusate temperatures and recovered effluent
volumes. A range of
conditioning protocols were evaluated over a 24-hour postpartum period, and
the cellular
composition of the effluent fluid was analyzed by flow cytometry, light
microscopy and
colony forming unit assays.
5.2.2. PLACENTAL CONDITIONING
The donor placentas were processed at room temperature within 12 to 24 hours
after
delivery. Before processing, the membranes wexe removed and the maternal site
washed
clean of residual blood. The umbilical vessels were cannulated with catheters
made from 20
gauge Butterfly needles use for blood sample collection.
The donor placentas were maintained under varying conditions such as
maintenance
at 5-37 ° 5% CO2, pH 7.2 to 7.5, preferably pH 7.45, in an attempt to
simulate and sustain a
physiologically compatible environment for the proliferation and recruitment
of residual
embryonic-like stem cells. The cannula was flushed with IMDM serum-free medium
(GibcoBRL, NY) containing 2U/ml heparin (Elkins-Sinn, NJ). Perfusion of the
placenta
continued at a rate of 50 ml per minute until approximately 150 ml of
perfusate was
collected. This volume of perfusate was labeled "early fraction." Continued
perfusion of
the placenta at the same rate resulted in the collection of a second fraction
of approximately
150 ml and was labeled "late fraction." During the course of the procedure,
the placenta
was gently massaged to aid in the perfusion process and assist in the recovery
of cellular
material. Effluent fluid was collected from the perfusion circuit by both
gravity drainage
and aspiration through the arterial cannula.
Placentas were then perfused with heparinized (2U/ml) Dulbecco's modified
Eagle
Medium (H.DMEM) at the rate of 15 ml/minute for 10 minutes and the perfusates
wexe
collected from the maternal sites within one hour and the nucleated cells
counted. The
perfusion and collection procedures were repeated once or twice until the
number of
recovered nucleated cells fell below 100/ml. The perfusates were pooled and
subjected to
light centrifugation to xemove platelets, debris and de-nucleated cell
membranes. The
nucleated cells were then isolated by Ficoll-Hypaque density gradient
centrifugation and
after washing, resuspended in H.DMEM. For isolation of the adherent cells,
aliquots of S-
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x 10~ cells were placed in each of several T-75 flasks and cultured with
commercially
available Mesenchymal Stem Cell Growth Medium (MSCGM) obtained from
BioWhittaker, and placed in a tissue culture incubator (37°C, 5% COZ).
After 10 to 15 days,
the non-adherent cells were removed by washing with PBS, which was then
replaced by
MSCGM. The flasks were examined daily for the presence of various adherent
cell types
and in particular, for identification and expansion of clusters of
fibroblastoid cells.
5.2.3. CELL RECOVERY AND ISOLATION
Cells were recovered from the perfusates by centrifugation at 5000 x g for 15
10 minutes at room temperature. This procedure served to separate cells from
contaminating
debris and platelets. The cell pellets were resuspended in IMDM serum-free
medium
containing 2U/ml heparin and ZmM EDTA (GibcoBRL, NY). The total mononuclear
cell
fraction was isolated using Lyrnphoprep (Nycomed Pharma, Oslo, Norway)
according to the
manufacturer's recommended procedure and the mononuclear cell fraction was
resuspended. Cells were counted using a hemocytometer. Viability was evaluated
by trypan
blue exclusion. Isolation of mesenchymal cells was achieved by "differential
trypsinization," using a solution of 0.05% trypsin with 0.2% EDTA (Sigma, St.
Louis MO).
Differential trypsinization was possible because fibroblastoid cells detached
from plastic
surfaces within about five minutes whereas the other adherent populations
required more
than 20-30 minutes incubation. The detached fibroblastoid cells were harvested
following
trypsinization and trypsin neutralization, using Trypsin Neutralizing Solution
(TNS,
BioWhittaker). The cells were washed in H.DMEM and resuspended in MSCGM.
Flow cytometry was carried out using a Becton-Dickinson FACSCalibur instrument
and FITC and PE labeled monoclonal antibodies (mAbs), selected on the basis of
known
markers for bone marrow-derived MSC (mesenchymal stem cells), were purchased
from
B.D. and Caltag laboratories (South San Francisco, CA.), and SH2, SH3 and SH4
antibody
producing hybridomas were obtained from and reactivities of the mAbs in their
cultured
supernatants were detected by FITC or PE labeled F(ab)'2 goat anti-mouse
antibodies.
Lineage differentiation was carried out using coixnnercially available
induction and
maintenance culture media (BioWhittaker), used as per manufacturer's
instructions.
5.2.4. ISOLATION OF PLACENTAL EMBRYONIC-LIKE STEM
CELLS
Microscopic examination of the adherent cells in the culture flasks revealed
morphologically different cell types. Spindle-shaped cells, round cells with
large nuclei and
n~erous perinuclear small vacuoles, and star-shaped cells with several
projections
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(through one of wluch star-shaped cells were attached to the flask) were
observed adhering
to the culture flasks. Although no attempts were made to further characterize
these adherent
cells, similar cells were observed in the culture of bone marrow, cord and
peripheral blood,
and therefore considered to be non-stem cell-like in nature. The fibroblastoid
cells,
appearing last as clusters, were candidates for being MSC (mesenchymal stem
cells) and
were isolated by differential trypsinization and subcultured in secondary
flasks. Phase
microscopy of the rounded cells, after trypsinization, revealed that the cells
were highly
granulated; indistinguishable from the bone marrow-derived MSC produced in the
laboratory or purchased from BioWhittaker. When subcultured, the placenta-
derived
embryonic-like stem cells, in contrast to their earlier phase, adhered within
hours, assumed
characteristic fibroblastoid shape, and formed a growth pattern identical to
the reference
bone marrow-derived MSC. During subculturing and refeeding, moreover, the
loosely
bound mononuclear cells were washed out and the cultures remained homogeneous
and
devoid of any visible non-fibroblastoid cell contaminants.
5.2.5. RESULTS
The expression of CD-34, CD-38, and other stem cell-associated surface markers
on
early and late fraction purified mononuclear cells was assessed by flow
cytometry.
Recovered, sorted cells were washed in PBS and then double-stained with
antiCD34
phycoerythrin and anti-CD38 fluorescein isothiocyanate (Becton Dickinson,
Mountain
View, CA).
Cell isolation was achieved by using magnetic cell separation, such as for
example,
Auto Macs (Miltenyi). Preferably, CD 34+ cell isolation is performed first.
5.3. EXAMPLE 3: PERFUSION MEDIUM
The following example provides a formula of the preferred perfusate solution
for the
cultivation of isolated placentas.
Chemical Source Stock Final 500 ml
ConcentrationConcentration
DMEM-LG GibcoBRLl 1885- 300 ml
084
MCDB201 Sigma M-6770 dissolved pH to 7.2. 200 ml
in
H20 filter
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FCS Hyclone 100% 2% 1 Oml
ITS Sigma I-3146 100x lx Sml
or
GibcoBRL41400-
045
Pen&Strep GibcoBRL15140- 100x lx 5 ml
122
LA+BSA Sigma+GibcoBRL 100x(1 ~,g/ml lOng/ml S mI
of of LA
BSA LA
DexamethasonSigma D-2915 0.25mM in H20 0.05 ~,M 100 ~,l
a
L-Ascorbic Sigma A-8960 1000x(100mM) 1x(0.1 mM) 500 ~,1
Acid
PDGF (SO R&D 220BD 10 ~g/ml in 10 ng/ml S00 ~1
~,g)
4mM HCl +
0.1% BSA
EGF (200 Sigma E-9644 10 ~g/ml in 10 ng/ml 500 ~,1
fig)
1 OmM HAc +
0.1 % BSA
The above-composition is a perfusate that may be used at a variety of
temperatures
to perfuse placenta. It should be noted that additional components such as
antibiotics,
anticoagulant and other growth factors may be used in the perfusate or culture
media.
5.4 EXAMPLE 4: INDUCTION OF DIFFERENTIATION INTO
PARTICULAR CELL TYPES
Cord blood cells and/or embryonic-like stem cells are induced to differentiate
into a
particular cell type by exposure to a growth factor. Growth factors that are
used to induce
induction include, but are not limited to: GM-CSF, TL-4, Flt3L, CD40L, IFN-
alpha, TNF-
alpha, IFN-gamma, IL-2, IL-6, retinoic acid, basic fibroblast growth factor,
TGF-beta-1,
TGF-beta-3, hepatocyte growth factor, epidermal growth factor, cardiotropin-1,
angiotensinogen, angiotensin I (AT), angiotensin II (AII), All ATZ type 2
receptor agonists,
or analogs or fragments thereof.
5.4.1 Induction Of Differentiation Into Neurons
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This example describes the induction of cord blood cells and/or embryonic-like
stem
cells to differentiate into neurons. The following protocol is employed to
induce neuronal
differentiation:
g 1. Placental stem cells are grown for 24 hr in preinduction media consisting
of
DMEM/20% FBS and 1 mM beta-mercaptoethanol.
2. Preinduction media is removed and cells are washed with PBS.
3. Neuronal induction media consisting of DMEM and 1-10 mM betamercaptoethanol
is added. Alternatively, induction media consisting of DMEM/2% DMSO/200 ~M
butylated hydroxyanisole may be used to enhance neuronal differentiation
efficiency.
4. In certain embodiments, morphologic and molecular changes may occur as
early as
60 minutes after exposure to serum-free media and betamercaptoethanol
(Woodbury
et al., J. Neurosci. Res., 61:364-370). RT/PCR may be used to assess the
expression
of e.g., nerve growth factor receptor and neurofilament heavy chain genes.
5.4.2 Induction Of Differentiation Into Ad~ocytes
This example describes the induction of cord blood cells and/or embryonic-like
stem
cells to differentiate into adipocytes. The following protocol is employed to
induce
adipogenic differentiation:
1. Placental stem cells are grown in MSCGM (Bio Whittaker) or DMEM
supplemented with 15% cord blood serum.
2. Three cycles of induction/maintenance are used. Each cycle consists of
feeding the
placental stem cells with Adipogenesis Induction Medium (Bio Whittaker) and
culturing the cells for 3 days (at 37°C, 5% COz), followed by 1-3 days
of culture in
Adipogenesis Maintenance Medium (Bio Whittaker). An induction medium is used
that contains 1 ~M dexamethasone, 0.2 mM indomethacin, 0.01 mg/ml insulin, 0.5
mM IBMX, DMEM-high glucose, FBS, and antibiotics.
3. After 3 complete cycles of induction/maintenance, the cells are cultured
for an
additional 7 days in adipogenesis maintenance medium, replacing the medium
every
2-3 days.
4. Adipogenesis may be assessed by the development of multiple
intracytoplasmic lipid
vesicles that can be easily observed using the lipophilic stain oil red O.
RT/PCR
assays are employed to examine the expression of lipase and fatty acid binding
protein genes.
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5.4.3 Induction Of Differentiation Into Chondroc es
This example describes the induction of cord blood cells and/or embryonic-like
stem
cells to differentiate into chondrocytes. The following protocol is employed
to induce
chondrogenic differentiation:
1. Placental stem cells are maintained in MSCGM (Bio Whittaker) or DMEM
supplemented with 15% cord blood serum.
2. Placental stem cells are aliquoted into a sterile polypropylene tube. The
cells are
centrifuged (150 x g for 5 minutes), and washed twice in Incomplete
Chondrogenesis Medium (Bio Whittaker).
3. After the last wash, the cells are resuspended in Complete Chondrogenesis
Medium
(Bio Whittaker) containing 0.01 ~,g/ml TGF-beta-3 at a concentration of 5 x
10(5)
cells/ml.
4. 0.5 rnl of cells is aliquoted into a 15 ml polypropylene culture tube. The
cells are
pelleted at 150 x g for 5 minutes. The pellet is left intact in the medium.
5. Loosely capped tubes are incubated at 37°C, 5% COZ for 24 hours.
6. The cell pellets are fed every 2-3 days with freshly prepared complete
chondrogenesis medium.
7. Pellets are maintained suspended in medium by daily agitation using a low
speed
2p vortex.
8. Chondrogenic cell pellets are harvested after 14-28 days in culture.
9. Chondrogenesis may be characterized by e.g., observation of production of
esoinophilic ground substance, assessing cell morphology, an/or RT/PCR for
examining collagen 2 and collagen 9 gene expression.
5.4.4 Induction Of Differentiation Into Osteoc. es
This example describes the induction of cord blood cells and/or embryonic-like
stem
cells to differentiate into osteocytes. The following protocol is employed to
induce
osteogenc differentiation:
1. Adherent cultures of placental stem cells are cultured in MSCGM (Bio
Whittaker) or
DMEM supplemented with 15% cord blood serum.
2. Cultures are rested for 24 hours in tissue culture flasks.
3. Osteogenic differentiation is induced by replacing MSCGM with Osteogenic
Induction Medium (Bio Whittaker) containing 0.1 ~,M dexamethasone, 0.05 mM
ascorbic acid-2-phosphate, 10 mM beta glycerophosphate.
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4. Cells are fed every 3-4 days for 2-3 weeks with Osteogenic Induction
Medium.
5. Differentiation is assayed using a calcium-specific stain and RT/PCR for
alkaline
phosphatase and osteopontin gene expression.
5.4.5 Induction Of Differentiation Into Hepatoc. es
This example describes the induction of cord blood cells and/or embryonic-like
stem
cells to differentiate into hepatocytes. The following protocol is employed to
induce
hepatogenic differentiation:
1. Placental stem cells are cultured in DMEM/20% CBS supplemented with
hepatocyte
growth factor, 20 ng/ml; and epidermal growth factor, 100 ng/ml. TW ockOut
Serum
Replacement may be used in lieu of FBS.
2. IL-6 50 ng/ml is added to induction flasks.
5.4.6 Induction Of Differentiation Into Pancreatic Cells
This example describes the induction of cord blood cells and/or embryonic-like
stem
cells to differentiate into pancreatic cells. The following protocol is
employed to induce
pancreatic differentiation:
1. Placental stem cells are cultured in DMEM/20% CBS, supplemented with basic
fibroblast growth factor, 10 ng/ml; and transforming growth factor beta-1, 2
ng/ml.
Knockout Serum Replacement may be used in lieu of CBS.
2. Conditioned media from nestin-positive neuronal cell cultures is added to
media at a
50/50 concentration.
3. Cells are cultured for 14-28 days, refeeding every 3-4 days.
4. Differentiation is characterized by assaying for insulin protein or insulin
gene
expression by RT/PCR.
5.4.7 Induction Of Differentiation Into Cardiac Cells
This example describes the induction of cord blood cells and/or embryonic-like
stem
cells to differentiate into cardiac cells. The following protocol is employed
to induce
myogenic differentiation:
1. Placental stem cells are cultured in DMEM/20% CBS, supplemented with
retinoic
acid, 1 ~M; basic fibroblast growth factor, 10 ng/ml; and transforming growth
factor
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beta-1, 2 ng/ml; and epidermal growth factor, 100 ng/m1. Knockout Serum
Replacement may be used in lieu of CBS.
2. Alternatively, placental stem cells are cultured in DMEM/20% CBS
supplemented
with 50 ng/mI Cardiotropin-1 for 24 hours.
3. Alternatively, placental stem cells are maintained in protein-free media
for 5-7 days,
then stimulated with human myocardium extract (escalating dose analysis).
Myocardium extract is produced by homogenizing 1 gm human myocardium in 1
HEPES buffer supplemented with 1% cord blood serum. The suspension is
incubated for 60 minutes, then centrifuged and the supernatant collected.
4. Cells are cultured for 10-14 days, refeeding every 3-4 days.
5. Differentiation is assessed using cardiac actin RTIPCR gene expression
assays.
5.4.8 Characterization of Cord Blood Cells and/or Embryonic-Like Stem Cells
Prior to and/or After Differentiation
The embryonic-like stem cells, the cord blood cells and/or the populations of
cord
blood cells spiked with embryonic-like stem cells are characterized prior to
and/or after
differentiation by measuring changes in morphology and cell surface markers
using
techniques such as flow cytometry and irmnunocytochemistry, and measuring
changes in
gene expression using techniques, such as PCR. Cells that have been exposed to
growth
factors and/or that have differentiated are characterized by the presence or
absence of the
following cell surface markers: CD10+, CD29+, CD34-, CD38-, CD44+, CD45-,
CD54+,
CD90+, SH2+, SH3+, SH4+, SSEA3-, SSEA4-, OCT-4+, and ABC-p+. Preferably, the
embryonic-like stem cell are characterized, prior to differentiation, by the
presence of cell
surface markers OCT-4+, APC-p+, CD34- and CD38-. Stem cells bearing these
markers
are as versatile (e.g., pluripotent) as human embryonic stem cells. Cord blood
cells are
characterized, prior to differentiation, by the presence of cell surface
markers CD34+ and
CD38+. Differentiated cells derived from embryonic-like stem cells, cord blood
cells
and/or a populations of cord blood cells spiked with embryonic-like stem cells
preferably do
not express these markers.
5.5 EXAMPLE 5: TREATMENT OF INDIVIDUALS HAVING
AMYLOTROPHIC LATERAL SCLEROSIS WITH EMBRYONIC-
LIKE STEM CELLS
Amyotrophic Lateral Sclerosis (ALS), also called Lou Gehrig's disease, is a
fatal
neurodegenerative disease affecting motor neurons of the cortex, brain stem
and spinal cord.
ALS affects as many as 20,000 Americans with 5,000 new cases occurring in the
US each
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year. The majority of ALS cases are sporadic (S-ALS) while ~ 5-10% are
hereditary
(familial - F-ALS). ALS occurs when specific nerve cells in the brain and
spinal cord that
control voluntary movement gradually degenerate. The cardinal feature of ALS
is the loss
of spinal motor neurons which causes the muscles under their control to weaken
and waste
away leading to paralysis. ALS manifests itself in different ways, depending
on which
muscles weaken first. ALS strikes in mid-life with men being one-and-a-half
times more
likely to have the disease as women. ALS is usually fatal within five years
after diagnosis.
ALS has both familial and sporadic forms, and the familial forms have now been
linked to several distinct genetic loci. Only about 5-10% of ALS cases are
familial. Of
these, 15-20% are due to mutations in the gene encoding Cu/Zn superoxide
dismutase 1
(SOD1). These appear to be "gain-of function" mutations that confer toxic
properties on
the enzyme. The discovery of SOD mutations as a cause for ALS has paved the
way for
some progress in the understanding of the disease; animal models for the
disease are now
available and hypotheses are being developed and tested concerning the
molecular events
leading to cell death.
Presented below is an example method of treating an individual having ALS with
embryonic-like stem cells derived from placenta. The method involves
intravenous infusion
through a peripheral, temporary angiocatheter.
An individual having ALS is first assessed by the performance of standard
laboratory analyses. Such analyses may include a metabolic profile; CDC with
differential;
lipid profile; fibrinogen level; ABO rH typing of the blood; liver function
tests; and
determination of BUN/creatine levels. Individuals are instructed the day prior
to the
transplant to take the following medications: diphenhydramine (BenadrylTM), 25
mg t.i.d,
and prednisone, 10 mg.
The embryonic-like stem cells, either alone or spiked into cord blood, are
taken from
cryopreserved stock, thawed, and maintained for approximately two days prior
to
transplantation at a temperature of approximately 5°C.
The individual is transplanted at an outpatient clinical center which has all
facilities
necessary for intravenous infusion, physiological monitoring and physical
observation.
Approximately one hour prior to transplantation, the individual receives
diphenhydramine
(BenadrylTM), 25 mg x 1 P.O., and prednisone, 10 mg x 1 P.O. This is
precautionary, and is
meant to reduce the likelihood of an acute allergic reaction. At the time of
transfusion, an
18 G indwelling peripheral venous line is places into one of the individual's
extremities,
and is maintained open by infusion of DS 1/2 normal saline + 20 mEq KCl at a
TKO rate.
The individual is examined prior to transplantation, specifically to note
heart rate,
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respiratory rate, temperature. Other monitoring may be performed, such as an
electrocardiogram and blood pressure measurement.
Embryonic-like stem cells are then infused at a rate of 1 unit per hour in a
total
delivered fluid volume of 60 ml, where a unit is approximately 1-2 109 total
nucleated cells.
Alternatively, the unit of embryonic-like stem cells is delivered in cord
blood having a total
fluid volume of 60 ml. In this case, the ratio of the number of embryonic-like
stem cells to
stem cells in the cord blood is at least 2:1. The administered unit may also
consist of cord
blood alone. Based upon data from pre-clinical studies in mice, a total of 2.0-
2.5 x 108 cells
per kilogram of body weight should be administered. For example, a 70 kilogram
individual would receive approximately 14-18 x 109 total nucleated cells. The
individual
should be monitored for signs of allergic response or hypersensitivity, which
are signals for
immediate cessation of infusion.
Post-infusion, the individual should be montored in a recumbent position for
at
least 60 minutes, whereupon he or she may resume normal activities.
5.6 EXAMPLE 6: TREATMENT OF INDIVIDUALS HAVING
ATHEROSCLEROSIS USING EMBRYONIC-LIKE STEM CELLS
The infusion protocol outlined in Example 5 may be used to administer the
embryonic-like stem cells, either alone or spiked into umbilical cord blood,
to a. patient
having atherosclerosis. The embryonic-like stem cells or supplemented cell
populations
may be achninstered to asymptomatic individuals, individuals that are
candidates for
angioplasty, or to patients that have recently (within one week) undergone
cardiac surgery.
The present invention is not to be limited in scope by the specific
embodiments
described herein. Indeed, various modifications of the invention in addition
to those
described herein will become apparent to those skilled in the art from the
foregoing
description. Such modifications are intended to fall within the scope of the
appended
claims.
All references cited herein are incorporated herein by reference in their
entirety and
for all purposes to the same extent as if each individual publication, patent
or patent
application was specifically and individually indicated to be incorporated by
reference in its
entirety for all purposes.
The citation of any publication is for its disclosure prior to the filing date
and should
not be construed as an admission that the present invention is not entitled to
antedate such
publication by virtue of prior invention.
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