U.S. patent application number 13/293037 was filed with the patent office on 2012-05-17 for production of oligodendrocytes from placenta-derived stem cells.
Invention is credited to Mohammad HEIDARAN.
Application Number | 20120121550 13/293037 |
Document ID | / |
Family ID | 37709411 |
Filed Date | 2012-05-17 |
United States Patent
Application |
20120121550 |
Kind Code |
A1 |
HEIDARAN; Mohammad |
May 17, 2012 |
PRODUCTION OF OLIGODENDROCYTES FROM PLACENTA-DERIVED STEM CELLS
Abstract
The present invention provides methods and compositions for the
production of glial cells and oligodendrocytes from placenta stem
cells. The invention further provides for the use of these glia and
oligodendrocytes in the treatment of, and intervention in, for
example, trauma, ischemia and degenerative disorders of the central
nervous system (CNS), particularly in the treatment of
demyelinating diseases such as multiple sclerosis.
Inventors: |
HEIDARAN; Mohammad;
(Chatham, NJ) |
Family ID: |
37709411 |
Appl. No.: |
13/293037 |
Filed: |
November 9, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11580625 |
Oct 13, 2006 |
8071376 |
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13293037 |
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60727601 |
Oct 13, 2005 |
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Current U.S.
Class: |
424/93.7 ;
435/377 |
Current CPC
Class: |
C12N 2506/02 20130101;
A61P 43/00 20180101; A61K 35/12 20130101; A61P 25/28 20180101; C12N
2501/999 20130101; C12N 5/0622 20130101; A61P 25/00 20180101 |
Class at
Publication: |
424/93.7 ;
435/377 |
International
Class: |
A61K 35/30 20060101
A61K035/30; A61P 25/28 20060101 A61P025/28; C12N 5/079 20100101
C12N005/079 |
Claims
1. A method of producing an oligodendrocyte, comprising culturing a
placenta-derived stem cell under conditions and for a time
sufficient for said stem cell to exhibit a characteristic of an
oligodendrocyte.
2. The method of claim 1, wherein said characteristic is the
production of myelin oligodendrocyte specific protein or expression
of a gene encoding myelin oligodendrocyte specific protein.
3. The method of claim 1, wherein said culturing comprises
contacting said stem cell with isobutylmethylxanthine (IBMX).
4. An oligodendrocyte produced by the method of claim 1.
5. A method of treating a subject having a disease, disorder or
condition associated with abnormal myelination, comprising
introducing the oligodendrocyte of claim 4 to said subject.
6. The method of claim 5, wherein said disease, disorder or
condition is multiple sclerosis.
Description
1. FIELD OF THE INVENTION
[0001] The present invention provides methods and compositions for
the production of glial cells and oligodendrocytes from
placenta-derived stem cells (referred to hereafter as PDSCs). The
invention further provides for the use of these glia and
oligodendrocytes in the treatment of, and intervention in, for
example, trauma, ischemia and degenerative disorders of the central
nervous system (CNS).
2. BACKGROUND OF THE INVENTION
[0002] Embryonic stem cells capable of generating CNS glia can
promote functional recovery after trauma to the spinal cord, and
have potential for repair in demyelinating and dysmyelinating
diseases such as multiple sclerosis. However, the use of embryonic
stem cells for clinical therapy raises ethical concerns that cannot
be easily addressed.
[0003] Somatic stem cells have also been proposed for therapeutic
applications. For example, in animal models of cell replenishment
therapy. The therapeutic potential of grafted stem cells can only
be translated to clinical use if an ethically acceptable source of
autologous stem cells is available, and if control of self renewal
and fate decisions that program stem cell maturation into specific
cell types is achieved.
[0004] Neurodegenerative disorders increasingly account for
significant morbidity and mortality. Destruction of myelin
underlies the most common neurological disorder in young adults,
multiple sclerosis, and myelin affects repair after traumatic
spinal cord injury, preventing regeneration of damaged neuronal
axons and affecting electrical conduction in proximal, undamaged
axons. Replacement of oligodendrocytes is thus a significant
clinical goal. While oligodendrocytes are obtainable from neural
stem cells, such stem cells are difficult to obtain.
3. SUMMARY OF THE INVENTION
[0005] The present invention provides methods and compositions for
the production of oligodendrocytes from placenta derived stem
cells, and methods of using such oligodendrocytes to treat
diseases, disorders or conditions, such as those involving trauma,
ischemia, or systemic disorders of the central nervous system. For
example, in one aspect, the present invention relates to use of
oligodendrocytes produced from placenta-derived stem cells in the
treatment of diseases, disorders or conditions associated with
abnormal myelination. In one embodiment, the invention provides a
method of producing an oligodendrocyte, comprising culturing a
placenta-derived stem cell under conditions and for a time
sufficient for said stem cell to exhibit a characteristic of an
oligodendrocyte. In a specific embodiment, said characteristic is
the production of myelin oligodendrocyte specific protein or
expression of a gene encoding myelin oligodendrocyte specific
protein. In another specific embodiment, said culturing comprises
contacting said stem cell with isobutylmethylxanthine (IBMX). In
another embodiment, the invention provides an oligodendrocyte
produced by differentiation of a placenta derived stem cell. The
invention also provides a method of treating a subject having a
disease, disorder or condition associated with abnormal
myelination, comprising introducing such an oligodendrocyte of into
said subject. In a more specific embodiment, the disease, disorder
or condition is multiple sclerosis.
4. BRIEF DESCRIPTION OF THE FIGURES
[0006] FIG. 1: Oligodendrocyte progenitor cell maturation (McMorris
& McKinnon, Brain Pathology 6:313-329 (1996)). The temporal
appearance of antigens marks progression from migratory "early"
(O2A) progenitors to non-migratory "late" (O4, pro-OLblasts) and
postmitotic OLs. Maturation can be reversibly inhibited (.xi.), or
reversed (7) with the indicated factors. Monoclonal antibodies and
target antigens are outlined in Table 1.
[0007] FIG. 2: Human placental stem cells. Left: placental stem
cell colony formed in primary culture. Right: placental stem cells
treated with isobutylmethylxanthine (IBMX, a nonspecific inhibitor
of phosphodiesterases that also possesses adenosine agonist
activity); immunostaining shows the presence of neural lineage
markers including neural stem cell markers (vimentin, GFAP,
nestin), as well as markers for both neuronal (neurofilament,
neuron specific enolase) and glial (myelin oligodendrocyte specific
protein (MOSP)) lineage progression.
5. DETAILED DESCRIPTION OF THE INVENTION
5.1 Production of Oligodendrocytes
[0008] The present invention provides methods and compositions for
the production of oligodendrocytes from placenta-derived cells,
particularly placental stem cells, also referred to as
placenta-derived stem cells (PDSCs). Stem cells may be obtained
from a mammalian placenta by perfusion (see, e.g., Hariri, U.S.
Application Publication Nos. 2002/0123141 and 2003/0032179, which
are hereby incorporated herein in their entireties. Stem cells may
also be obtained from placenta by disruption (e.g., maceration) of
a placenta or part thereof (see., e.g., Section 6.2). Cells
displaying oligodendrocyte characteristics may be obtained from
placenta derived stem cells. These cells are useful in the
treatment of diseases, disorders or conditions associated with, for
example, demyelination or dysmyelination, such as multiple
sclerosis.
[0009] In one embodiment, differentiable cells, such as stem cells,
may be obtained from the placenta as follows. Primary cultures of
mononuclear cells (MNCs) are isolated from placentas, e.g., human
placenta perfusates. The placentas are obtained following birth of
full-term infants under informed consent of the donors. Briefly,
umbilical vessels are cannulated then connected to a
flow-controlled circuit, and the placenta is perfused at, e.g., 1
mL/min (room temperature, up to 24 hours) with Dulbecco's modified
Eagle's medium (DMEM, Gibco/BRL) containing high glucose, 1%
heparin and penicillin/streptomycin. Placenta perfusate (750 mL) is
then pooled, centrifuged, and the cell pellet resuspended in PBS
containing 1% fetal calf serum (FBS) then separated by differential
gradient density centrifugation through Lymphoprep.TM. (Gibco/BRL).
The buffy-coat interface containing mononucleated cells including
adherent PDSCs are recovered, resuspended in DMEM/10% FBS, plated
on fibronectin-coated (Sigma) Falcon plates and incubated at
37<C with 5% humidified CO.sub.2. After a 24-hour incubation the
nonadherent cells are discarded and the adherent cells are
maintained and expanded in fresh culture media; individual cell
colonies develop between 10 and 18 days and are expanded as PDSC
lines.
[0010] Human placenta-derived stem cells (PDSCs) display
fibroblast-like morphology in culture (FIG. 2a) and are HLA-class I
positive. Using FACS analysis these cells do not express the
hematopoietic markers CD34 or CD45. However, they do express the
multipotential surface markers CD10 (CALLA), CD29 (.beta..sub.1
integrin), CD54 (ICAM-1), CD90 (Thy-1) as well as SH2 and SH3.
Under standard growth conditions the doubling time for PDSCs is 18
to 36 hours, and the cells maintain this phenotype for greater than
40 population doublings in vitro.
[0011] A number of studies have described neural differentiation of
stem cells in vitro and in vivo, including embryonic, hematopoietic
and bone marrow stromal cells (Glaser et al., FASEB J. 2005
19(1):112-4 (2005); Rogister et al., Cellular Neuroscience
14:287-300 (1999); Rao and MayerProschel, Dev. Biol. 188:48-63
(1997); Anderson, Neuron 30:19-35 (2001); Rao, Stem Cells and
Development 13:452-455 (2004); Hermanson et al., Nature 419:934-939
(2002); Johe et al., Genes Dev. 10:3129-3140 (1996)).
[0012] Neural differentiation in vitro can be promoted using agents
that elevate intracellular cAMP. To determine whether cells derived
from human placenta are capable of generating neural lineages,
their was examined under similar conditions in vitro. Monolayer
PDSCs were harvested (0.25% trypsin, 1 mM EDTA) then replated at
(5.times.10.sup.3/mL) in culture medium containing 0.5 mM IBMX
(Sigma), and morphologic changes were monitored after 24-72 hours
by phase contrast microscopy, immunofluorescence and flow
cytometry. After 3 days in culture approximately 50% of cells had a
neural-like morphology with long processes and a pronounced
spherical cell soma, while control cultures remained
undifferentiated. IBMX-treated cultures also displayed
immunoreactivity for a number of neuroepithelial lineage markers
including neural progenitor markers (nestin, vimentin, GFAP),
neuronal markers (enolase, neurofilament), and cells that exhibited
glia markers (MOSP) (FIG. 2). To determine the temporal profile of
neural antigen expression flow cytometry was performed on both
treated and untreated PDSCs. A pronounced shift in antigenic
expression was apparent after day 1 with IBMX for all markers
tested, with no shift in untreated cells. Thus the changes observed
under these induction conditions reflect the rapid acquisition of
antigenic markers, consistent with neural differentiation rather
than a selective enrichment or survival.
[0013] Placenta-derived stem cells may be differentiated to
oligodendrocytes by culturing in culture medium comprising IBMX,
neural stem cell maturation factors (e.g., EGF, FGF), and/or
oligodendrocyte progenitor cell mitogens (e.g., FGF, PDGF).
Oligodendrocytes can be produced from placenta derived stem cells
as described above, and maintained or cultured as described in
Section 6.1. Oligodendrocyte differentiation can be assessed using
immunohistochemistry and PCR as described in Section 6.3 and flow
cytometry as described in Section 6.5. Oligodendrocyte
proliferation, migration, and survival can be assessed as described
in Section 6.4.
5.2 Placental Stem Cells and Placental Stem Cell Populations
[0014] The methods of immunosuppression of the present invention
use placental stem cells, that is, stem cells obtainable from a
placenta or part thereof, that (1) adhere to a tissue culture
substrate; (2) have the capacity to differentiate into
non-placental cell types; and (3) have, in sufficient numbers, the
capacity to detectably suppress an immune function, e.g.,
proliferation of CD4.sup.+ and/or CD8.sup.+ stem cells in a mixed
lymphocyte reaction assay. Placental stem cells are not derived
from blood, e.g., placental blood or umbilical cord blood. The
placental stem cells used in the methods and compositions of the
present invention have the capacity, and are selected for their
capacity, to suppress the immune system of an individual.
[0015] Placental stem cells can be either fetal or maternal in
origin (that is, can have the genotype of either the mother or
fetus). Populations of placental stem cells, or populations of
cells comprising placental stem cells, can comprise placental stem
cells that are solely fetal or maternal in origin, or can comprise
a mixed population of placental stem cells of both fetal and
maternal origin. The placental stem cells, and populations of cells
comprising the placental stem cells, can be identified and selected
by the morphological, marker, and culture characteristics discussed
below.
[0016] 5.2.1 Physical and Morphological Characteristics
[0017] The placental stem cells used in the present invention, when
cultured in primary cultures or in cell culture, adhere to the
tissue culture substrate, e.g., tissue culture container surface
(e.g., tissue culture plastic). Placental stem cells in culture
assume a generally fibroblastoid, stellate appearance, with a
number of cyotplasmic processes extending from the central cell
body. The placental stem cells are, however, morphologically
differentiable from fibroblasts cultured under the same conditions,
as the placental stem cells exhibit a greater number of such
processes than do fibroblasts. Morphologically, placental stem
cells are also differentiable from hematopoietic stem cells, which
generally assume a more rounded, or cobblestone, morphology in
culture.
[0018] 5.2.2 Cell Surface, Molecular and Genetic Markers
[0019] Placental stem cells, and populations of placental stem
cells, useful in the methods and compositions of the present
invention, express a plurality of markers that can be used to
identify and/or isolate the stem cells, or populations of cells
that comprise the stem cells. The placental stem cells, and stem
cell populations of the invention (that is, two or more placental
stem cells) include stem cells and stem cell-containing cell
populations obtained directly from the placenta, or any part
thereof (e.g., amnion, chorion, placental cotyledons, and the
like). Placental stem cell populations also includes populations of
(that is, two or more) placental stem cells in culture, and a
population in a container, e.g., a bag. Placental stem cells are
not, however, trophoblasts.
[0020] Placental stem cells generally express the markers CD73,
CD105, CD200, HLA-G, and/or OCT-4, and do not express CD34, CD38,
or CD45. Placental stem cells can also express HLA-ABC (MHC-1) and
HLA-DR. These markers can be used to identify placental stem cells,
and to distinguish placental stem cells from other stem cell types.
Because the placental stem cells can express CD73 and CD105, they
can have mesenchymal stem cell-like characteristics. However,
because the placental stem cells can express CD200 and HLA-G, a
fetal-specific marker, they can be distinguished from mesenchymal
stem cells, e.g., bone marrow-derived mesenchymal stem cells, which
express neither CD200 nor HLA-G. In the same manner, the lack of
expression of CD34, CD38 and/or CD45 identifies the placental stem
cells as non-hematopoietic stem cells.
[0021] In one embodiment, the invention provides an isolated cell
population comprising a plurality of immunosuppressive placental
stem cells that are CD200.sup.+, HLA-G.sup.+, wherein said
plurality detectably suppresses T cell proliferation in a mixed
lymphocyte reaction (MLR) assay. In a specific embodiment of the
isolated populations, said stem cells are also CD73.sup.+ and
CD105.sup.+. In another specific embodiment, said stem cells are
also CD34.sup.-, CD38.sup.- or CD45.sup.-. In a more specific
embodiment, said stem cells are also CD34.sup.-, CD38.sup.-,
CD45.sup.-, CD73.sup.+ and CD105.sup.+. In another embodiment, said
isolated population produces one or more embryoid-like bodies when
cultured under conditions that allow the formation of embryoid-like
bodies.
[0022] In another embodiment, the invention provides an isolated
cell population comprising a plurality of immunosuppressive
placental stem cells that are CD73.sup.+, CD105.sup.+, CD200.sup.+,
wherein said plurality detectably suppress T cell proliferation in
a mixed lymphocyte reaction (MLR) assay. In a specific embodiment
of said populations, said stem cells are HLA-G.sup.+. In another
specific embodiment, said stem cells are CD34.sup.-, CD38.sup.- or
CD45.sup.-. In another specific embodiment, said stem cells are
CD34.sup.-, CD38.sup.- and CD45.sup.-. In a more specific
embodiment, said stem cells are CD34.sup.-, CD38.sup.-, CD45.sup.-,
and HLA-G.sup.+. In another specific embodiment, said population of
cells produces one or more embryoid-like bodies when cultured under
conditions that allow the formation of embryoid-like bodies.
[0023] The invention also provides an isolated cell population
comprising a plurality of immunosuppressive placental stem cells
that are CD200.sup.+, OCT-4.sup.+, wherein said plurality
detectably suppresses T cell proliferation in a mixed lymphocyte
reaction (MLR) assay. In a specific embodiment, said stem cells are
CD73.sup.+ and CD105.sup.+. In another specific embodiment, said
stem cells are HLA-G.sup.+. In another specific embodiment, said
stem cells are CD34.sup.-, CD38.sup.- and CD45.sup.-. In a more
specific embodiment, said stem cells are CD34.sup.-, CD38.sup.-,
CD45.sup.-, CD73.sup.+, CD105.sup.+ and HLA-G.sup.+. In another
specific embodiment, the population produces one or more
embryoid-like bodies when cultured under conditions that allow the
formation of embryoid-like bodies.
[0024] The invention also provides an isolated cell population
comprising a plurality of immunosuppressive placental stem cells
that are CD73.sup.+, CD105.sup.+ and HLA-G.sup.+, wherein said
plurality detectably suppresses T cell proliferation in a mixed
lymphocyte reaction (MLR) assay. In a specific embodiment of the
above plurality, said stem cells are also CD34.sup.-, CD38.sup.- or
CD45.sup.-. In another specific embodiment, said stem cells are
also CD34.sup.-, CD38.sup.- and CD45.sup.-. In another specific
embodiment, said stem cells are also OCT-4.sup.+. In another
specific embodiment, said stem cells are also CD200.sup.+. In a
more specific embodiment, said stem cells are also CD34.sup.-,
CD38.sup.-, CD45.sup.-, OCT-4.sup.+ and CD200.sup.+.
[0025] The invention also provides an isolated cell population
comprising a plurality of immunosuppressive placental stem cells
that are CD73.sup.+, CD105.sup.+ stem cells, wherein said plurality
forms one or more embryoid-like bodies under conditions that allow
formation of embryoid-like bodies, and wherein said plurality
detectably suppresses T cell proliferation in a mixed lymphocyte
reaction (MLR) assay. In a specific embodiment, said stem cells are
also CD34.sup.-, CD38.sup.- or CD45.sup.-. In another specific
embodiment, said stem cells are also CD34.sup.-, CD38.sup.- and
CD45.sup.-. In another specific embodiment, said stem cells are
also OCT-4.sup.+. In a more specific embodiment, said stem cells
are also OCT-4.sup.+, CD34.sup.-, CD38.sup.- and CD45.sup.-.
[0026] The invention also provides an isolated cell population
comprising a plurality of immunosuppressive placental stem cells
that are OCT-4.sup.+ stem cells, wherein said population forms one
or more embryoid-like bodies when cultured under conditions that
allow the formation of embryoid-like bodies, and wherein said
plurality detectably suppresses T cell proliferation in a mixed
lymphocyte reaction (MLR) assay. In various embodiments, at least
10%, at least 20%, at least 30%, at least 40%, at least 50% at
least 60%, at least 70%, at least 80%, at least 90%, or at least
95% of said isolated placental cells are OCT4.sup.+ stem cells. In
a specific embodiment of the above populations, said stem cells are
CD73.sup.+ and CD105.sup.+. In another specific embodiment, said
stem cells are CD34.sup.-, CD38.sup.-, or CD45.sup.-. In another
specific embodiment, said stem cells are CD200.sup.+. In a more
specific embodiment, said stem cells are CD73.sup.+, CD105.sup.+,
CD200.sup.+, CD34.sup.-, CD38.sup.-, and CD45.sup.-. In another
specific embodiment, said population has been expanded, for
example, passaged at least once, at least three times, at least
five times, at least 10 times, at least 15 times, or at least 20
times.
[0027] In another embodiment, the invention provides an isolated
cell population comprising a plurality of immunosuppressive
placental stem cells that are CD29.sup.+, CD44.sup.+, CD73.sup.+,
CD90.sup.+, CD105.sup.+, CD200.sup.+, CD34.sup.- and
CD133.sup.-.
[0028] In a specific embodiment of the above-mentioned placental
stem cells, the placental stem cells constitutively secrete IL-6,
IL-8 and monocyte chemoattractant protein (MCP-1).
[0029] Each of the above-referenced pluralities of placental stem
cells can comprise placental stem cells obtained and isolated
directly from a mammalian placenta, or placental stem cells that
have been cultured and passaged at least 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 12, 14, 16, 18, 20, 25, or more times, or a combination
thereof.
[0030] The immunosuppressive pluralities of placental stem cells
described above can comprise about, at least, or no more than,
1.times.10.sup.5, 5.times.10.sup.5, 1.times.10.sup.6,
5.times.10.sup.6, 1.times.10.sup.7, 5.times.10.sup.7,
1.times.10.sup.8, 5.times.10.sup.8, 1.times.10.sup.9,
5.times.10.sup.9, 1.times.10.sup.10, 5.times.10.sup.10,
1.times.10.sup.11 or more placental stem cells.
[0031] 5.2.3 Selecting and Producing Placental Stem Cell
Populations
[0032] In another embodiment, the invention also provides a method
of selecting a plurality of immunosuppressive placental stem cells
from a plurality of placental cells, comprising selecting a
population of placental cells wherein at least 10%, at least 20%,
at least 30%, at least 40%, at least 50% at least 60%, at least
70%, at least 80%, at least 90%, or at least 95% of said cells are
CD200.sup.+, HLA-G.sup.+ placental stem cells, and wherein said
placental stem cells detectably suppresses T cell proliferation in
a mixed lymphocyte reaction (MLR) assay. In a specific embodiment,
said selecting comprises selecting stem cells that are also
CD73.sup.+ and CD105.sup.+. In another specific embodiment, said
selecting comprises selecting stem cells that are also CD34.sup.-,
CD38.sup.- or CD45.sup.-. In another specific embodiment, said
selecting comprises selecting placental stem cells that are also
CD34.sup.-, CD38.sup.-, CD45.sup.-, CD73.sup.+ and CD105.sup.+. In
another specific embodiment, said selecting also comprises
selecting a plurality of placental stem cells that forms one or
more embryoid-like bodies when cultured under conditions that allow
the formation of embryoid-like bodies.
[0033] In another embodiment, the invention also provides a method
of selecting a plurality of immunosuppressive placental stem cells
from a plurality of placental cells, comprising selecting a
plurality of placental cells wherein at least 10%, at least 20%, at
least 30%, at least 40%, at least 50% at least 60%, at least 70%,
at least 80%, at least 90%, or at least 95% of said cells are
CD73.sup.+, CD105.sup.+, CD200.sup.+ placental stem cells, and
wherein said placental stem cells detectably suppresses T cell
proliferation in a mixed lymphocyte reaction (MLR) assay. In a
specific embodiment, said selecting comprises selecting stem cells
that are also HLA-G.sup.+. In another specific embodiment, said
selecting comprises selecting placental stem cells that are also
CD34.sup.-, CD38.sup.- or CD45.sup.-. In another specific
embodiment, said selecting comprises selecting placental stem cells
that are also CD34.sup.-, CD38.sup.- and CD45.sup.-. In another
specific embodiment, said selecting comprises selecting placental
stem cells that are also CD34.sup.-, CD38.sup.-, CD45.sup.-, and
HLA-G.sup.+. In another specific embodiment, said selecting
additionally comprises selecting a population of placental cells
that produces one or more embryoid-like bodies when the population
is cultured under conditions that allow the formation of
embryoid-like bodies.
[0034] In another embodiment, the invention also provides a method
of selecting a plurality of immunosuppressive placental stem cells
from a plurality of placental cells, comprising selecting a
plurality of placental cells wherein at least 10%, at least 20%, at
least 30%, at least 40%, at least 50% at least 60%, at least 70%,
at least 80%, at least 90%, or at least 95% of said cells are
CD200.sup.+, OCT-4.sup.+ placental stem cells, and wherein said
placental stem cells detectably suppresses T cell proliferation in
a mixed lymphocyte reaction (MLR) assay. In a specific embodiment,
said selecting comprises selecting placental stem cells that are
also CD73.sup.+ and CD105.sup.+. In another specific embodiment,
said selecting comprises selecting placental stem cells that are
also HLA-G.sup.+. In another specific embodiment, said selecting
comprises selecting placental stem cells that are also CD34.sup.-,
CD38.sup.- and CD45.sup.-. In another specific embodiment, said
selecting comprises selecting placental stem cells that are also
CD34.sup.-, CD38.sup.-, CD45.sup.-, CD73.sup.+, CD105.sup.+ and
HLA-G.sup.+.
[0035] In another embodiment, the invention also provides a method
of selecting a plurality of immunosuppressive placental stem cells
from a plurality of placental cells, comprising selecting a
plurality of placental cells wherein at least 10%, at least 20%, at
least 30%, at least 40%, at least 50% at least 60%, at least 70%,
at least 80%, at least 90%, or at least 95% of said cells are
CD73.sup.+, CD105.sup.+ and HLA-G.sup.+ placental stem cells, and
wherein said placental stem cells detectably suppresses T cell
proliferation in a mixed lymphocyte reaction (MLR) assay. In a
specific embodiment, said selecting comprises selecting placental
stem cells that are also CD34.sup.-, CD38.sup.- or CD45.sup.-. In
another specific embodiment, said selecting comprises selecting
placental stem cells that are also CD34.sup.-, CD38.sup.- and
CD45.sup.-. In another specific embodiment, said selecting
comprises selecting placental stem cells that are also CD200.sup.+.
In another specific embodiment, said selecting comprises selecting
placental stem cells that are also CD34.sup.-, CD38.sup.-,
CD45.sup.-, OCT-4.sup.+ and CD200.sup.+.
[0036] In another embodiment, the invention also provides a method
of selecting a plurality of immunosuppressive placental stem cells
from a plurality of placental cells, comprising selecting a
plurality of placental cells wherein at least 10%, at least 20%, at
least 30%, at least 40%, at least 50% at least 60%, at least 70%,
at least 80%, at least 90%, or at least 95% of said cells are
CD73.sup.+, CD105.sup.+ placental stem cells, and wherein said
plurality forms one or more embryoid-like bodies under conditions
that allow formation of embryoid-like bodies. In a specific
embodiment, said selecting comprises selecting placental stem cells
that are also CD34.sup.-, CD38.sup.- or CD45.sup.-. In another
specific embodiment, said selecting comprises selecting placental
stem cells that are also CD34.sup.-, CD38.sup.- and CD45.sup.-. In
another specific embodiment, said selecting comprises selecting
placental stem cells that are also OCT-4.sup.+. In a more specific
embodiment, said selecting comprises selecting placental stem cells
that are also OCT-4.sup.+, CD34.sup.-, CD38.sup.- and
CD45.sup.-.
[0037] In another embodiment, the invention also provides a method
of selecting a plurality of immunosuppressive placental stem cells
from a plurality of placental cells, comprising selecting a
plurality of placental cells wherein at least 10%, at least 20%, at
least 30%, at least 40%, at least 50% at least 60%, at least 70%,
at least 80%, at least 90%, or at least 95% of said isolated
placental cells are OCT4.sup.+ stem cells, and wherein said
plurality forms one or more embryoid-like bodies under conditions
that allow formation of embryoid-like bodies. In a specific
embodiment, said selecting comprises selecting placental stem cells
that are also CD73.sup.+ and CD105.sup.+. In another specific
embodiment, said selecting comprises selecting placental stem cells
that are also CD34.sup.-, CD38.sup.-, or CD45.sup.-. In another
specific embodiment, said selecting comprises selecting placental
stem cells that are also CD200.sup.+. In a more specific
embodiment, said selecting comprises selecting placental stem cells
that are also CD73.sup.+, CD105.sup.+, CD200.sup.+, CD34.sup.-,
CD38.sup.-, and CD45.sup.-.
[0038] The invention also provides methods of producing
immunosuppressive populations, or pluralities, of placental stem
cells. For example, the invention provides a method of producing a
cell population, comprising selecting any of the pluralities of
placental stem cells described above, and isolating the plurality
of placental stem cells from other cells, e.g., other placental
cells. In a specific embodiment, the invention provides a method of
producing a cell population comprising selecting placental cells,
wherein said placental cells (a) adhere to a substrate, (b) express
CD200 and HLA-G, or express CD73, CD105, and CD200, or express
CD200 and OCT-4, or express CD73, CD105, and HLA-G, or express CD73
and CD105 and facilitate the formation of one or more embryoid-like
bodies in a population of placental cells that comprise the stem
cell, when said population is cultured under conditions that allow
formation of embryoid-like bodies, or express OCT-4 and facilitate
the formation of one or more embryoid-like bodies in a population
of placental cells that comprise the stem cell, when said
population is cultured under conditions that allow formation of
embryoid-like bodies; and (c) detectably suppress CD4.sup.+ or
CD8.sup.+ T cell proliferation in an MLR (mixed lymphocyte
reaction); and isolating said placental cells from other cells to
form a cell population.
[0039] In a more specific embodiment, the invention provides a
method of producing a cell population comprising selecting
placental stem cells that (a) adhere to a substrate, (b) express
CD200 and HLA-G, and (c) detectably suppress CD4.sup.+ or CD8.sup.+
T cell proliferation in an MLR (mixed lymphocyte reaction); and
isolating said placental stem cells from other cells to form a cell
population. In another specific embodiment, the invention provides
a method of producing a cell population comprising selecting
placental stem cells that (a) adhere to a substrate, (b) express
CD73, CD105, and CD200, and (c) detectably suppress CD4.sup.+ or
CD8.sup.+ T cell proliferation in an MLR; and isolating said
placental stem cells from other cells to form a cell population. In
another specific embodiment, the invention provides a method of
producing a cell population comprising selecting placental stem
cells that (a) adhere to a substrate, (b) express CD200 and OCT-4,
and (c) detectably suppress CD4.sup.+ or CD8.sup.+ T cell
proliferation in an MLR; and isolating said placental stem cells
from other cells to form a cell population. In another specific
embodiment, the invention provides a method of producing a cell
population comprising selecting placental stem cells that (a)
adhere to a substrate, (b) express CD73 and CD105, (c) form
embryoid-like bodies when cultured under conditions allowing the
formation of embryoid-like bodies, and (d) detectably suppress
CD4.sup.+ or CD8.sup.+ T cell proliferation in an MLR; and
isolating said placental stem cells from other cells to form a cell
population. In another specific embodiment, the invention provides
a method of producing a cell population comprising selecting
placental stem cells that (a) adhere to a substrate, (b) express
CD73, CD105, and HLA-G, and (c) detectably suppress CD4.sup.+ or
CD8.sup.+ T cell proliferation in an MLR; and isolating said
placental stem cells from other cells to form a cell population. A
method of producing a cell population comprising selecting
placental stem cells that (a) adhere to a substrate, (b) express
OCT-4, (c) form embryoid-like bodies when cultured under conditions
allowing the formation of embryoid-like bodies, and (d) detectably
suppress CD4.sup.+ or CD8.sup.+ T cell proliferation in an MLR; and
isolating said placental stem cells from other cells to form a cell
population.
[0040] In a specific embodiment of the methods of producing an
immunosuppressive placental stem cell population, said T cells and
said placental cells are present in said MLR at a ratio of about
5:1. The placental cells used in the method can be derived from the
whole placenta, or primarily from amnion, or amnion and chorion. In
another specific embodiment, the placental cells suppress CD4.sup.+
or CD8.sup.+ T cell proliferation by at least 50%, at least 75%, at
least 90%, or at least 95% in said MLR compared to an amount of T
cell proliferation in said MLR in the absence of said placental
cells. The method can additionally comprise the selection and/or
production of a placental stem cell population capable of
immunomodulation, e.g., suppression of the activity of, other
immune cells, e.g., an activity of a natural killer (NK) cell.
[0041] 5.2.4 Growth in Culture
[0042] The growth of the placental stem cells described herein, as
for any mammalian cell, depends in part upon the particular medium
selected for growth. Under optimum conditions, placental stem cells
typically double in number in 3-5 days. During culture, the
placental stem cells of the invention adhere to a substrate in
culture, e.g. the surface of a tissue culture container (e.g.,
tissue culture dish plastic, fibronectin-coated plastic, and the
like) and form a monolayer.
[0043] Populations of isolated placental cells that comprise the
placental stem cells of the invention, when cultured under
appropriate conditions, form embryoid-like bodies, that is,
three-dimensional clusters of cells grow atop the adherent stem
cell layer. Cells within the embryoid-like bodies express markers
associated with very early stem cells, e.g., OCT-4, Nanog, SSEA3
and SSEA4. Cells within the embryoid-like bodies are typically not
adherent to the culture substrate, as are the placental stem cells
described herein, but remain attached to the adherent cells during
culture. Embryoid-like body cells are dependent upon the adherent
placental stem cells for viability, as embryoid-like bodies do not
form in the absence of the adherent stem cells. The adherent
placental stem cells thus facilitate the growth of one or more
embryoid-like bodies in a population of placental cells that
comprise the adherent placental stem cells. Without wishing to be
bound by theory, the cells of the embryoid-like bodies are thought
to grow on the adherent placental stem cells much as embryonic stem
cells grow on a feeder layer of cells. Mesenchymal stem cells,
e.g., bone marrow-derived mesenchymal stem cells, do not develop
embryoid-like bodies in culture.
[0044] 5.2.5 Differentiation
[0045] The placental stem cells, useful in the methods of the
present invention, are differentiable into different committed cell
lineages. For example, the placental stem cells can be
differentiated into cells of an adipogenic, chondrogenic,
neurogenic, or osteogenic lineage. Such differentiation can be
accomplished by any method known in the art for differentiating,
e.g., bone marrow-derived mesenchymal stem cells into similar cell
lineages.
5.3 Methods of Obtaining Placental Stem Cells
[0046] 5.3.1 Stem Cell Collection Composition
[0047] The present invention further provides methods of collecting
and isolating placental stem cells. Generally, stem cells are
obtained from a mammalian placenta using a
physiologically-acceptable solution, e.g., a stem cell collection
composition. A stem cell collection composition is described in
detail in related U.S. Provisional Application No. 60/754,969,
entitled "Improved Composition for Collecting and Preserving
Placental Stem Cells and Methods of Using the Composition" filed on
Dec. 29, 2005.
[0048] The stem cell collection composition can comprise any
physiologically-acceptable solution suitable for the collection
and/or culture of stem cells, for example, a saline solution (e.g.,
phosphate-buffered saline, Kreb's solution, modified Kreb's
solution, Eagle's solution, 0.9% NaCl. etc.), a culture medium
(e.g., DMEM, H.DMEM, etc.), and the like.
[0049] The stem cell collection composition can comprise one or
more components that tend to preserve placental stem cells, that
is, prevent the placental stem cells from dying, or delay the death
of the placental stem cells, reduce the number of placental stem
cells in a population of cells that die, or the like, from the time
of collection to the time of culturing. Such components can be,
e.g., an apoptosis inhibitor (e.g., a caspase inhibitor or JNK
inhibitor); a vasodilator (e.g., magnesium sulfate, an
antihypertensive drug, atrial natriuretic peptide (ANP),
adrenocorticotropin, corticotropin-releasing hormone, sodium
nitroprusside, hydralazine, adenosine triphosphate, adenosine,
indomethacin or magnesium sulfate, a phosphodiesterase inhibitor,
etc.); a necrosis inhibitor (e.g.,
2-(1H-Indol-3-yl)-3-pentylamino-maleimide, pyrrolidine
dithiocarbamate, or clonazepam); a TNF-.alpha. inhibitor; and/or an
oxygen-carrying perfluorocarbon (e.g., perfluorooctyl bromide,
perfluorodecyl bromide, etc.).
[0050] The stem cell collection composition can comprise one or
more tissue-degrading enzymes, e.g., a metalloprotease, a serine
protease, a neutral protease, an RNase, or a DNase, or the like.
Such enzymes include, but are not limited to, collagenases (e.g.,
collagenase I, II, III or IV, a collagenase from Clostridium
histolyticum, etc.); dispase, thermolysin, elastase, trypsin,
LIBERASE, hyaluronidase, and the like.
[0051] The stem cell collection composition can comprise a
bacteriocidally or bacteriostatically effective amount of an
antibiotic. In certain non-limiting embodiments, the antibiotic is
a macrolide (e.g., tobramycin), a cephalosporin (e.g., cephalexin,
cephradine, cefuroxime, cefprozil, cefaclor, cefixime or
cefadroxil), a clarithromycin, an erythromycin, a penicillin (e.g.,
penicillin V) or a quinolone (e.g., ofloxacin, ciprofloxacin or
norfloxacin), a tetracycline, a streptomycin, etc. In a particular
embodiment, the antibiotic is active against Gram(+) and/or Gram(-)
bacteria, e.g., Pseudomonas aeruginosa, Staphylococcus aureus, and
the like.
[0052] The stem cell collection composition can also comprise one
or more of the following compounds: adenosine (about 1 mM to about
50 mM); D-glucose (about 20 mM to about 100 mM); magnesium ions
(about 1 mM to about 50 mM); a macromolecule of molecular weight
greater than 20,000 daltons, in one embodiment, present in an
amount sufficient to maintain endothelial integrity and cellular
viability (e.g., a synthetic or naturally occurring colloid, a
polysaccharide such as dextran or a polyethylene glycol present at
about 25 g/l to about 100 g/l, or about 40 g/l to about 60 g/l); an
antioxidant (e.g., butylated hydroxyanisole, butylated
hydroxytoluene, glutathione, vitamin C or vitamin E present at
about 25 .mu.M to about 100 .mu.M); a reducing agent (e.g.,
N-acetylcysteine present at about 0.1 mM to about 5 mM); an agent
that prevents calcium entry into cells (e.g., verapamil present at
about 2 .mu.M to about 25 .mu.M); nitroglycerin (e.g., about 0.05
g/L to about 0.2 g/L); an anticoagulant, in one embodiment, present
in an amount sufficient to help prevent clotting of residual blood
(e.g., heparin or hirudin present at a concentration of about 1000
units/1 to about 100,000 units/1); or an amiloride containing
compound (e.g., amiloride, ethyl isopropyl amiloride, hexamethylene
amiloride, dimethyl amiloride or isobutyl amiloride present at
about 1.0 .mu.M to about 5 .mu.M).
[0053] 5.3.2 Collection and Handling of Placenta
[0054] Generally, a human placenta is recovered shortly after its
expulsion after birth. In a preferred embodiment, the placenta is
recovered from a patient after informed consent and after a
complete medical history of the patient is taken and is associated
with the placenta. Preferably, the medical history continues after
delivery. Such a medical history can be used to coordinate
subsequent use of the placenta or the stem cells harvested
therefrom. For example, human placental stem cells can be used, in
light of the medical history, for personalized medicine for the
infant associated with the placenta, or for parents, siblings or
other relatives of the infant.
[0055] Prior to recovery of placental stem cells, the umbilical
cord blood and placental blood are removed. In certain embodiments,
after delivery, the cord blood in the placenta is recovered. The
placenta can be subjected to a conventional cord blood recovery
process. Typically a needle or cannula is used, with the aid of
gravity, to exsanguinate the placenta (see, e.g., Anderson, U.S.
Pat. No. 5,372,581; Hessel et al., U.S. Pat. No. 5,415,665). The
needle or cannula is usually placed in the umbilical vein and the
placenta can be gently massaged to aid in draining cord blood from
the placenta. Such cord blood recovery may be performed
commercially, e.g., LifeBank Inc., Cedar Knolls, N.J., ViaCord,
Cord Blood Registry and Cryocell. Preferably, the placenta is
gravity drained without further manipulation so as to minimize
tissue disruption during cord blood recovery.
[0056] Typically, a placenta is transported from the delivery or
birthing room to another location, e.g., a laboratory, for recovery
of cord blood and collection of stem cells by, e.g., perfusion or
tissue dissociation. The placenta is preferably transported in a
sterile, thermally insulated transport device (maintaining the
temperature of the placenta between 20-28.degree. C.), for example,
by placing the placenta, with clamped proximal umbilical cord, in a
sterile zip-lock plastic bag, which is then placed in an insulated
container. In another embodiment, the placenta is transported in a
cord blood collection kit substantially as described in pending
U.S. patent application Ser. No. 11/230,760, filed Sep. 19, 2005.
Preferably, the placenta is delivered to the laboratory four to
twenty-four hours following delivery. In certain embodiments, the
proximal umbilical cord is clamped, preferably within 4-5 cm
(centimeter) of the insertion into the placental disc prior to cord
blood recovery. In other embodiments, the proximal umbilical cord
is clamped after cord blood recovery but prior to further
processing of the placenta.
[0057] The placenta, prior to stem cell collection, can be stored
under sterile conditions and at either room temperature or at a
temperature of 5 to 25.degree. C. (centigrade). The placenta may be
stored for a period of longer than forty eight hours, and
preferably for a period of four to twenty-four hours prior to
perfusing the placenta to remove any residual cord blood. The
placenta is preferably stored in an anticoagulant solution at a
temperature of 5 to 25.degree. C. (centigrade). Suitable
anticoagulant solutions are well known in the art. For example, a
solution of heparin or warfarin sodium can be used. In a preferred
embodiment, the anticoagulant solution comprises a solution of
heparin (e.g., 1% w/w in 1:1000 solution). The exsanguinated
placenta is preferably stored for no more than 36 hours before
placental stem cells are collected.
[0058] The mammalian placenta or a part thereof, once collected and
prepared generally as above, can be treated in any art-known
manner, e.g., can be perfused or disrupted, e.g., digested with one
or more tissue-disrupting enzymes, to obtain stem cells.
[0059] 5.3.3 Physical Disruption and Enzymatic Digestion of
Placental Tissue
[0060] In one embodiment, stem cells are collected from a mammalian
placenta by physical disruption, e.g., enzymatic digestion, of the
organ. For example, the placenta, or a portion thereof, may be,
e.g., crushed, sheared, minced, diced, chopped, macerated or the
like, while in contact with the stem cell collection composition of
the invention, and the tissue subsequently digested with one or
more enzymes. The placenta, or a portion thereof, may also be
physically disrupted and digested with one or more enzymes, and the
resulting material then immersed in, or mixed into, the stem cell
collection composition of the invention. Any method of physical
disruption can be used, provided that the method of disruption
leaves a plurality, more preferably a majority, and more preferably
at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the cells in said
organ viable, as determined by, e.g., trypan blue exclusion.
[0061] The placenta can be dissected into components prior to
physical disruption and/or enzymatic digestion and stem cell
recovery. For example, placental stem cells can be obtained from
the amniotic membrane, chorion, placental cotyledons, or any
combination thereof. Preferably, placental stem cells are obtained
from placental tissue comprising amnion and chorion. Typically,
placental stem cells can be obtained by disruption of a small block
of placental tissue, e.g., a block of placental tissue that is
about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80,
90, 100, 200, 300, 400, 500, 600, 700, 800, 900 or about 1000 cubic
millimeters in volume.
[0062] A preferred stem cell collection composition comprises one
or more tissue-disruptive enzyme(s). Enzymatic digestion preferably
uses a combination of enzymes, e.g., a combination of a matrix
metalloprotease and a neutral protease, for example, a combination
of collagenase and dispase. In one embodiment, enzymatic digestion
of placental tissue uses a combination of a matrix metalloprotease,
a neutral protease, and a mucolytic enzyme for digestion of
hyaluronic acid, such as a combination of collagenase, dispase, and
hyaluronidase or a combination of LIBERASE (Boehringer Mannheim
Corp., Indianapolis, Ind.) and hyaluronidase. Other enzymes that
can be used to disrupt placenta tissue include papain,
deoxyribonucleases, serine proteases, such as trypsin,
chymotrypsin, or elastase. Serine proteases may be inhibited by
alpha 2 microglobulin in serum and therefore the medium used for
digestion is usually serum-free. EDTA and DNase are commonly used
in enzyme digestion procedures to increase the efficiency of cell
recovery. The digestate is preferably diluted so as to avoid
trapping stem cells within the viscous digest.
[0063] Any combination of tissue digestion enzymes can be used.
Typical concentrations for tissue digestion enzymes include, e.g.,
50-200 U/mL for collagenase I and collagenase IV, 1-10 U/mL for
dispase, and 10-100 U/mL for elastase. Proteases can be used in
combination, that is, two or more proteases in the same digestion
reaction, or can be used sequentially in order to liberate
placental stem cells. For example, in one embodiment, a placenta,
or part thereof, is digested first with an appropriate amount of
collagenase I at 2 mg/ml for 30 minutes, followed by digestion with
trypsin, 0.25%, for 10 minutes, at 37.degree. C. Serine proteases
are preferably used consecutively following use of other
enzymes.
[0064] In another embodiment, the tissue can further be disrupted
by the addition of a chelator, e.g., ethylene glycol
bis(2-aminoethyl ether)-N,N,N'N'-tetraacetic acid (EGTA) or
ethylenediaminetetraacetic acid (EDTA) to the stem cell collection
composition comprising the stem cells, or to a solution in which
the tissue is disrupted and/or digested prior to isolation of the
stem cells with the stem cell collection composition.
[0065] It will be appreciated that where an entire placenta, or
portion of a placenta comprising both fetal and maternal cells (for
example, where the portion of the placenta comprises the chorion or
cotyledons), the placental stem cells collected will comprise a mix
of placental stem cells derived from both fetal and maternal
sources. Where a portion of the placenta that comprises no, or a
negligible number of, maternal cells (for example, amnion), the
placental stem cells collected will comprise almost exclusively
fetal placental stem cells.
[0066] 5.3.4 Placental Perfusion
[0067] Placental stem cells can also be obtained by perfusion of
the mammalian placenta. Methods of perfusing mammalian placenta to
obtain stem cells are disclosed, e.g., in Hariri, U.S. Application
Publication No. 2002/0123141, and in related U.S. Provisional
Application No. 60/754,969, entitled "Improved Composition for
Collecting and Preserving Placental Stem Cells and Methods of Using
the Composition" filed on Dec. 29, 2005.
[0068] Placental stem cells can be collected by perfusion, e.g.,
through the placental vasculature, using, e.g., a stem cell
collection composition as a perfusion solution. In one embodiment,
a mammalian placenta is perfused by passage of perfusion solution
through either or both of the umbilical artery and umbilical vein.
The flow of perfusion solution through the placenta may be
accomplished using, e.g., gravity flow into the placenta.
Preferably, the perfusion solution is forced through the placenta
using a pump, e.g., a peristaltic pump. The umbilical vein can be,
e.g., cannulated with a cannula, e.g., a TEFLON.RTM. or plastic
cannula, that is connected to a sterile connection apparatus, such
as sterile tubing. The sterile connection apparatus is connected to
a perfusion manifold.
[0069] In preparation for perfusion, the placenta is preferably
oriented (e.g., suspended) in such a manner that the umbilical
artery and umbilical vein are located at the highest point of the
placenta. The placenta can be perfused by passage of a perfusion
fluid, e.g., the stem cell collection composition of the invention,
through the placental vasculature, or through the placental
vasculature and surrounding tissue. In one embodiment, the
umbilical artery and the umbilical vein are connected
simultaneously to a pipette that is connected via a flexible
connector to a reservoir of the perfusion solution. The perfusion
solution is passed into the umbilical vein and artery. The
perfusion solution exudes from and/or passes through the walls of
the blood vessels into the surrounding tissues of the placenta, and
is collected in a suitable open vessel from the surface of the
placenta that was attached to the uterus of the mother during
gestation. The perfusion solution may also be introduced through
the umbilical cord opening and allowed to flow or percolate out of
openings in the wall of the placenta which interfaced with the
maternal uterine wall. In another embodiment, the perfusion
solution is passed through the umbilical veins and collected from
the umbilical artery, or is passed through the umbilical artery and
collected from the umbilical veins.
[0070] In one embodiment, the proximal umbilical cord is clamped
during perfusion, and more preferably, is clamped within 4-5 cm
(centimeter) of the cord's insertion into the placental disc.
[0071] The first collection of perfusion fluid from a mammalian
placenta during the exsanguination process is generally colored
with residual red blood cells of the cord blood and/or placental
blood. The perfusion fluid becomes more colorless as perfusion
proceeds and the residual cord blood cells are washed out of the
placenta. Generally from 30 to 100 ml (milliliter) of perfusion
fluid is adequate to initially exsanguinate the placenta, but more
or less perfusion fluid may be used depending on the observed
results.
[0072] The volume of perfusion liquid used to collect placental
stem cells may vary depending upon the number of stem cells to be
collected, the size of the placenta, the number of collections to
be made from a single placenta, etc. In various embodiments, the
volume of perfusion liquid may be from 50 mL to 5000 mL, 50 mL to
4000 mL, 50 mL to 3000 mL, 100 mL to 2000 mL, 250 mL to 2000 mL,
500 mL to 2000 mL, or 750 mL to 2000 mL. Typically, the placenta is
perfused with 700-800 mL of perfusion liquid following
exsanguination.
[0073] The placenta can be perfused a plurality of times over the
course of several hours or several days. Where the placenta is to
be perfused a plurality of times, it may be maintained or cultured
under aseptic conditions in a container or other suitable vessel,
and perfused with the stem cell collection composition, or a
standard perfusion solution (e.g., a normal saline solution such as
phosphate buffered saline ("PBS")) with or without an anticoagulant
(e.g., heparin, warfarin sodium, coumarin, bishydroxycoumarin),
and/or with or without an antimicrobial agent (e.g.,
.beta.-mercaptoethanol (0.1 mM); antibiotics such as streptomycin
(e.g., at 40-100 .mu.g/ml), penicillin (e.g., at 40 U/ml),
amphotericin B (e.g., at 0.5 .mu.g/ml). In one embodiment, an
isolated placenta is maintained or cultured for a period of time
without collecting the perfusate, such that the placenta is
maintained or cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or 2 or 3
or more days before perfusion and collection of perfusate. The
perfused placenta can be maintained for one or more additional
time(s), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24 or more hours, and perfused a
second time with, e.g., 700-800 mL perfusion fluid. The placenta
can be perfused 1, 2, 3, 4, 5 or more times, for example, once
every 1, 2, 3, 4, 5 or 6 hours. In a preferred embodiment,
perfusion of the placenta and collection of perfusion solution,
e.g., stem cell collection composition, is repeated until the
number of recovered nucleated cells falls below 100 cells/ml. The
perfusates at different time points can be further processed
individually to recover time-dependent populations of cells, e.g.,
stem cells. Perfusates from different time points can also be
pooled.
[0074] Without wishing to be bound by any theory, after
exsanguination and a sufficient time of perfusion of the placenta,
placental stem cells are believed 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. Perfusing the
isolated placenta not only serves to remove residual cord blood but
also provide the placenta with the appropriate nutrients, including
oxygen. The placenta may be cultivated and perfused with a similar
solution which was used to remove the residual cord blood cells,
preferably, without the addition of anticoagulant agents.
[0075] Perfusion according to the methods of the invention results
in the collection of significantly more placental stem cells than
the number obtainable from a mammalian placenta not perfused with
said solution, and not otherwise treated to obtain stem cells
(e.g., by tissue disruption, e.g., enzymatic digestion). In this
context, "significantly more" means at least 10% more. Perfusion
according to the methods of the invention yields significantly more
placental stem cells than, e.g., the number of placental stem cells
obtainable from culture medium in which a placenta, or portion
thereof, has been cultured.
[0076] Stem cells can be isolated from placenta by perfusion with a
solution comprising one or more proteases or other
tissue-disruptive enzymes. In a specific embodiment, a placenta or
portion thereof (e.g., amniotic membrane, amnion and chorion,
placental lobule or cotyledon, or combination of any of the
foregoing) is brought to 25-37.degree. C., and is incubated with
one or more tissue-disruptive enzymes in 200 mL of a culture medium
for 30 minutes. Cells from the perfusate are collected, brought to
4.degree. C., and washed with a cold inhibitor mix comprising 5 mM
EDTA, 2 mM dithiothreitol and 2 mM beta-mercaptoethanol. The stem
cells are washed after several minutes with a cold (e.g., 4.degree.
C.) stem cell collection composition of the invention.
[0077] It will be appreciated that perfusion using the pan method,
that is, whereby perfusate is collected after it has exuded from
the maternal side of the placenta, results in a mix of fetal and
maternal cells. As a result, the cells collected by this method
comprise a mixed population of placental stem cells of both fetal
and maternal origin. In contrast, perfusion solely through the
placental vasculature, whereby perfusion fluid is passed through
one or two placental vessels and is collected solely through the
remaining vessel(s), results in the collection of a population of
placental stem cells almost exclusively of fetal origin.
[0078] 5.3.5 Isolation, Sorting, and Characterization of Placental
Stem Cells
[0079] Stem cells from mammalian placenta, whether obtained by
perfusion or enyzmatic digestion, can initially be purified from
(i.e., be isolated from) other cells by Ficoll gradient
centrifugation. Such centrifugation can follow any standard
protocol for centrifugation speed, etc. In one embodiment, for
example, cells collected from the placenta are recovered from
perfusate by centrifugation at 5000.times.g for 15 minutes at room
temperature, which separates cells from, e.g., contaminating debris
and platelets. In another embodiment, placental perfusate is
concentrated to about 200 ml, gently layered over Ficoll, and
centrifuged at about 1100.times.g for 20 minutes at 22.degree. C.,
and the low-density interface layer of cells is collected for
further processing.
[0080] Cell pellets can be resuspended in fresh stem cell
collection composition, or a medium suitable for stem cell
maintenance, e.g., IMDM serum-free medium containing 2 U/ml heparin
and 2 mM EDTA (GibcoBRL, NY). The total mononuclear cell fraction
can be isolated, e.g., using Lymphoprep (Nycomed Pharma, Oslo,
Norway) according to the manufacturer's recommended procedure.
[0081] As used herein, "isolating" placental stem cells means to
remove at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%
of the cells with which the stem cells are normally associated in
the intact mammalian placenta. A stem cell from an organ is
"isolated" when it is present in a population of cells that
comprises fewer than 50% of the cells with which the stem cell is
normally associated in the intact organ.
[0082] Placental cells obtained by perfusion or digestion can, for
example, be further, or initially, isolated by differential
trypsinization using, e.g., a solution of 0.05% trypsin with 0.2%
EDTA (Sigma, St. Louis Mo.). Differential trypsinization is
possible because placental stem cells typically detach from plastic
surfaces within about five minutes whereas other adherent
populations typically require more than 20-30 minutes incubation.
The detached placental stem cells can be harvested following
trypsinization and trypsin neutralization, using, e.g., Trypsin
Neutralizing Solution (TNS, Cambrex). In one embodiment of
isolation of adherent cells, aliquots of, for example, about
5-10.times.10.sup.6 cells are placed in each of several T-75
flasks, preferably fibronectin-coated T75 flasks. In such an
embodiment, the cells can be cultured with commercially available
Mesenchymal Stem Cell Growth Medium (MSCGM) (Cambrex), and placed
in a tissue culture incubator (37.degree. C., 5% CO.sub.2). After
10 to 15 days, non-adherent cells are removed from the flasks by
washing with PBS. The PBS is then replaced by MSCGM. Flasks are
preferably examined daily for the presence of various adherent cell
types and in particular, for identification and expansion of
clusters of fibroblastoid cells.
[0083] The number and type of cells collected from a mammalian
placenta can be monitored, for example, by measuring changes in
morphology and cell surface markers using standard cell detection
techniques such as flow cytometry, cell sorting,
immunocytochemistry (e.g., staining with tissue specific or
cell-marker specific antibodies) fluorescence activated cell
sorting (FACS), magnetic activated cell sorting (MACS), by
examination of the morphology of cells using light or confocal
microscopy, and/or by measuring changes in gene expression using
techniques well known in the art, such as PCR and gene expression
profiling. These techniques can be used, too, to identify cells
that are positive for one or more particular markers. For example,
using antibodies to CD34, one can determine, using the techniques
above, whether a cell comprises a detectable amount of CD34; if so,
the cell is CD34.sup.+. Likewise, if a cell produces enough OCT-4
RNA to be detectable by RT-PCR, or significantly more OCT-4 RNA
than an adult cell, the cell is OCT-4.sup.+ Antibodies to cell
surface markers (e.g., CD markers such as CD34) and the sequence of
stem cell-specific genes, such as OCT-4, are well-known in the
art.
[0084] Placental cells, particularly cells that have been isolated
by Ficoll separation, differential adherence, or a combination of
both, may be sorted using a fluorescence activated cell sorter
(FACS). Fluorescence activated cell sorting (FACS) is a well-known
method for separating particles, including cells, based on the
fluorescent properties of the particles (Kamarch, 1987, Methods
Enzymol, 151:150-165). Laser excitation of fluorescent moieties in
the individual particles results in a small electrical charge
allowing electromagnetic separation of positive and negative
particles from a mixture. In one embodiment, cell surface
marker-specific antibodies or ligands are labeled with distinct
fluorescent labels. Cells are processed through the cell sorter,
allowing separation of cells based on their ability to bind to the
antibodies used. FACS sorted particles may be directly deposited
into individual wells of 96-well or 384-well plates to facilitate
separation and cloning.
[0085] In one sorting scheme, stem cells from placenta are sorted
on the basis of expression of the markers CD34, CD38, CD44, CD45,
CD73, CD105, OCT-4 and/or HLA-G. This can be accomplished in
connection with procedures to select stem cells on the basis of
their adherence properties in culture. For example, an adherence
selection stem can be accomplished before or after sorting on the
basis of marker expression. In one embodiment, for example, cells
are sorted first on the basis of their expression of CD34;
CD34.sup.- cells are retained, and cells that are CD200.sup.+
HLA-G.sup.+, are separated from all other CD34.sup.- cells. In
another embodiment, cells from placenta are based on their
expression of markers CD200 and/or HLA-G; for example, cells
displaying either of these markers are isolated for further use.
Cells that express, e.g., CD200 and/or HLA-G can, in a specific
embodiment, be further sorted based on their expression of CD73
and/or CD105, or epitopes recognized by antibodies SH2, SH3 or SH4,
or lack of expression of CD34, CD38 or CD45. For example, in one
embodiment, placental cells are sorted by expression, or lack
thereof, of CD200, HLA-G, CD73, CD105, CD34, CD38 and CD45, and
placental cells that are CD200.sup.+, HLA-G.sup.+, CD73.sup.+,
CD105.sup.+, CD34.sup.-, CD38.sup.- and CD45.sup.- are isolated
from other placental cells for further use.
[0086] In another embodiment, magnetic beads can be used to
separate cells. The cells may be sorted using a magnetic activated
cell sorting (MACS) technique, a method for separating particles
based on their ability to bind magnetic beads (0.5-100 .mu.m
diameter). A variety of useful modifications can be performed on
the magnetic microspheres, including covalent addition of antibody
that specifically recognizes a particular cell surface molecule or
hapten. The beads are then mixed with the cells to allow binding.
Cells are then passed through a magnetic field to separate out
cells having the specific cell surface marker. In one embodiment,
these cells can then isolated and re-mixed with magnetic beads
coupled to an antibody against additional cell surface markers. The
cells are again passed through a magnetic field, isolating cells
that bound both the antibodies. Such cells can then be diluted into
separate dishes, such as microtiter dishes for clonal
isolation.
[0087] Placental stem cells can also be characterized and/or sorted
based on cell morphology and growth characteristics. For example,
placental stem cells can be characterized as having, and/or
selected on the basis of, e.g., a fibroblastoid appearance in
culture. Placental stem cells can also be characterized as having,
and/or be selected, on the basis of their ability to form
embryoid-like bodies. In one embodiment, for example, placental
cells that are fibroblastoid in shape, express CD73 and CD105, and
produce one or more embryoid-like bodies in culture are isolated
from other placental cells. In another embodiment, OCT-4.sup.+
placental cells that produce one or more embryoid-like bodies in
culture are isolated from other placental cells.
[0088] In another embodiment, placental stem cells can be
identified and characterized by a colony forming unit assay. Colony
forming unit assays are commonly known in the art, such as Mesen
Cult.TM. medium (Stem Cell Technologies, Inc., Vancouver British
Columbia)
[0089] Placental stem cells can be assessed for viability,
proliferation potential, and longevity using standard techniques
known in the art, such as trypan blue exclusion 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.
[0090] Placental stem cells can also be separated from other
placental cells using other techniques known in the art, e.g.,
selective growth of desired cells (positive selection), selective
destruction of unwanted cells (negative selection); separation
based upon differential cell agglutinability in the mixed
population as, for example, with soybean agglutinin; freeze-thaw
procedures; filtration; conventional and zonal centrifugation;
centrifugal elutriation (counter-streaming centrifugation); unit
gravity separation; countercurrent distribution; electrophoresis;
and the like.
5.4 Culture of Placental Stem Cells
[0091] 5.4.1 Culture Media
[0092] Isolated placental stem cells, or placental stem cell
population, or cells or placental tissue from which placental stem
cells grow out, can be used to initiate, or seed, cell cultures.
Cells are generally transferred to sterile tissue culture vessels
either uncoated or coated with extracellular matrix or ligands such
as laminin, collagen (e.g., native or denatured), gelatin,
fibronectin, ornithine, vitronectin, and extracellular membrane
protein (e.g., MATRIGEL (BD Discovery Labware, Bedford,
Mass.)).
[0093] Placental stem cells can be cultured in any medium, and
under any conditions, recognized in the art as acceptable for the
culture of stem cells. Preferably, the culture medium comprises
serum. Placental stem cells can be cultured in, for example,
DMEM-LG (Dulbecco's Modified Essential Medium, low glucose) IMCDB
201 (chick fibroblast basal medium) containing ITS
(insulin-transferrin-selenium), LA+BSA (linoleic acid-bovine serum
albumin), dextrose, L-ascorbic acid, PDGF, EGF, IGF-1, and
penicillin/streptomycin; DMEM-HG (high glucose) comprising 10%
fetal bovine serum (FBS); DMEM-HG comprising 15% FBS; IMDM
(Iscove's modified Dulbecco's medium) comprising 10% FBS, 10% horse
serum, and hydrocortisone; M199 comprising 10% FBS, EGF, and
heparin; .alpha.-MEM (minimal essential medium) comprising 10% FBS,
GlutaMAX.TM. and gentamicin; DMEM comprising 10% FBS, GlutaMAX.TM.
and gentamicin, etc. A preferred medium is DMEM-LG/MCDB-201
comprising 2% FBS, ITS, LA+BSA, dextrose, L-ascorbic acid, PDGF,
EGF, and penicillin/streptomycin.
[0094] Other media in that can be used to culture placental stem
cells include DMEM (high or low glucose), Eagle's basal medium,
Ham's F10 medium (F10), Ham's F-12 medium (F12), Iscove's modified
Dulbecco's medium, Mesenchymal Stem Cell Growth Medium (MSCGM),
Liebovitz's L-15 medium, MCDB, DMIEM/F12, RPMI 1640, advanced DMEM
(Gibco), DMEM/MCDB201 (Sigma), and CELL-GRO FREE.
[0095] The culture medium can be supplemented with one or more
components including, for example, serum (e.g., fetal bovine serum
(FBS), preferably about 2-15% (v/v); equine (horse) serum (ES);
human serum (HS)); beta-mercaptoethanol (BME), preferably about
0.001% (v/v); one or more growth factors, for example,
platelet-derived growth factor (PDGF), epidermal growth factor
(EGF), basic fibroblast growth factor (bFGF), insulin-like growth
factor-1 (IGF-1), leukemia inhibitory factor (LIF), vascular
endothelial growth factor (VEGF), and erythropoietin (EPO); amino
acids, including L-valine; and one or more antibiotic and/or
antimycotic agents to control microbial contamination, such as, for
example, penicillin G, streptomycin sulfate, amphotericin B,
gentamicin, and nystatin, either alone or in combination.
[0096] 5.4.2 Expansion and Proliferation of Placental Stem
Cells
[0097] Once an isolated placental stem cell, or isolated population
of stem cells (e.g., a stem cell or population of stem cells
separated from at least 50% of the placental cells with which the
stem cell or population of stem cells is normally associated in
vivo), the stem cell or population of stem cells can be
proliferated and expanded in vitro. For example, a population of
placental stem cells can be cultured in tissue culture containers,
e.g., dishes, flasks, multiwell plates, or the like, for a
sufficient time for the stem cells to proliferate to 70-90%
confluence, that is, until the stem cells and their progeny occupy
70-90% of the culturing surface area of the tissue culture
container.
[0098] Placental stem cells can be seeded in culture vessels at a
density that allows cell growth. For example, the cells may be
seeded at low density (e.g., about 1,000 to about 5,000
cells/cm.sup.2) to high density (e.g., about 50,000 or more
cells/cm.sup.2). In a preferred embodiment, the cells are cultured
at about 0 to about 5 percent by volume CO.sub.2 in air. In some
preferred embodiments, the cells are cultured at about 2 to about
25 percent O.sub.2 in air, preferably about 5 to about 20 percent
O.sub.2 in air. The cells preferably are cultured at about
25.degree. C. to about 40.degree. C., preferably 37.degree. C. The
cells are preferably cultured in an incubator. The culture medium
can be static or agitated, for example, using a bioreactor.
Placental stem cells preferably are grown under low oxidative
stress (e.g., with addition of glutathione, ascorbic acid,
catalase, tocopherol, N-acetylcysteine, or the like).
[0099] Once 70%-90% confluence is obtained, the cells may be
passaged. For example, the cells can be enzymatically treated,
e.g., trypsinized, using techniques well-known in the art, to
separate them from the tissue culture surface. After removing the
cells by pipetting and counting the cells, about 20,000-100,000
stem cells, preferably about 50,000 stem cells, are passaged to a
new culture container containing fresh culture medium. Typically,
the new medium is the same type of medium from which the stem cells
were removed. The invention encompasses populations of placental
stem cells that have been passaged at least 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 12, 14, 16, 18, or 20 times, or more.
[0100] 5.4.3 Placental Stem Cell Populations
[0101] The invention provides populations of placental stem cells.
Placental stem cell population can be isolated directly from one or
more placentas; that is, the placental stem cell population can be
a population of placental cells, comprising placental stem cells,
obtained from, or contained within, perfusate, or obtained from, or
contained within, digestate (that is, the collection of cells
obtained by enzymatic digestion of a placenta or part thereof).
Isolated placental stem cells of the invention can also be cultured
and expanded to produce placental stem cell populations.
Populations of placental cells comprising placental stem cells can
also be cultured and expanded to produce placental stem cell
populations.
[0102] Placental stem cell populations of the invention comprise
placental stem cells, for example, placental stem cells as
described herein. In various embodiments, at least 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the cells in an
isolated placental stem cell population are placental stem cells.
That is, a placental stem cell population can comprise, e.g., as
much as 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%
non-stem cells.
[0103] The invention provides methods of producing isolated
placental stem cell population by, e.g., selecting placental stem
cells, whether derived from enzymatic digestion or perfusion, that
express particular markers and/or particular culture or
morphological characteristics. In one embodiment, for example, the
invention provides a method of producing a cell population
comprising selecting placental cells that (a) adhere to a
substrate, and (b) express CD200 and HLA-G; and isolating said
cells from other cells to form a cell population. In another
embodiment, the method of producing a cell population comprises
selecting placental cells that (a) adhere to a substrate, and (b)
express CD73, CD105, and CD200; and isolating said cells from other
cells to form a cell population. In another embodiment, the method
of producing a cell population comprises selecting placental cells
that (a) adhere to a substrate and (b) express CD200 and OCT-4; and
isolating said cells from other cells to form a cell population. In
another embodiment, the method of producing a cell population
comprises selecting placental cells that (a) adhere to a substrate,
(b) express CD73 and CD105, and (c) facilitate the formation of one
or more embryoid-like bodies in a population of placental cells
comprising said stem cell when said population is cultured under
conditions that allow for the formation of an embryoid-like body;
and isolating said cells from other cells to form a cell
population. In another embodiment, the method of producing a cell
population comprises selecting placental cells that (a) adhere to a
substrate, and (b) express CD73, CD105 and HLA-G; and isolating
said cells from other cells to form a cell population. In another
embodiment, the method of producing a cell population comprises
selecting placental cells that (a) adhere to a substrate, (b)
express OCT-4, and (c) facilitate the formation of one or more
embryoid-like bodies in a population of placental cells comprising
said stem cell when said population is cultured under conditions
that allow for the formation of an embryoid-like body; and
isolating said cells from other cells to form a cell population. In
any of the above embodiments, the method can additionally comprise
selecting placental cells that express ABC-p (a placenta-specific
ABC transporter protein; see, e.g., Allikmets et al., Cancer Res.
58(23):5337-9 (1998)). The method can also comprise selecting cells
exhibiting at least one characteristic specific to, e.g., a
mesenchymal stem cell, for example, expression of CD29, expression
of CD44, expression of CD90, or expression of a combination of the
foregoing.
[0104] In the above embodiments, the substrate can be any surface
on which culture and/or selection of cells, e.g., placental stem
cells, can be accomplished. Typically, the substrate is plastic,
e.g., tissue culture dish or multiwell plate plastic. Tissue
culture plastic can be coated with a biomolecule, e.g., laminin or
fibronectin.
[0105] Cells, e.g., placental stem cells, can be selected for a
placental stem cell population by any means known in the art of
cell selection. For example, cells can be selected using an
antibody or antibodies to one or more cell surface markers, for
example, in flow cytometry or FACS. Selection can be accomplished
using antibodies in conjunction with magnetic beads. Antibodies
that are specific for certain stem cell-related markers are known
in the art. For example, antibodies to OCT-4 (Abeam, Cambridge,
Mass.), CD200 (Abeam), HLA-G (Abeam), CD73 (BD Biosciences
Pharrningen, San Diego, Calif.), CD105 (Abeam; BioDesign
International, Saco, Me.), etc. Antibodies to other markers are
also available commercially, e.g., CD34, CD38 and CD45 are
available from, e.g., StemCell Technologies or BioDesign
International.
[0106] The isolated placental stem cell population can comprise
placental cells that are not stem cells, or cells that are not
placental cells.
[0107] Isolated placental stem cell populations can be combined
with one or more populations of non-stem cells or non-placental
cells. For example, an isolated population of placental stem cells
can be combined with blood (e.g., placental blood or umbilical cord
blood), blood-derived stem cells (e.g., stem cells derived from
placental blood or umbilical cord blood), populations of
blood-derived nucleated cells, bone marrow-derived mesenchymal
cells, bone-derived stem cell populations, crude bone marrow, adult
(somatic) stem cells, populations of stem cells contained within
tissue, cultured stem cells, populations of fully-differentiated
cells (e.g., chondrocytes, fibroblasts, amniotic cells,
osteoblasts, muscle cells, cardiac cells, etc.) and the like. Cells
in an isolated placental stem cell population can be combined with
a plurality of cells of another type in ratios of about 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; 1:200; 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; 1:50,000,000; or about
1:100,000,000, comparing numbers of total nucleated cells in each
population. Cells in an isolated placental stem cell population can
be combined with a plurality of cells of a plurality of cell types,
as well.
[0108] In one, an isolated population of placental stem cells is
combined with a plurality of hematopoietic stem cells. Such
hematopoietic stem cells can be, for example, contained within
unprocessed placental, umbilical cord blood or peripheral blood; in
total nucleated cells from placental blood, umbilical cord blood or
peripheral blood; in an isolated population of CD34.sup.+ cells
from placental blood, umbilical cord blood or peripheral blood; in
unprocessed bone marrow; in total nucleated cells from bone marrow;
in an isolated population of CD34.sup.+ cells from bone marrow, or
the like.
5.5 Preservation of Placental Stem Cells
[0109] Placental stem cells can be preserved, that is, placed under
conditions that allow for long-term storage, or conditions that
inhibit cell death by, e.g., apoptosis or necrosis.
[0110] Placental stem cells can be preserved using, e.g., a
composition comprising an apoptosis inhibitor, necrosis inhibitor
and/or an oxygen-carrying perfluorocarbon, as described in related
U.S. Provisional Application No. 60/754,969, entitled "Improved
Composition for Collecting and Preserving Placental Stem Cells and
Methods of Using the Composition" filed on Dec. 25, 2005. In one
embodiment, the invention provides a method of preserving a
population of stem cells comprising contacting said population of
stem cells with a stem cell collection composition comprising an
inhibitor of apoptosis and an oxygen-carrying perfluorocarbon,
wherein said inhibitor of apoptosis is present in an amount and for
a time sufficient to reduce or prevent apoptosis in the population
of stem cells, as compared to a population of stem cells not
contacted with the inhibitor of apoptosis. In a specific
embodiment, said inhibitor of apoptosis is a caspase inhibitor. In
another specific embodiment, said inhibitor of apoptosis is a JNK
inhibitor. In a more specific embodiment, said JNK inhibitor does
not modulate differentiation or proliferation of said stem cells.
In another embodiment, said stem cell collection composition
comprises said inhibitor of apoptosis and said oxygen-carrying
perfluorocarbon in separate phases. In another embodiment, said
stem cell collection composition comprises said inhibitor of
apoptosis and said oxygen-carrying perfluorocarbon in an emulsion.
In another embodiment, the stem cell collection composition
additionally comprises an emulsifier, e.g., lecithin. In another
embodiment, said apoptosis inhibitor and said perfluorocarbon are
between about 0.degree. C. and about 25.degree. C. at the time of
contacting the stem cells. In another more specific embodiment,
said apoptosis inhibitor and said perfluorocarbon are between about
2.degree. C. and 10.degree. C., or between about 2.degree. C. and
about 5.degree. C., at the time of contacting the stem cells. In
another more specific embodiment, said contacting is performed
during transport of said population of stem cells. In another more
specific embodiment, said contacting is performed during freezing
and thawing of said population of stem cells.
[0111] In another embodiment, the invention provides a method of
preserving a population of placental stem cells comprising
contacting said population of stem cells with an inhibitor of
apoptosis and an organ-preserving compound, wherein said inhibitor
of apoptosis is present in an amount and for a time sufficient to
reduce or prevent apoptosis in the population of stem cells, as
compared to a population of stem cells not contacted with the
inhibitor of apoptosis. In a specific embodiment, the
organ-preserving compound is UW solution (described in U.S. Pat.
No. 4,798,824; also known as ViaSpan; see also Southard et al.,
Transplantation 49(2):251-257 (1990)) or a solution described in
Stem et al., U.S. Pat. No. 5,552,267. In another embodiment, said
organ-preserving compound is hydroxyethyl starch, lactobionic acid,
raffinose, or a combination thereof. In another embodiment, the
stem cell collection composition additionally comprises an
oxygen-carrying perfluorocarbon, either in two phases or as an
emulsion.
[0112] In another embodiment of the method, placental stem cells
are contacted with a stem cell collection composition comprising an
apoptosis inhibitor and oxygen-carrying perfluorocarbon,
organ-preserving compound, or combination thereof, during
perfusion. In another embodiment, said stem cells are contacted
during a process of tissue disruption, e.g., enzymatic digestion.
In another embodiment, placental stem cells are contacted with said
stem cell collection compound after collection by perfusion, or
after collection by tissue disruption, e.g., enzymatic
digestion.
[0113] Typically, during placental cell collection, enrichment and
isolation, it is preferable to minimize or eliminate cell stress
due to hypoxia and mechanical stress. In another embodiment of the
method, therefore, a stem cell, or population of stem cells, is
exposed to a hypoxic condition during collection, enrichment or
isolation for less than six hours during said preservation, wherein
a hypoxic condition is a concentration of oxygen that is less than
normal blood oxygen concentration. In a more specific embodiment,
said population of stem cells is exposed to said hypoxic condition
for less than two hours during said preservation. In another more
specific embodiment, said population of stem cells is exposed to
said hypoxic condition for less than one hour, or less than thirty
minutes, or is not exposed to a hypoxic condition, during
collection, enrichment or isolation. In another specific
embodiment, said population of stem cells is not exposed to shear
stress during collection, enrichment or isolation.
[0114] The placental stem cells of the invention can be
cryopreserved, e.g., in cryopreservation medium in small
containers, e.g., ampoules. Suitable cryopreservation medium
includes, but is not limited to, culture medium including, e.g.,
growth medium, or cell freezing medium, for example commercially
available cell freezing medium, e.g., C2695, C2639 or C6039
(Sigma). Cryopreservation medium preferably comprises DMSO
(dimethylsulfoxide), at a concentration of, e.g., about 10% (v/v).
Cryopreservation medium may comprise additional agents, for
example, methylcellulose and/or glycerol. Placental stem cells are
preferably cooled at about 1.degree. C./min during
cryopreservation. A preferred cryopreservation temperature is about
-80.degree. C. to about -180.degree. C., preferably about
-125.degree. C. to about -140.degree. C. Cryopreserved cells can be
transferred to liquid nitrogen prior to thawing for use. In some
embodiments, for example, once the ampoules have reached about
-90.degree. C., they are transferred to a liquid nitrogen storage
area. Cryopreserved cells preferably are thawed at a temperature of
about 25.degree. C. to about 40.degree. C., preferably to a
temperature of about 37.degree. C.
5.6 Uses of Placental Stem Cells
[0115] 5.6.1 Compositions Comprising Placental Stem Cells
[0116] The methods of immunosuppression of the present invention
can use compositions comprising placental stem cells, or
biomolecules therefrom. In the same manner, the pluralities and
populations of placental stem cells of the present invention can be
combined with any physiologically-acceptable or
medically-acceptable compound, composition or device for use in,
e.g., research or therapeutics.
[0117] 5.6.1.1 Cryopreserved Placental Stem Cells
[0118] The immunosuppressive placental stem cell populations of the
invention can be preserved, for example, cryopreserved for later
use. Methods for cryopreservation of cells, such as stem cells, are
well known in the art. Placental stem cell populations can be
prepared in a form that is easily administrable to an individual.
For example, the invention provides a placental stem cell
population that is contained within a container that is suitable
for medical use. Such a container can be, for example, a sterile
plastic bag, flask, jar, or other container from which the
placental stem cell population can be easily dispensed. For
example, the container can be a blood bag or other plastic,
medically-acceptable bag suitable for the intravenous
administration of a liquid to a recipient. The container is
preferably one that allows for cryopreservation of the combined
stem cell population.
[0119] Cryopreserved immunosuppressive placental stem cell
populations can comprise placental stem cells derived from a single
donor, or from multiple donors. The placental stem cell population
can be completely HLA-matched to an intended recipient, or
partially or completely HLA-mismatched.
[0120] Thus, in one embodiment, the invention provides a
composition comprising an immunosuppressive placental stem cell
population in a container. In a specific embodiment, the stem cell
population is cryopreserved. In another specific embodiment, the
container is a bag, flask, or jar. In more specific embodiment,
said bag is a sterile plastic bag. In a more specific embodiment,
said bag is suitable for, allows or facilitates intravenous
administration of said placental stem cell population. The bag can
comprise multiple lumens or compartments that are interconnected to
allow mixing of the placental stem cells and one or more other
solutions, e.g., a drug, prior to, or during, administration. In
another specific embodiment, the composition comprises one or more
compounds that facilitate cryopreservation of the combined stem
cell population. In another specific embodiment, said placental
stem cell population is contained within a
physiologically-acceptable aqueous solution. In a more specific
embodiment, said physiologically-acceptable aqueous solution is a
0.9% NaCl solution. In another specific embodiment, said placental
stem cell population comprises placental cells that are HLA-matched
to a recipient of said stem cell population. In another specific
embodiment, said combined stem cell population comprises placental
cells that are at least partially HLA-mismatched to a recipient of
said stem cell population. In another specific embodiment, said
placental stem cells are derived from a plurality of donors.
5.6.1.2 Pharmaceutical Compositions
[0121] Immunosuppressive populations of placental stem cells, or
populations of cells comprising placental stem cells, can be
formulated into pharmaceutical compositions for use in vivo. Such
pharmaceutical compositions comprise a population of placental stem
cells, or a population of cells comprising placental stem cells, in
a pharmaceutically-acceptable carrier, e.g., a saline solution or
other accepted physiologically-acceptable solution for in vivo
administration. Pharmaceutical compositions of the invention can
comprise any of the placental stem cell populations, or placental
stem cell types, described elsewhere herein. The pharmaceutical
compositions can comprise fetal, maternal, or both fetal and
maternal placental stem cells. The pharmaceutical compositions of
the invention can further comprise placental stem cells obtained
from a single individual or placenta, or from a plurality of
individuals or placentae.
[0122] The pharmaceutical compositions of the invention can
comprise any immunosuppressive number of placental stem cells. For
example, a single unit dose of placental stem cells can comprise,
in various embodiments, about, at least, or no more than
1.times.10.sup.5, 5.times.10.sup.5, 1.times.10.sup.6,
5.times.10.sup.6, 1.times.10.sup.7, 5.times.10.sup.7,
1.times.10.sup.8, 5.times.10.sup.8, 1.times.10.sup.9,
5.times.10.sup.9, 1.times.10.sup.10, 5.times.10.sup.10,
1.times.10.sup.11 or more placental stem cells.
[0123] The pharmaceutical compositions of the invention comprise
populations of cells that comprise 50% viable cells or more (that
is, at least 50% of the cells in the population are functional or
living). Preferably, at least 60% of the cells in the population
are viable. More preferably, at least 70%, 80%, 90%, 95%, or 99% of
the cells in the population in the pharmaceutical composition are
viable.
[0124] The pharmaceutical compositions of the invention can
comprise one or more compounds that, e.g., facilitate engraftment
(e.g., anti-T-cell receptor antibodies, an immunosuppressant, or
the like); stabilizers such as albumin, dextran 40, gelatin,
hydroxyethyl starch, and the like.
5.6.1.3 Placental Stem Cell Conditioned Media
[0125] The placental stem cells of the invention can be used to
produce conditioned medium that is immunosuppressive, that is,
medium comprising one or more biomolecules secreted or excreted by
the stem cells that have a detectable immunosuppressive effect on a
plurality of one or more types of immune cells. In various
embodiments, the conditioned medium comprises medium in which
placental stem cells have grown for at least 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14 or more days. In other embodiments, the
conditioned medium comprises medium in which placental stem cells
have grown to at least 30%, 40%, 50%, 60%, 70%, 80%, 90%
confluence, or up to 100% confluence. Such conditioned medium can
be used to support the culture of a separate population of
placental stem cells, or stem cells of another kind. In another
embodiment, the conditioned medium comprises medium in which
placental stem cells have been differentiated into an adult cell
type. In another embodiment, the conditioned medium of the
invention comprises medium in which placental stem cells and
non-placental stem cells have been cultured.
[0126] Thus, in one embodiment, the invention provides a
composition comprising culture medium from a culture of placental
stem cells, wherein said placental stem cells (a) adhere to a
substrate; (b) express CD200 and HLA-G, or express CD73, CD105, and
CD200, or express CD200 and OCT-4, or express CD73, CD105, and
HLA-G, or express CD73 and CD105 and facilitate the formation of
one or more embryoid-like bodies in a population of placental cells
that comprise the placental stem cells, when said population is
cultured under conditions that allow formation of embryoid-like
bodies, or express OCT-4 and facilitate the formation of one or
more embryoid-like bodies in a population of placental cells that
comprise the placental stem cells when said population is cultured
under conditions that allow formation of embryoid-like bodies; and
(c) detectably suppress CD4.sup.+ or CD8.sup.+ T cell proliferation
in an MLR (mixed lymphocyte reaction), wherein said culture of
placental stem cells has been cultured in said medium for 24 hours
or more. In a specific embodiment, the composition further
comprises a plurality of said placental stem cells. In another
specific embodiment, the composition comprises a plurality of
non-placental cells. In a more specific embodiment, said
non-placental cells comprise CD34.sup.+ cells, e.g., hematopoietic
progenitor cells, such as peripheral blood hematopoietic progenitor
cells, cord blood hematopoietic progenitor cells, or placental
blood hematopoietic progenitor cells. The non-placental cells can
also comprise other stem cells, such as mesenchymal stem cells,
e.g., bone marrow-derived mesenchymal stem cells. The non-placental
cells can also be one or more types of adult cells or cell lines.
In another specific embodiment, the composition comprises an
anti-proliferative agent, e.g., an anti-MIP-1.alpha. or
anti-MIP-1.beta. antibody.
5.6.1.4 Matrices Comprising Placental Stem Cells
[0127] The invention further comprises matrices, hydrogels,
scaffolds, and the like that comprise an immunosuppresive
population of placental stem cells.
[0128] Placental stem cells of the invention can be seeded onto a
natural matrix, e.g., a placental biomaterial such as an amniotic
membrane material. Such an amniotic membrane material can be, e.g.,
amniotic membrane dissected directly from a mammalian placenta;
fixed or heat-treated amniotic membrane, substantially dry (i.e.,
<20% H.sub.2O) amniotic membrane, chorionic membrane,
substantially dry chorionic membrane, substantially dry amniotic
and chorionic membrane, and the like. Preferred placental
biomaterials on which placental stem cells can be seeded are
described in Hariri, U.S. Application Publication No.
2004/0048796.
[0129] Placental stem cells of the invention can be suspended in a
hydrogel solution suitable for, e.g., injection. Suitable hydrogels
for such compositions include self-assembling peptides, such as
RAD16. In one embodiment, a hydrogel solution comprising the cells
can be allowed to harden, for instance in a mold, to form a matrix
having cells dispersed therein for implantation. Placental stem
cells in such a matrix can also be cultured so that the cells are
mitotically expanded prior to implantation. The hydrogel is, e.g.,
an organic polymer (natural or synthetic) that is cross-linked via
covalent, ionic, or hydrogen bonds to create a three-dimensional
open-lattice structure that entraps water molecules to form a gel.
Hydrogel-forming materials include polysaccharides such as alginate
and salts thereof, peptides, polyphosphazines, and polyacrylates,
which are crosslinked ionically, or block polymers such as
polyethylene oxide-polypropylene glycol block copolymers which are
crosslinked by temperature or pH, respectively. In some
embodiments, the hydrogel or matrix of the invention is
biodegradable.
[0130] In some embodiments of the invention, the formulation
comprises an in situ polymerizable gel (see., e.g., U.S. Patent
Application Publication 2002/0022676; Anseth et al., J. Control
Release, 78(1-3):199-209 (2002); Wang et al., Biomaterials,
24(22):3969-80 (2003).
[0131] In some embodiments, the polymers are at least partially
soluble in aqueous solutions, such as water, buffered salt
solutions, or aqueous alcohol solutions, that have charged side
groups, or a monovalent ionic salt thereof. Examples of polymers
having acidic side groups that can be reacted with cations are
poly(phosphazenes), poly(acrylic acids), poly(methacrylic acids),
copolymers of acrylic acid and methacrylic acid, poly(vinyl
acetate), and sulfonated polymers, such as sulfonated polystyrene.
Copolymers having acidic side groups formed by reaction of acrylic
or methacrylic acid and vinyl ether monomers or polymers can also
be used. Examples of acidic groups are carboxylic acid groups,
sulfonic acid groups, halogenated (preferably fluorinated) alcohol
groups, phenolic OH groups, and acidic OH groups.
[0132] The placental stem cells of the invention or co-cultures
thereof can be seeded onto a three-dimensional framework or
scaffold and implanted in vivo. Such a framework can be implanted
in combination with any one or more growth factors, cells, drugs or
other components that stimulate tissue formation or otherwise
enhance or improve the practice of the invention.
[0133] Examples of scaffolds that can be used in the present
invention include nonwoven mats, porous foams, or self assembling
peptides. Nonwoven mats can be formed using fibers comprised of a
synthetic absorbable copolymer of glycolic and lactic acids (e.g.,
PGA/PLA) (VICRYL, Ethicon, Inc., Somerville, N.J.). Foams, composed
of, e.g., poly(.epsilon.-caprolactone)/poly(glycolic acid)
(PCL/PGA) copolymer, formed by processes such as freeze-drying, or
lyophilization (see, e.g., U.S. Pat. No. 6,355,699), can also be
used as scaffolds.
[0134] Placental stem cells of the invention can also be seeded
onto, or contacted with, a physiologically-acceptable ceramic
material including, but not limited to, mono-, di-, tri-,
alpha-tri-, beta-tri-, and tetra-calcium phosphate, hydroxyapatite,
fluoroapatites, calcium sulfates, calcium fluorides, calcium
oxides, calcium carbonates, magnesium calcium phosphates,
biologically active glasses such as BIOGLASS.RTM., and mixtures
thereof. Porous biocompatible ceramic materials currently
commercially available include SURGIBONE.RTM. (CanMedica Corp.,
Canada), ENDOBON.RTM. (Merck Biomaterial France, France),
CEROS.RTM. (Mathys, AG, Bettlach, Switzerland), and mineralized
collagen bone grafting products such as HEALOS.TM. (DePuy, Inc.,
Raynham, Mass.) and VITOSS.RTM., RHAKOSS.TM., and CORTOSS.RTM.
(Orthovita, Malvern, Pa.). The framework can be a mixture, blend or
composite of natural and/or synthetic materials.
[0135] In another embodiment, placental stem cells can be seeded
onto, or contacted with, a felt, which can be, e.g., composed of a
multifilament yarn made from a bioabsorbable material such as PGA,
PLA, PCL copolymers or blends, or hyaluronic acid.
[0136] The placental stem cells of the invention can, in another
embodiment, be seeded onto foam scaffolds that may be composite
structures. Such foam scaffolds can be molded into a useful shape,
such as that of a portion of a specific structure in the body to be
repaired, replaced or augmented. In some embodiments, the framework
is treated, e.g., with 0.1M acetic acid followed by incubation in
polylysine, PBS, and/or collagen, prior to inoculation of the cells
of the invention in order to enhance cell attachment. External
surfaces of a matrix may be modified to improve the attachment or
growth of cells and differentiation of tissue, such as by
plasma-coating the matrix, or addition of one or more proteins
(e.g., collagens, elastic fibers, reticular fibers), glycoproteins,
glycosaminoglycans (e.g., heparin sulfate, chondroitin-4-sulfate,
chondroitin-6-sulfate, dermatan sulfate, keratin sulfate, etc.), a
cellular matrix, and/or other materials such as, but not limited
to, gelatin, alginates, agar, agarose, and plant gums, and the
like.
[0137] In some embodiments, the scaffold comprises, or is treated
with, materials that render it non-thrombogenic. These treatments
and materials may also promote and sustain endothelial growth,
migration, and extracellular matrix deposition. Examples of these
materials and treatments include but are not limited to natural
materials such as basement membrane proteins such as laminin and
Type IV collagen, synthetic materials such as EPTFE, and segmented
polyurethaneurea silicones, such as PURSPAN.TM. (The Polymer
Technology Group, Inc., Berkeley, Calif.). The scaffold can also
comprise anti-thrombotic agents such as heparin; the scaffolds can
also be treated to alter the surface charge (e.g., coating with
plasma) prior to seeding with placental stem cells.
[0138] 5.6.2 Immortalized Placental Stem Cell Lines
[0139] Mammalian placental cells can be conditionally immortalized
by transfection with any suitable vector containing a
growth-promoting gene, that is, a gene encoding a protein that,
under appropriate conditions, promotes growth of the transfected
cell, such that the production and/or activity of the
growth-promoting protein is regulatable by an external factor. In a
preferred embodiment the growth-promoting gene is an oncogene such
as, but not limited to, v-myc, N-myc, c-myc, p53, SV40 large T
antigen, polyoma large T antigen, E1a adenovirus or E7 protein of
human papillomavirus.
[0140] External regulation of the growth-promoting protein can be
achieved by placing the growth-promoting gene under the control of
an externally-regulatable promoter, e.g., a promoter the activity
of which can be controlled by, for example, modifying the
temperature of the transfected cells or the composition of the
medium in contact with the cells. in one embodiment, a tetracycline
(tet)-controlled gene expression system can be employed (see Gossen
et al., Proc. Natl. Acad. Sci. USA 89:5547-5551, 1992; Hoshimaru et
al., Proc. Natl. Acad. Sci. USA 93:1518-1523, 1996). In the absence
of tet, a tet-controlled transactivator (tTA) within this vector
strongly activates transcription from ph.sub.CMV*-1, a minimal
promoter from human cytomegalovirus fused to tet operator
sequences. tTA is a fusion protein of the repressor (tetR) of the
transposon-10-derived tet resistance operon of Escherichia coli and
the acidic domain of VP16 of herpes simplex virus. Low, non-toxic
concentrations of tet (e.g., 0.01-1.0 .mu.g/mL) almost completely
abolish transactivation by tTA.
[0141] In one embodiment, the vector further contains a gene
encoding a selectable marker, e.g., a protein that confers drug
resistance. The bacterial neomycin resistance gene (neo.sup.R) is
one such marker that may be employed within the present invention.
Cells carrying neo.sup.R may be selected by means known to those of
ordinary skill in the art, such as the addition of, e.g., 100-200
.mu.g/mL G418 to the growth medium.
[0142] Transfection can be achieved by any of a variety of means
known to those of ordinary skill in the art including, but not
limited to, retroviral infection. In general, a cell culture may be
transfected by incubation with a mixture of conditioned medium
collected from the producer cell line for the vector and DMEM/F12
containing N2 supplements. For example, a placental cell culture
prepared as described above may be infected after, e.g., five days
in vitro by incubation for about 20 hours in one volume of
conditioned medium and two volumes of DMEM/F12 containing N2
supplements. Transfected cells carrying a selectable marker may
then be selected as described above.
[0143] Following transfection, cultures are passaged onto a surface
that permits proliferation, e.g., allows at least 30% of the cells
to double in a 24 hour period. Preferably, the substrate is a
polyornithine/laminin substrate, consisting of tissue culture
plastic coated with polyornithine (10 .mu.g/mL) and/or laminin (10
.mu.g/mL), a polylysine/laminin substrate or a surface treated with
fibronectin. Cultures are then fed every 3-4 days with growth
medium, which may or may not be supplemented with one or more
proliferation-enhancing factors. Proliferation-enhancing factors
may be added to the growth medium when cultures are less than 50%
confluent.
[0144] The conditionally-immortalized placental stem cell lines can
be passaged using standard techniques, such as by trypsinization,
when 80-95% confluent. Up to approximately the twentieth passage,
it is, in some embodiments, beneficial to maintain selection (by,
for example, the addition of G418 for cells containing a neomycin
resistance gene). Cells may also be frozen in liquid nitrogen for
long-term storage.
[0145] Clonal cell lines can be isolated from a
conditionally-immortalized human placental stem cell line prepared
as described above. In general, such clonal cell lines may be
isolated using standard techniques, such as by limit dilution or
using cloning rings, and expanded. Clonal cell lines may generally
be fed and passaged as described above.
[0146] Conditionally-immortalized human placental stem cell lines,
which may, but need not, be clonal, may generally be induced to
differentiate by suppressing the production and/or activity of the
growth-promoting protein under culture conditions that facilitate
differentiation. For example, if the gene encoding the
growth-promoting protein is under the control of an
externally-regulatable promoter, the conditions, e.g., temperature
or composition of medium, may be modified to suppress transcription
of the growth-promoting gene. For the tetracycline-controlled gene
expression system discussed above, differentiation can be achieved
by the addition of tetracycline to suppress transcription of the
growth-promoting gene. In general, 1 .mu.g/mL tetracycline for 4-5
days is sufficient to initiate differentiation. To promote further
differentiation, additional agents may be included in the growth
medium.
[0147] 5.6.3 Assays
[0148] The placental stem cells for the present invention can be
used in assays to determine the influence of culture conditions,
environmental factors, molecules (e.g., biomolecules, small
inorganic molecules. etc.) and the like on stem cell proliferation,
expansion, and/or differentiation, compared to placental stem cells
not exposed to such conditions.
[0149] In a preferred embodiment, the placental stem cells of the
present invention are assayed for changes in proliferation,
expansion or differentiation upon contact with a molecule. In one
embodiment, for example, the invention provides a method of
identifying a compound that modulates the proliferation of a
plurality of placental stem cells, comprising contacting said
plurality of stem cells with said compound under conditions that
allow proliferation, wherein if said compound causes a detectable
change in proliferation of said plurality of stem cells compared to
a plurality of stem cells not contacted with said compound, said
compound is identified as a compound that modulates proliferation
of placental stem cells. In a specific embodiment, said compound is
identified as an inhibitor of proliferation. In another specific
embodiment, said compound is identified as an enhancer of
proliferation.
[0150] In another embodiment, the invention provides a method of
identifying a compound that modulates the expansion of a plurality
of placental stem cells, comprising contacting said plurality of
stem cells with said compound under conditions that allow
expansion, wherein if said compound causes a detectable change in
expansion of said plurality of stem cells compared to a plurality
of stem cells not contacted with said compound, said compound is
identified as a compound that modulates expansion of placental stem
cells. In a specific embodiment, said compound is identified as an
inhibitor of expansion. In another specific embodiment, said
compound is identified as an enhancer of expansion.
[0151] In another embodiment, the invention provides a method of
identifying a compound that modulates the differentiation of a
placental stem cell, comprising contacting said stem cells with
said compound under conditions that allow differentiation, wherein
if said compound causes a detectable change in differentiation of
said stem cells compared to a stem cell not contacted with said
compound, said compound is identified as a compound that modulates
proliferation of placental stem cells. In a specific embodiment,
said compound is identified as an inhibitor of differentiation. In
another specific embodiment, said compound is identified as an
enhancer of differentiation.
[0152] 5.6.4 Placental Stem Cell Bank
[0153] Stem cells from postpartum placentas can be cultured in a
number of different ways to produce a set of lots, e.g., a set of
individually-administrable doses, of placental stem cells. Such
lots can, for example, be obtained from stem cells from placental
perfusate or from enzyme-digested placental tissue. Sets of lots of
placental stem cells, obtained from a plurality of placentas, can
be arranged in a bank of placental stem cells for, e.g., long-term
storage. Generally, adherent stem cells are obtained from an
initial culture of placental material to form a seed culture, which
is expanded under controlled conditions to form populations of
cells from approximately equivalent numbers of doublings. Lots are
preferably derived from the tissue of a single placenta, but can be
derived from the tissue of a plurality of placentas.
[0154] In one embodiment, stem cell lots are obtained as follows.
Placental tissue is first disrupted, e.g., by mincing, digested
with a suitable enzyme, e.g., collagenase (see Section 5.2.3,
above). The placental tissue preferably comprises, e.g., the entire
amnion, entire chorion, or both, from a single placenta, but can
comprise only a part of either the amnion or chorion. The digested
tissue is cultured, e.g., for about 1-3 weeks, preferably about 2
weeks. After removal of non-adherent cells, high-density colonies
that form are collected, e.g., by trypsinization. These cells are
collected and resuspended in a convenient volume of culture medium,
and defined as Passage 0 cells.
[0155] Passage 0 cells are then used to seed expansion cultures.
Expansion cultures can be any arrangement of separate cell culture
apparatuses, e.g., a Cell Factory by NUNC.TM.. Cells in the Passage
0 culture can be subdivided to any degree so as to seed expansion
cultures with, e.g., 1.times.10.sup.3, 2.times.10.sup.3,
3.times.10.sup.3, 4.times.10.sup.3, 5.times.10.sup.3,
6.times.10.sup.3, 7.times.10.sup.3, 8.times.10.sup.3,
9.times.10.sup.3, 1.times.10.sup.4, 1.times.10.sup.4,
2.times.10.sup.4, 3.times.10.sup.4, 4.times.10.sup.4,
5.times.10.sup.4, 6.times.10.sup.4, 7.times.10.sup.4,
8.times.10.sup.4, 9.times.10.sup.4, or 10.times.10.sup.4 stem
cells. Preferably, from about 2.times.10.sup.4 to about
3.times.10.sup.4 Passage 0 cells are used to seed each expansion
culture. The number of expansion cultures can depend upon the
number of Passage 0 cells, and may be greater or fewer in number
depending upon the particular placenta(s) from which the stem cells
are obtained.
[0156] Expansion cultures are grown until the density of cells in
culture reaches a certain value, e.g., about 1.times.10.sup.5
cells/cm.sup.2. Cells can either be collected and cryopreserved at
this point, or passaged into new expansion cultures as described
above. Cells can be passaged, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19 or 20 times prior to use. A record
of the cumulative number of population doublings is preferably
maintained during expansion culture(s). The cells from a Passage 0
culture can be expanded for 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16,
18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 or 40 doublings, or up
to 60 doublings. Preferably, however, the number of population
doublings, prior to dividing the population of cells into
individual doses, is between about 15 and about 30, preferably
about 20 doublings. The cells can be culture continuously
throughout the expansion process, or can be frozen at one or more
points during expansion.
[0157] Cells to be used for individual doses can be frozen, e.g.,
cryopreserved for later use. Individual doses can comprise, e.g.,
about 1 million to about 100 million cells per ml, and can comprise
between about 10.sup.6 and about 10.sup.9 cells in total.
[0158] In a specific embodiment, of the method, Passage 0 cells are
cultured for approximately 4 doublings, then frozen in a first cell
bank. Cells from the first cell bank are frozen and used to seed a
second cell bank, the cells of which are expanded for about another
eight doublings. Cells at this stage are collected and frozen and
used to seed new expansion cultures that are allowed to proceed for
about eight additional doublings, bringing the cumulative number of
cell doublings to about 20. Cells at the intermediate points in
passaging can be frozen in units of about 100,000 to about 10
million cells per ml, preferably about 1 million cells per ml for
use in subsequent expansion culture. Cells at about 20 doublings
can be frozen in individual doses of between about 1 million to
about 100 million cells per ml for administration or use in making
a stem cell-containing composition.
[0159] In a preferred embodiment, the donor from which the placenta
is obtained (e.g., the mother) is tested for at least one pathogen.
If the mother tests positive for a tested pathogen, the entire lot
from the placenta is discarded. Such testing can be performed at
any time during production of placental stem cell lots, including
before or after establishment of Passage 0 cells, or during
expansion culture. Pathogens for which the presence is tested can
include, without limitation, hepatitis A, hepatitis B, hepatitis C,
hepatitis D, hepatitis E, human immunodeficiency virus (types I and
II), cytomegalovirus, herpesvirus, and the like.
[0160] 5.6.5 Treatment of Multiple Sclerosis
[0161] In another aspect, the invention provides a method of
treating an individual having multiple sclerosis, or a symptom
associated with multiple sclerosis, comprising administering to the
individual a plurality of placental stem cells in an amount and for
a time sufficient to detectably modulate, e.g., suppress an immune
response in the individual.
[0162] Multiple sclerosis (MS) is a chronic, recurrent inflammatory
disease of the central nervous system. The disease results in
injury to the myelin sheaths surrounding CNS and PNS axons,
oligodendrocytes, and the nerve cells themselves. The disease is
mediated by autoreactive T cells, particularly CD4.sup.+ T cells,
that proliferate, cross the blood-brain barrier, and enter the CNS
under the influence of cellular adhesion molecules and
pro-inflammatory cytokines. The symptoms of MS include sensory
disturbances in the limbs, optic nerve dysfunction, pyramidal tract
dysfunction, bladder dysfunction, bowel dysfunction, sexual
dysfunction, ataxia, and diplopia.
[0163] Four different types or clinical courses of MS have been
identified. The first, relapsing/remitting MS (RRMS) is
characterized by self-limiting attacks of neurological dysfunction
that manifest acutely, over the course of days to weeks, followed
by a period of recovery, sometimes incomplete, over several months.
The second type, secondary progressive MS (SPMS), begins as RRMS
but changes such that the clinical course becomes characterized by
a steady deterioration in function unrelated to acute attacks. The
third, primary progressive MS (PPMS), is characterized by a steady
decline in function from onset, with no acute attacks. The fourth
type, progressive/relapsing MS (PRMS), also begins with a
progressive course, with occasional attacks superimposed on the
progressive decline in function.
[0164] Persons having MS are generally evaluated using a motor
skills assessment, optionally with an MRI. For example, one motor
skills assessment, the expanded disability status scale, scores
gradations in an affected individual's abilities, as follows:
[0165] 0.0 Normal neurological examination [0166] 1.0 No
disability, minimal signs in one FS [0167] 1.5 No disability,
minimal signs in more than one FS [0168] 2.0 Minimal disability in
one FS [0169] 2.5 Mild disability in one FS or minimal disability
in two FS [0170] 3.0 Moderate disability in one FS, or mild
disability in three or four FS. Fully ambulatory. [0171] 3.5 Fully
ambulatory but with moderate disability in one FS and more than
minimal disability in several others [0172] 4.0 Fully ambulatory
without aid, self-sufficient, up and about some 12 hours a day
despite relatively severe disability; able to walk without aid or
rest some 500 meters [0173] 4.5 Fully ambulatory without aid, up
and about much of the day, able to work a full day, may otherwise
have some limitation of full activity or require minimal
assistance; characterized by relatively severe disability; able to
walk without aid or rest some 300 meters. [0174] 5.0 Ambulatory
without aid or rest for about 200 meters; disability severe enough
to impair full daily activities (work a full day without special
provisions) [0175] 5.5 Ambulatory without aid or rest for about 100
meters; disability severe enough to preclude full daily activities
[0176] 6.0 Intermittent or unilateral constant assistance (cane,
crutch, brace) required to walk about 100 meters with or without
resting [0177] 6.5 Constant bilateral assistance (canes, crutches,
braces) required to walk about 20 meters without resting [0178] 7.0
Unable to walk beyond approximately five meters even with aid,
essentially restricted to wheelchair; wheels self in standard
wheelchair and transfers alone; up and about in wheelchair some 12
hours a day [0179] 7.5 Unable to take more than a few steps;
restricted to wheelchair; may need aid in transfer; wheels self but
cannot carry on in standard wheelchair a full day; [0180] May
require motorized wheelchair [0181] 8.0 Essentially restricted to
bed or chair or perambulated in wheelchair, but may be out of bed
itself much of the day; retains many self-care functions; generally
has effective use of arms [0182] 8.5 Essentially restricted to bed
much of day; has some effective use of arms retains some self care
functions [0183] 9.0 Confined to bed; can still communicate and
eat. [0184] 9.5 Totally helpless bed patient; unable to communicate
effectively or eat/swallow [0185] 10.0 Death due to MS
[0186] In the above scoring system, "FS" refers to the eight
functional systems measured, including pyramidal, cerebellar,
brainstem, sensory, bowel and bladder, visual, cerebral, and other
systems.
[0187] Other, similar scoring systems are known, including the
Scripps neurological rating scale, the ambulatory index, and the
multiple sclerosis functional composite score (MSFC).
[0188] The progress of MS has also been assessed by a determination
of the attack rate.
[0189] The progress of MS has also been assessed by magnetic
resonance imaging, which can detect neural lesions associated with
MS (e.g., new lesions, enhancing lesions, or combined unique active
lesions).
[0190] Thus, in one embodiment, the invention provides a method of
treating an individual having MS, e.g., and individual who has been
diagnosed with MS, comprising administering to the individual a
plurality of placental stem cells, wherein the placental stem cells
are capable of differentiating into olidogdndrocytes, e.g.,
differentiate to oligodendrocytes within the individual. In a
specific embodiment, the administering detectably improves one or
more symptoms of MS in the individual. In more specific
embodiments, the symptom is, e.g., one or more of a sensory
disturbance in the limbs, an optic nerve dysfunction, a pyramidal
tract dysfunction, a bladder dysfunction, a bowel dysfunction, a
sexual dysfunction, ataxia, or diplopia. In another specific
embodiment, said administering results in an improvement on the
EDSS scale of at least one half point. In another specific
embodiment, said administering results in an improvement on the
EDSS scale of at least one point. In another specific embodiment,
said administering results in an improvement on the EDSS scale of
at least two points. In other specific embodiments, said
administering results in a detectable improvement on a multiple
sclerosis assessment scale or on an MRI.
[0191] MS has been treated with other therapeutic agents, for
example immunomodulatory or immunosuppressive agents, e.g.,
interferon beta (IFN.beta.), including IFN.beta.-1a and IFN-1b;
gliatriamer acetate (Copaxone); cyclophosphamide; methotrexate;
azathioprine (Imuran); cladribine (Leustatin); cyclosporine;
mitoxantrone; and the like. MS has also been treated with
anti-inflammatory therapeutic agents, such as glucocorticoids,
including adrenocorticotropic hormone (ACTH), methylprednisolone,
dexamethasone, and the like. MS has also been treated with other
types of therapeutic agents, such as intravenous immunoglobulin,
plasma exchange, or sulfasalazine.
[0192] Thus, the invention further provides for the treatment of an
individual having MS, e.g., an individual who has been diagnosed as
having MS, comprising administering to the individual a plurality
of placental stem cells, wherein the administering detectably
improves one or more symptoms of MS in the individual, and one or
more therapeutic agents, and wherein the placental stem cells are
capable of differentiating into olidogdndrocytes, e.g.,
differentiate to oligodendrocytes within the individual. In one
embodiment, the therapeutic agent is a glucocorticoid. In specific
embodiments, the glucocorticoid is adrenocorticotropic hormone
(ACTH), methylprednisolone, or dexamethasone. In another
embodiment, the therapeutic agent is an immunomodulatory or
immunosuppressive agent. In various specific embodiments, the
immunomodulatory or immunosuppressive agent is IFN.beta.-1a,
IFN-1b, gliatriamer acetate, cyclophosphamide, methotrexate,
azathioprine, cladribine, cyclosporine or mitoxantrone. In other
embodiments, the therapeutic agent is intravenous immunoglobulin,
plasma exchange, or sulfasalazine. In another embodiment, the
individual is administered any combination of the foregoing
therapeutic agents.
[0193] An individual having MS, e.g., an individual diagnosed with
MS, can be treated with a plurality of placental stem cells, and,
optionally, one or more therapeutic agents, at any time during the
progression of the disease. For example, the individual can be
treated immediately after diagnosis, or within 1, 2, 3, 4, 5, 6
days of diagnosis, or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,
25, 30, 35, 40, 45, 50 or more weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9,
10 or more years after diagnosis. The individual can be treated
once, or multiple times during the clinical course of the disease.
The individual can be treated, as appropriate, during an acute
attack, during remission, or during a chronic degenerative phase.
In another embodiment, the placental stem cells are administered to
a female having MS, post-partum, to maintain the state of remission
or reduced occurrence of relapse experienced during pregnancy. In
one embodiment, the individual is administered a dose of about 300
million placental stem cells. Dosage, however, can vary according
to the individual's physical characteristics, e.g., weight, and can
range from 1 million to 10 billion placental stem cells per does,
preferably between 10 million and 1 billion per dose, or between
100 million and 50 million placental stem cells per dose. The
administration is preferably intravenous, but can be by any
art-accepted route for the administration of live cells. In one
embodiment, the placental stem cells are from a cell bank.
6. EXAMPLES
6.1 Example 1
Oligodendrocyte Maintenance Medium
[0194] A representative medium for maintaining oligodendrocytes is
as follows. Preferred medium is a serum-free formulation optimized
for the maintenance of rodent OL lineage cells. A base media
(R1236) comprises DMEM high glucose supplemented with (Sigma), 1 mM
Na pyruvate, antibiotics (penicillin-streptomycin), 0.05 .mu.g/mL
insulin (to stimulate the glucose transporter), 100 .mu.g/mL
transferrin (iron uptake), 30 nM selenium (metabolic co-factor), 10
.mu.M forskolin (cAMP), 60 .mu.g/mL N-acetyl cystein (Redox,
survival), and 5 .mu.g/mL bovine serum albumin (carrier protein).
Rodent oligodendrocyte progenitor cells are maintained using R1236
supplemented with mitogens to promote proliferation and self
renewal (10 ng/mL PDGF-AA plus 5 ng/mL FGF2, or 20% v:v B104
conditioned media). To promote oligodendrocyte differentiation
mitogen-containing medium is replaced with R1236 containing 10
.mu.g/mL bovine insulin plus 5 .mu.g/mL T3 (triiodothreonine), both
of which are survival and maturation factors for rodent
oligodendrocytes. All growth factors included in the medium are
recombinant (human) polypeptides (R&D Inc), and the B 104-cm is
prepared from neuroblastoma cells (available from the ATCC)
cultured at 50% confluence then exposed to R1236 for 48 hrs. This
conditioned media is then filtered and aliquots stored at
-30.degree. C. until use. B104 is one of a number of neural cell
lines established by Dave Schubert at the Salk Institute (Schubert
et al., Nature 249:224-227 (1974)), and secretes factors that
support the survival and self-renewal of rodent oligodendrocyte
progenitor cells.
6.2 Example 2
Obtaining Stem Cells from Placenta by Enzymatic Digestion
[0195] An exemplary protocol for obtaining stem cells from
placental tissue by enzymatic digestion is as follows. Frozen
placental tissue (three pieces of approximately
.about.1.times.1.times.0.5 cm each) is obtained. The tissue is
umbilical cord, maternal surface of the placenta, or amniotic
membrane. Digestive enzymes used include trypsin-EDTA (0.25%, GIBCO
BRL); collagenase IA (Sigma), collagenase I (Worthington),
collagenase 1A (Sigma)+Trypsin-EDTA, collagenase 1
(Worthington)+Trypsin-EDTA, or Elastase+Collagenase I+Collagenase
IV+Daspase (Worthington). Digestion of placental tissue is as
follows. Tissue is minced in the presence of enzymes (1 g in 10 ml
in 50 ml tube) at 37.degree. C., 250 rpm shaking, tube position at
45.degree. angle for 1 hr (C25 Incubator Shaker, New Brunswick
Scientific, Edison, N.J., USA). The supernatant is then discarded.
The pellet is washed with 20 ml Hank's+5% FCS (3 times), and
re-suspended in 12 ml culture medium. 3 ml of the resulting
suspension are aliquoted into T-75 flasks containing 10 ml culture
medium each (four flasks per digestion). Optionally, 10 ml
Trypsin/EDTA is added for 30 min at 37.degree. C., with shaking at
250 rpm, with recentrifugation and an additional wash with 10 ml
Hank's+5% FCS. Cells are plated and cultured, selecting for
adherent cells.
6.3 Example 3
Oligodendrocyte Progenitor Lineage Assays
[0196] The emergence, maturation and differentiation of OPCs can be
determined by immunochemistry and transcript expression. For
immunochemistry, cells growing on glass coverslips are incubated in
culture media containing specific concentrations of growth factors.
Coverslips removed after 1-7 days are fixed with 4%
para-formaldehyde then characterized using lineage-specific
antibodies (Table 1). Staining is detected using secondary
antibodies coupled to fluorescent tags (Alexa Fluors, Molecular
Probes Inc) and visualized by fluorescence microscopy. Secondary
antibodies alone are used as a negative control. The proportion of
cells that are immuno-reactive will be determined by counting up to
200 cells per coverslip.
TABLE-US-00001 TABLE 1 Immunohistochemical reagents: Stage;
Antibody Specificity Target Source Reference nSC: Nestin Ms IgG
filament DSHB (Johe et al., 1996) NRP: e-NCAM Ms IgG filament DSHB
GRP: A2B5 Ms IgM gangliosides cond. media (Eisenbarth et al., 1979)
(ATCC) OPC: Olig2 rabbit IgG bHLH factor (H. Yakoo, JP) (Sun et
al., 2001) NG2 rabbit IgG proteoglycan Chemicon (Nishiyama et al.,
1996) Pdgfra goat IgG PDGF receptor R&D Inc. (Matsui et al.,
1989) Unc5b goat IgG Netrin receptor R&D Inc. (Lu et al., 2004)
O4 Ms IgM sulfatide CM (Bansal et al., 1989) OL: O1 Ms IgM GalC CM
(Raff et al., 1978) CNPase Ms IgG myelin Sigma (Pfeiffer et al.,
1993) MBP rabbit IgG CNPase Chemicon Inc (Pfeiffer et al., 1993)
myelin basic neuron NF-H neurofilament Virginia Lee astrocyte GFAP
rabbit IgG glial filament (Pfeiffer et al., 1993) CM = conditioned
medium
[0197] Immune histochemical studies are extended by analysis of
transcript expression under specific culture conditions. RNA
analysis uses Northern blot (McKinnon et al., 1990) and RT-PCR
(McKinnon et al., 1993b). Cells growing in 60 mm plates are
recovered and RNA is harvested with TRIzol Reagent (Gibco). For
RT-PCR, analysis is performed with 1 .mu.g RNA reverse transcribed
into cDNA (MoMuLV reverse transcriptase; 1:1 yield of cDNA). 50-100
ng cDNA is then used as a template for PCR amplification with Taq
Polymerase and synthetic primers chosen using the Primers Selection
Program as described (see. e.g., McKinnon et al., Glia 7: 245-254
(1993)). Primers are constructed to hybridize to transcripts
encoding lineage-specific oligodendrocyte and oligodendrocyte
precursor proteins. For new primer pairs a gradient (.+-.10.degree.
C.) is used to establish optimal amplification parameters. PCR
fragments are resolved by electrophoresis, visualized by EtBr
staining, and their identity is confirmed by automated DNA sequence
analysis (DNA core facility).
6.4 Example 4
Proliferation, Migration and Survival Assays
[0198] Proliferation assay: The ability of oligodendrocyte
progenitors to generate a mitogenic response to specific ligands is
measured in a quantitative .sup.3H-thymidine incorporation assay.
Cells are exposed to growth factors, in a dose range that brackets
the maximal response to FOP and PDGF, in order to determine the
half-maximal response. Responses range from a background of
500-1000 cpm (no growth factor) to 10,000 cpm (recombinant
PDGF-AA), and the assay is sufficiently sensitive to accurately
detect partial mitogenic responses. All cell proliferation assays
are optionally performed at least three independent times. For
qualitative assays, cells are exposed to mitogens with 50 .mu.M
BrdU (Sigma) present for the final 4 hrs, and DNA synthesis is
monitored by dual immunofluorescence for BrdU (Osterhout et al., J.
Neurosci. 17:9122-9132 (1997)) and a second lineage marker.
[0199] Proliferation assays are performed on cells that have been
removed from mitogens for 24 hrs prior to exposure to recombinant
growth factors to reduce background levels of DNA synthesis. The
proliferation assay described herein measures the response to
mitogens by increased DNA synthesis, with incorporation of
.sup.3H-thymidine during the final 4 hrs of this assay dependant on
exposure to growth factors. Cells in 96 well plates (2,000
cells/well) are incubated for 24 hrs in R1236 medium lacking growth
factors, then for 24 hrs in the presence of specific concentrations
of factors, with 0.5 .mu.Ci/ml .sup.3H-thymidine (Amersham) present
for the final 4 hrs. Nucleic acid is recovered using an automatic
harvester (Brandel) and the incorporated radioactivity measured by
scintillation counting. Assays are run in triplicate (three wells)
for each growth factor concentration.
[0200] Migration assay: The ability of OPCs to migrate (chemotaxis)
and their directional response in response to growth factors is
measured by cinematography. Quantitative assays use a modified
Boyden chamber assay (Armstrong et al., 1990). In this assay,
PDGF-AA mediated chemotaxis (4,000 cells/mm.sup.2) can be
distinguished from background migration (1,000 cells/mm.sup.2) and
from chemokinesis (random motility) by adding attractants to both
upper and lower wells of the chemotaxis chamber (which abolishes
chemotaxis but not chemokinesis) (see Armstrong et al., J.
Neurosci. Res. 27:400-407 (1990)).
[0201] Cells are cultured for 16 hrs in media lacking mitogens then
transferred into the top wells of a microchemotaxis chamber (20,000
cells/well) in defined medium, with a polycarbonate filter
separating them from the lower chamber containing media plus
attractant. Growth factors are given in triplicate wells for each
concentration, and the cells are incubated for 16 hrs at 37.degree.
C. The number of cells migrated per mm.sup.2 on the lower side of
the filter is determined by counting GFP-tagged cells, and total
migration is determined after staining the membrane with Dip-Quik
(American Scientific).
[0202] Survival assays: The survival of individual OPCs is measured
using the MTT assay, (Mosmann, 1983) and chromatin fragmentation
(nucleosome laddering) using a modified TUNEL assay (Gavrieli et
al., J. Cell. Biol. 119:493-501 (1992)) as described (Yasuda et
al., J. Neurosci. Res. 40:306-317 (1995)). Cells are cultured for
24 hrs in media containing bFGF, PDGF-AA, or without growth
factors, and the number of cells which incorporate MTT, or the
level of nick end labeling, are compared between mutant and wild
type OL cultures. The PI3K inhibitor wortmannin is used as a
positive control for cell death (Ebner et al., J. Neurosci. Res.
62:336-345 (2000)).
[0203] The MTT assay is performed on cells growing in 96 well
plates, and the proportion of labeled cells will be determined by
counting stained cells as previously described (Barres et al., Cell
70:31-46 (1992)) The ability of bFGF and PDGF-AA to prevent DNA
fragmentation is determined by analysis of chromatin DNA from cells
growing in the presence or absence of increasing concentrations of
these factors. For quantitative analysis, cells growing in 96 well
plates are incubated in PBS containing 4 units terminal transferase
(Promega Biotech.), 2 .mu.Ci [.alpha.-.sup.32P]-dideoxyATP
(Amersham), and 0.3% Triton X-100 for 60 min at 37.degree. C., then
the cell lysates are harvested on Whatman GF/C filters (Brandel
Cell Harvester) and .sup.32P-incorporation into 3'-ends of DNA is
determined by liquid scintillation counting. The assay is enzyme
dependent and gives a background of 10,000 cpm and incorporation of
100,000 cpm in cells cultured for 72 hrs in the presence of 1 .mu.M
staurosporine (Ebner, 2000) For qualitative analysis of nucleosome
laddering, DNA is isolated from cells growing in 35 mm dishes, end
labeled with terminal transferase and .sup.32P-ddATP in vitro, size
separated on agarose gels to resolve nucleosome-sized fragments,
and the incorporation of radioactivity is determined by
densitometry.
6.5 Example 5
Flow Cytometry
[0204] To perform intracellular staining for flow cytometry,
approximately 5.times.10.sup.5 PDSCs are permeabilized with 0.5 mL
of Beckman Coulter IntraPrep reagent for 15 minutes. After rinsing
with PBS, cells are incubated with primary antibody (1 g) on ice
for 30 minutes followed by two washes. Cells are resuspended in a
1:100 of secondary antibody and incubated for 30 minutes. After
staining, cells are washed twice and analyzed immediately on a
BeckmanCoulter XL-MCL flow cytometer. To evaluate protein
expression in untreated and IBMX-induced cells,
1.5.times.10.times.10.sup.4 cells are collected using FL1 (FITC)
and FL2 (CY3) signals. Dead cells and debris are eliminated by
using a high forward and orthogonal light scatter window or by
propidium iodine (PI) exclusion.
[0205] Mouse, rabbit, and donkey primary antibodies and final
dilutions are as follows: rabbit anti-nestin, 1:100 (BD
PharMingen); mouse anti-neuron specific enolase, 1:100 (Chemicon);
mouse anti-myelin/oligodendrocyte specific protein, 1:100 (DAKO);
mouse anti-neurofilament-L, 1:100 (DAKO); rabbit anti-glial
fibrillary acidic protein, 1:200 (DAKO); mouse anti-vimentin, 1:100
(BD PharMingen). The following antibodies, which are used in flow
cytometry experiments, are available from Becton Dickinson and are
used at a 1:10 dilution:anti-CD45, anti-CD34, anti-CD29, anti-CD10,
anti-HLA-1, anti-CD54, anti-CD90, anti-SH2, and anti-SH3.
[0206] Cells are incubated in DMEM containing 10% FCS on
polyornithine-coated glass coverslips (Sigma). Cells are fixed with
4% paraformaldehyde in PBS for 10 minutes and permeabilized for 10
minutes with 0.2% Triton X-100 in PBS at RT. Cells are then
incubated with the primary antibody for 30 minutes at 37.degree. C.
Following three washes with PBS, cells are incubated with either
fluorescein (FITC)-conjugated donkey anti-mouse IgG (Jackson
Laboratories) or Cy3-conjugated goat anti-rabbit IgG (Jackson
Laboratories), both at a 1:50 dilution for 30 minutes at 37.degree.
C. in the dark. Labeled cells are washed and mounted with
Vectashield mounting medium (Vector Laboratories).
* * * * *