U.S. patent application number 13/252142 was filed with the patent office on 2012-01-26 for treatment of neurodegenerative disease using placental stem cells.
Invention is credited to Robert J. Hariri.
Application Number | 20120020936 13/252142 |
Document ID | / |
Family ID | 22953866 |
Filed Date | 2012-01-26 |
United States Patent
Application |
20120020936 |
Kind Code |
A1 |
Hariri; Robert J. |
January 26, 2012 |
TREATMENT OF NEURODEGENERATIVE DISEASE USING PLACENTAL STEM
CELLS
Abstract
The present invention provides a method of extracting and
recovering embryonic-like stem cells, including, but not limited to
pluripotent or multipotent stem cells, from an exsanguinated human
placenta. A placenta is treated to remove residual umbilical cord
blood by perfusing an exsanguinated placenta, preferably with an
anticoagulant solution, to flush out residual cells. The residual
cells and perfusion liquid from the exsanguinated placenta are
collected, and the embryonic-like stem cells are separated from the
residual cells and perfusion liquid. The invention also provides a
method of utilizing the isolated and perfused placenta as a
bioreactor in which to propagate endogenous cells, including, but
not limited to, embryonic-like stem cells. The invention also
provides methods for propagation of exogenous cells in a placental
bioreactor and collecting the propagated exogenous cells and
bioactive molecules therefrom.
Inventors: |
Hariri; Robert J.; (Florham
Park, NJ) |
Family ID: |
22953866 |
Appl. No.: |
13/252142 |
Filed: |
October 3, 2011 |
Related U.S. Patent Documents
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Oct 27, 2008 |
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Current U.S.
Class: |
424/93.7 |
Current CPC
Class: |
A61K 48/00 20130101;
C12N 5/0647 20130101; A61P 5/14 20180101; C12M 29/10 20130101; C12N
2509/00 20130101; C12M 47/04 20130101; C12N 2510/02 20130101; A61K
2035/124 20130101; A61P 25/00 20180101; C12N 5/0605 20130101; A61P
3/00 20180101; A61P 9/00 20180101; C12N 5/0668 20130101; A61P 13/00
20180101; C12N 2533/90 20130101; A61M 2202/0462 20130101; A61P
13/12 20180101; A61P 17/00 20180101; A61P 9/10 20180101; A61P 27/02
20180101; A61P 43/00 20180101; A61M 2210/1466 20130101; A61P 17/02
20180101; A61P 13/02 20180101; A61P 25/28 20180101; A61P 29/00
20180101; A61K 35/50 20130101; A61P 7/06 20180101; A61K 35/12
20130101; A61P 11/00 20180101; C12N 5/0603 20130101; A01N 1/021
20130101; A61M 2210/1458 20130101; A61P 21/00 20180101; A61P 25/16
20180101; A61P 27/06 20180101; A61M 1/02 20130101 |
Class at
Publication: |
424/93.7 |
International
Class: |
A61K 35/12 20060101
A61K035/12; A61P 25/28 20060101 A61P025/28; A61P 25/16 20060101
A61P025/16 |
Claims
1-59. (canceled)
60. A method of treating an individual who has a neurodegenerative
disease, comprising administering to the individual therapeutically
effective amount of isolated human placental stem cells, wherein
said placental stem cells are: CD34.sup.-, CD10.sup.+, CD29.sup.+,
CD44.sup.+, CD45.sup.-, CD54.sup.+, CD90.sup.+, SH3.sup.+,
SH4.sup.+, SSEA3.sup.-, SSEA4.sup.-, wherein OCT-4 is octamer
binding protein 4; OCT-4.sup.+, CD34.sup.-, SSEA3.sup.- and
SSEA4.sup.-; OCT-4.sup.+ and CD34.sup.-, and additionally SH3.sup.+
or SH4.sup.-; or CD34.sup.- and one or more of CD29.sup.+,
CD45.sup.-, CD90.sup.+, SH2.sup.+, SH3.sup.+, SH4.sup.+, or MHC
Class. II.sup.-.
61. The method of claim 60, wherein said placental stem cells are
CD34.sup.-, CD10.sup.+, CD29.sup.+, CD44.sup.+, CD45.sup.-,
CD54.sup.+, CD90.sup.+, SH3.sup.+, SH4.sup.+, SSEA3, SSEA4.
62. The method of claim 60, wherein said placental stem cells are
OCT-4.sup.+, CD34.sup.-, SSEA3.sup.- and SSEA4.sup.-.
63. The method of claim 60, wherein said placental stem cells are
OCT-4.sup.+ and CD34.sup.-, and additionally SH3.sup.+ or
SH4.sup.+.
64. The method of claim 63, wherein said isolated placental stem
cells have at least one of the following characteristics:
CD10.sup.+, CD29.sup.+, CD44.sup.+, CD45.sup.-, CD54.sup.+,
CD90.sup.+, SSEA3.sup.-, or SSEA4.sup.-.
65. The method of claim 63, wherein said isolated placental stem
cells have at least the following characteristics: CD10.sup.+,
CD29.sup.+, CD44.sup.+, CD45.sup.-, CD54.sup.-, CD90.sup.+,
SSEA3.sup.-, and SSEA4.sup.-.
66. The method of claim 60, wherein said placental stem cells are
CD34.sup.- and one or more of CD29.sup.+, CD45.sup.-, CD90.sup.+,
SH2.sup.+, SH3.sup.+, SH4.sup.+, or MHC Class II.sup.-.
67. The method of claim 60, wherein said neurodegenerative disease
is Alzheimer's disease.
68. The method of claim 60, wherein said neurodegenerative disease
is Parkinson's disease.
Description
1. INTRODUCTION
[0001] The present invention relates to methods of exsanguinating
and perfusing a placenta following expulsion from the uterus, e.g.,
after birth. The present invention relates to methods of treating
and culturing an isolated placenta for the propagation of
embryonic-like stem cells originating from the placenta and
exogenous sources. The present invention further relates to the use
of a cultured placenta as a bioreactor to produce biological
materials or culture cells, tissues and organoids. The present
invention also relates to stem cell collection and propagation, and
in particular, to the collection of embryonic-like stem cells and
other multipotent stem cells from placentas. The present invention
relates to embryonic-like stem cells originating from a post-partum
placenta.
2. BACKGROUND OF THE INVENTION
[0002] There is considerable interest in the identification,
isolation and generation of human stem cells. Human stem cells are
totipotential or pluripotential precursor cells capable of
generating a variety of mature human cell lineages. This ability
serves as the basis for the cellular differentiation and
specialization necessary for organ and tissue development.
[0003] Recent success at transplanting such stem cells have
provided new clinical tools to reconstitute and/or supplement bone
marrow after myeloablation due to disease, exposure to toxic
chemical and/or radiation. Further evidence exists that
demonstrates that stem cells can be employed to repopulate many, if
not all, tissues and restore physiologic and anatomic
functionality. The application of stem cells in tissue engineering,
gene therapy delivery and cell therapeutics is also advancing
rapidly.
[0004] Many different types of mammalian stem cells have been
characterized. For example, embryonic stem cells, embryonic germ
cells, adult stem cells or other committed stem cells or progenitor
cells are known. Certain stem cells have not only been isolated and
characterized but have also been cultured under conditions to allow
differentiation to a limited extent. A basic problem remains,
however, in that obtaining sufficient quantities and populations of
human stem cells which are capable of differentiating into all cell
types is near impossible. Stem cells are in critically short
supply. These are important for the treatment of a wide variety of
disorders, including malignancies, inborn errors of metabolism,
hemoglobinopathies, and immunodeficiences. It would be highly
advantageous to have a source of more embryonic stem cells.
[0005] Obtaining sufficient numbers of human stem cells has been
problematic for several reasons. First, isolation of normally
occurring populations of stem cells in adult tissues has been
technically difficult and costly due, in part, to very limited
quantity found in blood or tissue. Secondly, procurement of these
cells from embryos or fetal tissue, including abortuses, has raised
religious and ethical concerns. The widely held belief that the
human embryo and fetus constitute independent life has prompted
governmental restrictions on the use of such sources for all
purposes, including medical research. Alternative sources that do
not require the use of cells procured from embryonic or fetal
tissue are therefore essential for further progress in the use of
stem cells clinically. There are, however, few viable alternative
sources of stem cells, particularly human stem cells, and thus
supply is limited. Furthermore, harvesting of stem cells from
alternative sources in adequate amounts for therapeutic and
research purposes is generally laborious, involving, e.g.,
harvesting of cells or tissues from a donor subject or patient,
culturing and/or propagation of cells in vitro, dissection,
etc.
[0006] For example, Caplan et al. (U.S. Pat. No. 5,486,359 entitled
"Human mesenchymal stem cells," issued Jan. 23, 1996), discloses
human mesenchymal stem cell (hMSC) compositions derived from the
bone marrow that serve as the progenitors for mesenchymal cell
lineages. Caplan et al. discloses that hMSCs are identified by
specific cell surface markers that are identified with monoclonal
antibodies. Homogeneous hMSC compositions are obtained by positive
selection of adherent marrow or periosteal cells that are free of
markers associated with either hematopoietic cell or differentiated
mesenchymal cells. These isolated mesenchymal cell populations
display epitopic characteristics associated with mesenchymal stem
cells, have the ability to regenerate in culture without
differentiating, and have the ability to differentiate into
specific mesenchymal lineages when either induced in vitro or
placed in vivo at the site of damaged tissue. The drawback of such
methods, however, is that they require harvesting of marrow or
periosteal cells from a donor, from which the MSCs must be
subsequently isolated.
[0007] Hu et al. (WO 00/73421 entitled "Methods of isolation,
cryopreservation, and therapeutic use of human amniotic epithelial
cells," published Dec. 7, 2000) discloses human amniotic epithelial
cells derived from placenta at delivery that are isolated,
cultured, cryopreserved for future use, or induced to
differentiate. According to Hu et al., a placenta is harvested
immediately after delivery and the amniotic membrane separated from
the chorion, e.g., by dissection. Amniotic epithelial cells are
isolated from the amniotic membrane according to standard cell
isolation techniques. The disclosed cells can be cultured in
various media, expanded in culture, cryopreserved, or induced to
differentiate. Hu et al. discloses that amniotic epithelial cells
are multipotential (and possibly pluripotential), and can
differentiate into epithelial tissues such as corneal surface
epithelium or vaginal epithelium. The drawback of such methods,
however, is that they are labor-intensive and the yield of stem
cells is very low. For example, to obtain sufficient numbers of
stem cells for typical therapeutic or research purposes, amniotic
epithelial cells must be first isolated from the amnion by
dissection and cell separation techniques, then cultured and
expanded in vitro.
[0008] Umbilical cord blood (cord blood) is a known alternative
source of hematopoietic progenitor stem cells. Stem cells from cord
blood are routinely cryopreserved for use in hematopoietic
reconstitution, a widely used therapeutic procedure used in bone
marrow and other related transplantations (see e.g., Boyse et al.,
U.S. Pat. No. 5,004,681, "Preservation of Fetal and Neonatal
Hematopoietin Stem and Progenitor Cells of the Blood", Boyse et
al., U.S. Pat. No. 5,192,553, entitled "Isolation and preservation
of fetal and neonatal hematopoietic stem and progenitor cells of
the blood and methods of therapeutic use", issued Mar. 9, 1993).
Conventional techniques for the collection of cord blood are based
on the use of a needle or cannula, which is used with the aid of
gravity to drain cord blood from (i.e., exsanguinate) the placenta
(Boyse et al., U.S. Pat. No. 5,192,553, issued Mar. 9, 1993; Boyse
et U.S. Pat. No. 5,004,681, issued Apr. 2, 1991; Anderson, U.S.
Pat. No. 5,372,581, entitled Method and apparatus for placental
blood collection, issued Dec. 13, 1994; Hessel et al., U.S. Pat.
No. 5,415,665, entitled Umbilical cord clamping, cutting, and blood
collecting device and method, issued May 16, 1995). The needle or
cannula is usually placed in the umbilical vein and the placenta is
gently massaged to aid in draining cord blood from the placenta.
Thereafter, however, the drained placenta has been regarded as
having no further use and has typically been discarded. A major
limitation of stem cell procurement from cord blood, moreover, has
been the frequently inadequate volume of cord blood obtained,
resulting in insufficient cell numbers to effectively reconstitute
bone marrow after transplantation.
[0009] Naughton et al. (U.S. Pat. No. 5,962,325 entitled
"Three-dimensional stromal tissue cultures" issued Oct. 5, 1999)
discloses that fetal cells, including fibroblast-like cells and
chondrocyte-progenitors, may be obtained from umbilical cord or
placenta tissue or umbilical cord blood. Naughton et al. (U.S. Pat.
No. 5,962,325) discloses that such fetal stromal cells can be used
to prepare a "generic" stromal or cartilaginous tissue. Naughton et
al. also discloses that a "specific" stromal tissue may be prepared
by inoculating a three-dimensional matrix with fibroblasts derived
from a particular individual who is later to receive the cells
and/or tissues grown in culture in accordance with the disclosed
methods. The drawback of such an approach however, is that it is
labor intensive. According to the methods disclosed in Naughton et
al., to recover fetal stromal cells from the umbilical cord or
placenta requires dissection of these tissues, mincing of the
tissue into pieces and disaggregation. Furthermore, to obtain
adequate amounts of the fetal stromal cells from umbilical cord
blood, as well as the umbilical cord and placenta, requires further
expansion ex vivo.
[0010] Currently available methods for the ex vivo expansion of
cell populations are also labor-intensive. For example, Emerson et
al. (Emerson et al., U.S. Pat. No. 6,326,198 entitled "Methods and
compositions for the ex vivo replication of stem cells, for the
optimization of hematopoietic progenitor cell cultures, and for
increasing the metabolism, GM-CSF secretion and/or M-6 secretion of
human stromal cells", issued Dec. 4, 2001); discloses methods, and
culture media conditions for ex vivo culturing of human stem cell
division and/or the optimization of human hematopoietic progenitor
stem cells. According to the disclosed methods, human stem cells or
progenitor cells derived from bone marrow are cultured in a liquid
culture medium that is replaced, preferably perfused, either
continuously or periodically, at a rate of 1 ml of medium per ml of
culture per about 24 to about 48 hour period. Metabolic products
are removed and depleted nutrients replenished while maintaining
the culture under physiologically acceptable conditions.
[0011] Kraus et al. (Kraus et al., U.S. Pat. No. 6,338,942,
entitled "Selective expansion of target cell populations", issued
Jan. 15, 2002) discloses that a predetermined target population of
cells may be selectively expanded by introducing a starting sample
of cells from cord blood or peripheral blood into a growth medium,
causing cells of the target cell population to divide, and
contacting the cells in the growth medium with a selection element
comprising binding molecules with specific affinity (such as a
monoclonal antibody for CD34) for a predetermined population of
cells (such as CD34 cells), so as to select cells of the
predetermined target population from other cells in the growth
medium.
[0012] Rodgers et al. (U.S. Pat. No. 6,335,195 entitled "Method for
promoting hematopoietic and mesenchymal cell proliferation and
differentiation," issued Jan. 1, 2002) discloses methods for ex
vivo culture of hematopoietic and mesenchymal stem cells and the
induction of lineage-specific cell proliferation and
differentiation by growth in the presence of angiotens ogen,
angiotensin I (AI), AI analogues, AI fragments and analogues
thereof, angiotensin II (AII), AII analogues, All fragments or
analogues thereof or AII AT.sub.2 type 2 receptor agonists, either
alone or in combination with other growth factors and cytokines.
The stem cells are derived from bone marrow, peripheral blood or
umbilical cord blood. The drawback of such methods, however, is
that such ex vivo methods for inducing proliferation and
differentiation of stem cells are time-consuming, as discussed
above, and also result in low yields of stem cells.
[0013] Naughton et al., (U.S. Pat. No. 6,022,743 entitled
"Three-dimensional culture of pancreatic parenchymal cells cultured
living stromal tissue prepared in vitro," issued Feb. 8, 2000)
discloses a tissue culture system in which stem cells or progenitor
cells (e.g., stromal cells such as those derived from umbilical
cord cells, placental cells, mesenchymal stem cells or fetal cells)
are propagated on three-dimensional support rather than as a
two-dimensional monolayer in, e.g., a culture vessel such as a
flask or dish.
[0014] Because of restrictions on the collection and use of stem
cells, and the inadequate numbers of cells typically collected from
cord blood, stem cells are in critically short supply. Stem cells
have the potential to be used in the treatment of a wide variety of
disorders, including malignancies, inborn errors of metabolism,
hemoglobinopathies, and immunodeficiencies. There is a critical
need for a readily accessible source of large numbers of human stem
cells for a variety of therapeutic and other medically related
purposes. The present invention addresses that need and others.
3. SUMMARY OF THE INVENTION
[0015] The present invention relates to a mammalian placenta,
preferably human, which following expulsion from the uterus has
been treated and cultured to produce multipotent stem cells (e.g.,
committed progenitor cells), embryonic-like stem cells and other
biological materials. In particular, the present invention provides
methods of perfusing and exsanguinating a placenta post birth. The
present invention provides methods of exsanguinating and perfusing
a placenta under sterile conditions for a period of at least two to
greater than forty-eight hours following expulsion of the placenta
from the uterus. In a preferred embodiment, the placenta is
perfused with a solution containing factors to enhance the
exsanguination, such as anticoagulant factors. In another
embodiment, the placenta is perfused with a solution containing
factors to enhance the sterile conditions, such as antimicrobial
and antiviral agents. In a preferred embodiment, the placenta is
perfused with a solution containing growth factors. Such solutions
which contains growth factors and other culture components but
without anticoagulants are referred to as culture solution.
[0016] In another preferred embodiment of the invention, the
placenta is perfused to remove blood, residual cells, proteins and
any other residual material. The placenta may be further processed
to remove material debris. Perfusion is normally continued with an
appropriate perfusate for at least two to more than twenty-four
hours. In several additional embodiments, of the invention, the
placenta is perfused for at least 2, 4, 6, 8, 10, 12, 14, 16, 18,
20, and 22 hours prior to the collection of stem cells. The
perfusate collected from any of these time points may also provide
a source of embryonic-like stem cells. It should be understood that
the first collection of blood from the placenta is referred to as
cord blood which contains predominantly CD34+ and CD38+
hematopoietic progenitor cells. Within the first twenty-four hours
of post-partum perfusion, CD34+ and CD38- hematopoietic progenitor
cells can be isolated from the placenta along with CD34+ and CD38-
cells. After about twenty-four hours of perfusion, CD34- and CD38-
cells can be isolated from the placenta along with the
aforementioned cells.
[0017] The present invention relates to an isolated placenta that
has been exsanguinated and perfused under sterile conditions. In a
preferred embodiment, the invention provides an isolated placenta
that has been exsanguinated and perfused to remove all residual
cells and cultured for a period of two to twenty four hours
following expulsion from the uterus. The present invention also
provides an isolated placenta that has been treated and cultured to
result in a viable organ capable of producing embryonic-like stem
cells, progenitor cells and other biological materials.
[0018] The present invention relates to a stem cell producing
apparatus which comprises a post-partum mammalian placenta which
has been exsanguinated and perfused, a means for incubating or
culturing the placenta; and a means for detecting stem cells. In
another embodiment, the apparatus of the invention further
comprises a collection device and/or a means for separating the
collected cells. In another embodiment, the apparatus of the
invention further comprises a means for monitoring and adjusting
the culture conditions and collection of cells.
[0019] The present invention also provides methods of incubating
and culturing an isolated exsanguinated placenta under the
appropriate conditions to allow for the production of
embryonic-like stem cells that originate from the placenta. In
accordance with the present invention, embryonic-like stem cells
are obtained from a placenta following expulsion from the uterus.
The placenta is exsanguinated and perfused for a period of at least
two to twenty four hours to remove all residual cells. The
exsanguinated placenta is then cultured under the appropriate
conditions to allow for the production of endogenous stem cells
originating from the placenta, including, but not limited to
embryonic-like stem cells, and pluripotent or multipotent stem
cells. In a preferred embodiment, the exsanguinated placenta is
cultured in the presence of growth factors, such as PDGF and
EGF.
[0020] The present invention further provides methods of treating
and culturing an isolated placenta for use as a bioreactor for the
propagation of endogenous stem cells originating from the placenta.
The present invention provides methods of treating and culturing an
isolated placenta for use as a bioreactor for the propagation of
exogenous cells and biological materials, e.g., antibodies,
proteins, oligonucleotides, hormones, viruses, cytokines and
enzymes. The present invention also provides propagation and
collection of embryonic-like stem cells and other pluripotent and
multipotent stem cells from placentas. The cultured placenta may be
used repeatedly as a bioreactor and may be cultured over a period
of days, months and even years. The cultured placenta may be
maintained by periodically or continuously removing a portion of a
culture medium or perfusion fluid that is introduced into the
system and from which the propagated cells or produced biological
materials may be recovered, and replaced with fresh medium or
perfusate liquid.
[0021] In another embodiment, the invention provides a method of
utilizing the isolated and perfused placenta as a bioreactor in
which to propagate endogenous cells, including, but not limited to,
embryonic-like stem cells, progenitor cells, pluripotent cells and
multipotent cells. The endogenous cells propagated in the placental
bioreactor may be collected, and/or bioactive molecules recovered
from the perfusate, culture medium or from the placenta cells
themselves.
[0022] In another embodiment, the invention provides a method of
utilizing the isolated and perfused placenta as a bioreactor in
which to propagate exogenous cells. In accordance with this
embodiment, the invention relates to an isolated placenta which
contains a cell not derived from the placenta, wherein the
engraftment of said cell into the placenta may stimulate the
placenta to produce embryonic-like stem cells or wherein the
engrafted cell produces signals, such a cytokines and growth
factors, which may stimulate the placenta to produce stem cells. In
accordance with this embodiment, the placenta may be engrafted with
cells not placental in origin obtained from the infant associated
with the placenta. In another embodiment, the placenta may be
engrafted with cells not placental in origin obtained from the
parents or siblings of the infant associated with the placenta. The
exogenous cells propagated in the placental bioreactor may be
collected, and/or bioactive molecules recovered from the perfusate,
culture medium or from the placenta cells themselves.
[0023] The present invention provides embryonic-like stem cells
that originate from a placenta. The embryonic-like stem cells of
the invention may be characterized by measuring changes in
morphology and cell surface markers using techniques such as flow
cytometry and immunocytochemistry, and measuring changes in gene
expression using techniques, such as PCR. In one embodiment of the
invention, such embryonic-like stem cells may be characterized by
the presence of the following cell surface markers: CD10+, CD29+,
CD34-, CD38-, CD44+, CD45-, CD54+, CD90+, SH2+, SH3+, SH4+, SSEA3-,
SSEA4-, OCT-4+, and ABC-p+. In a preferred embodiment, such
embryonic-like stem cells may be characterized by the presence of
cell surface markers OCT-4+ and APC-p+. Embryonic-like stem cells
originating from placenta the have characteristics of embryonic
stem cells but are not derived from the embryo. In other words, the
invention encompasses OCT-4+ and ABC-p+ cells that are
undifferentiated stem cells that are isolated from post-partum
perfused placenta. Such cells are as versatile (e.g., pluripotent)
as human embryonic stem cells. As mentioned above, a number of
different pluripotent or multipotent stem cells can be isolated
from the perfused placenta at different time points e.g.,
CD34+/CD38+, CD34+/CD38-, and CD34-/CD38- hematopoietic cells.
According to the methods of the invention, human placenta is used
post-birth as the source of embryonic-like stem cells.
[0024] In another embodiment, the invention provides a method for
isolating other embryonic-like and/or multipotent or pluripotent
stem cells from an extractant or perfusate of a exsanguinated
placenta.
[0025] The present invention relates to pharmaceutical compositions
which comprise the embryonic-like stem cells of the invention. The
present invention further relates to an isolated homogenous
population of human placental stem cells which has the potential to
differentiate into all cell types. The invention also encompasses
pharmaceutical compositions have high concentrations (or larger
populations) of homogenous hematopoietic stem cells including but
not limited to CD34+/CD38- cells; and CD34-/CD38- cells one or more
of these cell populations can be used with or as a mixture with
cord blood hematopoietic cells i.e., CD34+/CD38+ hematopoietic
cells for transplantation and other uses.
[0026] The stem cells obtained by the methods of the invention have
a multitude of uses in transplantation to treat or prevent disease.
In one embodiment of the invention, they are used to renovate and
repopulate tissues and organs, thereby replacing or repairing
diseased tissues, organs or portions thereof.
3.1. Definitions
[0027] As used herein, the term "bioreactor" refers to an ex vivo
system for propagating cells, producing or expressing biological
materials and growing or culturing cells tissues, organoids,
viruses, proteins, polynucleotides and microorganisms.
[0028] As used herein, the term "embryonic stem cell" refers to a
cell that is derived from the inner cell mass of a blastocyst
(e.g., a 4- to 5-day-old human embryo) and that is pluripotent.
[0029] As used herein, the term "embryonic-like stem cell" refers
to a cell that is not derived from the inner cell mass of a
blastocyst. As used herein, an "embryonic-like stem cell" may also
be referred to as a "placental stem cell." An embryonic-like stem
cell is preferably pluripotent. However, the stem cells which may
be obtained from the placenta include embryonic-like stem cells,
multipotent cells, and committed progenitor cells. According to the
methods of the invention, embryonic-like stem cells derived from
the placenta may be collected from the isolated placenta once it
has been exsanguinated and perfused for a period of time sufficient
to remove residual cells.
[0030] As used herein, the term "exsanguinated" or
"exsanguination," when used with respect to the placenta, refers to
the removal and/or draining of substantially all cord blood from
the placenta. In accordance with the present invention,
exsanguination of the placenta can be achieved by, for example, but
not by way of limitation, draining, gravity induced efflux,
massaging, squeezing, pumping, etc. In a preferred embodiment,
exsanguination of the placenta may further be achieved by
perfusing, rinsing or flushing the placenta with a fluid that may
or may not contain agents, such as anticoagulants, to aid in the
exsanguination of the placenta.
[0031] As used herein, the term "perfuse" or "perfusion" refers to
the act of pouring or passaging a fluid over or through an organ or
tissue, preferably the passage of fluid through an organ or tissue
with sufficient force or pressure to remove any residual cells,
e.g., non-attached cells from the organ or tissue. As used herein,
the term "perfusate" refers to the fluid collected following its
passage through an organ or tissue. In a preferred embodiment, the
perfusate contains one or more anticoagulants.
[0032] As used herein, the term "exogenous cell" refers to a
"foreign" cell, i.e., a heterologous cell (i.e., a "non-self` cell
derived from a source other than the placental donor) or autologous
cell (i.e., a "self' cell derived from the placental donor) that
is-derived from an organ or tissue other than the placenta.
[0033] As used herein, the term "organoid" refers to an aggregation
of one or more cell types assembled in superficial appearance or in
actual structure as any organ or gland of a mammalian body,
preferably the human body.
[0034] As used herein, the term "multipotent cell" refers to a cell
that has the capacity to grow into any of subset of the mammalian
body's approximately 260 cell types. Unlike a pluripotent cell, a
multipotent cell does not have the capacity to form all of the cell
types.
[0035] As used herein, the term "pluripotent cell" refers to a cell
that has complete differentiation versatility, i.e., the capacity
to grow into any of the mammalian body's approximately 260 cell
types. A pluripotent cell can be self-renewing, and can remain
dormant or quiescent within a tissue. Unlike a totipotent cell
(e.g., a fertilized, diploid egg cell), an embryonic stem cell
cannot usually form a new blastocyst.
[0036] As used herein, the term "progenitor cell" refers to a cell
that is committed to differentiate into a specific type of cell or
to form a specific type of tissue.
[0037] As used herein, the term "stem cell" refers to a master cell
that can reproduce indefinitely to form the specialized cells of
tissues and organs. A stem cell is a developmentally pluripotent or
multipotent cell. A stem cell can divide to produce two daughter
stem cells, or one daughter stem cell and one progenitor
("transit") cell, which then proliferates into the tissue's mature,
fully formed cells.
[0038] As used herein, the term "totipotent cell" refers to a cell
that is able to form a complete embryo (e.g., a blastocyst).
4. BRIEF DESCRIPTION OF THE FIGURES
[0039] FIG. 1 is a cross-sectional view of the cannulation of the
vein and artery of a placenta to perfuse the placenta and then
collect the perfusate.
[0040] FIGS. 2a-e are schematics showing the collection, clamping,
perfusion, collection and storage of an exsanguinated and perfused
placenta.
[0041] FIG. 3 is a cross-sectional schematic of a perfused placenta
in a device for use as a bioreactor.
[0042] FIG. 4 is a selection scheme for sorting cells, including
embryonic-like stem cells, retrieved from a perfused placenta.
5. DETAILED DESCRIPTION OF THE INVENTION
[0043] The applicant has unexpectedly discovered that the placenta
after birth contains quiescent cells which can be activated if the
placenta is properly processed after birth. For example, after
expulsion from the womb, the placenta is exsanguinated as quickly
as possible to prevent or minimize apoptosis. Subsequently, as soon
as possible after exsanguination the placenta is perfused to remove
blood, residual cells, proteins, factors and any other materials
present in the organ. Materials debris may also be removed from the
placenta. Perfusion is normally continued with an appropriate
perfusate for at least two to more than twenty-four hours. In
several additional embodiments the placenta is perfused for at
least 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22 hours. In other
words, this invention is based at least in part on the discovery
that the cells of a post-partum placenta can be activated by
exsanguination and perfusion for a sufficient amount of time.
Therefore, the placenta can readily be used as a rich and abundant
source of embryonic-like stem cells, which cells can be used for
research, including drug discovery, treatment and prevention of
diseases, in particular transplantation surgeries or therapies, and
the generation of committed cells, tissues and organoids.
[0044] Further, surprisingly and unexpectedly the human placental
stem cells produced by the exsanguinated, perfused and/or cultured
placenta are pluripotent stem cells that can readily be
differentiated into any desired cell type.
[0045] The present invention relates to methods of treating and
culturing an isolated placenta for use as a bioreactor for the
production and propagation of embryonic-like stem cells originating
from the placenta or from exogenous sources. The present invention
also relates to the use of a cultured placenta as a bioreactor to
produce biological materials, including, but not limited to,
antibodies, hormones, cytokines, growth factors and viruses. The
present invention also relates to methods of collecting and
isolating the stem cells and biological materials from the cultured
placenta.
[0046] The present invention relates to methods of perfusing and
exsanguinating an isolated placenta once it has been expunged from
a uterus, to remove all residual cells. The invention further
relates to culturing the isolated and exsanguinated placenta under
the appropriate conditions to allow for the production and
propagation of embryonic-like stem cells.
[0047] The present invention provides a method of extracting and
recovering embryonic-like stem cells, including, but not limited to
pluripotent or multipotent stem cells, from an exsanguinated human
placenta. Embryonic-like stem cells have characteristics of
embryonic stem cells but are not derived from the embryo. Such
cells are as versatile (e.g., pluripotent) as human embryonic stem
cells. According to the methods of the invention, human placenta is
used post-birth as the source of embryonic-like stem cells.
[0048] According to the methods of the invention embryonic-like
stem cells are extracted from a drained placenta by means of a
perfusion technique that utilizes either or both of the umbilical
artery and umbilical vein. The placenta is preferably drained by
exsanguination and collection of residual blood (e.g., residual
umbilical cord blood). The drained placenta is then processed in
such a mariner as to establish an ex vivo, natural bioreactor
environment in which resident embryonic-like stem cells within the
parenchyma and extravascular space are recruited. The
embryonic-like stem cells migrate into the drained, empty
microcirculation where, according to the methods of the invention,
they are collected, preferably by washing into a collecting vessel
by perfusion.
5.1. Methods of Isolating and Culturing Placenta
5.1.1. Pretreatment of Placenta
[0049] According to the methods of the invention, a human placenta
is recovered shortly after its expulsion after birth and, in
certain embodiments, the cord blood in the placenta is recovered.
In certain embodiments, the placenta is subjected to a conventional
cord blood recovery process. Such cord blood recovery may be
obtained commercially, e.g., LifeBank Inc., Cedar Knolls, N.J.,
ViaCord, Cord Blood Registry and Cryocell. The cord blood can be
drained shortly after expulsion of the placenta.
[0050] Postpartum the placenta is drained of cord blood. The
placenta stored may be 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.
[0051] Typically, a placenta is transported from the delivery or
birthing room to another location, e.g., a laboratory, for recovery
of the cord blood and/or drainage and perfusion. 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, as shown in FIGS.
2a-e. Preferably, the placenta is delivered to the laboratory four
to twenty-four hours following delivery.
[0052] The placenta is preferably recovered after expulsion under
aseptic conditions, and 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 (1% w/w in 1:1000 solution). The drained placenta is
preferably stored for no more than 36 hours before the
embryonic-like stem cells are collected. The solution which is used
to perfuse the placenta to remove residual cells can be the same
solution used to perfuse and culture the placenta for the recovery
of stem cells. Any of these perfusates may be collected and used as
a source of embryonic-like stem cells.
[0053] 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.
[0054] Conventional techniques for the collection of cord blood may
be used. Typically a needle or cannula is used, with the aid of
gravity, to drain cord blood from (i.e., exsanguinate) the placenta
(Boyse et al., U.S. Pat. No. 5,192,553, issued Mar. 9, 1993; Boyse
et al., U.S. Pat. No. 5,004,681, issued Apr. 2, 1991; Anderson,
U.S. Pat. No. 5,372,581, entitled Method and apparatus for
placental blood collection, issued Dec. 13, 1994; Hessel et al.,
U.S. Pat. No. 5,415,665, entitled Umbilical cord clamping, cutting,
and blood collecting device and method, issued May 16, 1995). The
needle or cannula is usually placed in the umbilical vein and the
placenta is gently massaged to aid in draining cord blood from the
placenta.
[0055] In a preferred embodiment, the placenta is recovered from a
patient by informed consent and a complete medical history of the
patient prior to, during and after pregnancy is also taken and is
associated with the placenta. These medical records can be used to
coordinate subsequent use of the placenta or the stem cells
harvested therefrom. For example, the human placental stem cells
can then easily be used for personalized medicine for the infant in
question, the parents, siblings or other relatives. Indeed, the
human placental stem cells are more versatile than cord blood.
However, it should be noted that the invention includes the
addition of human placental stem cells produced by the
exsanguinated, perfused and/or cultured placenta to cord blood from
the same or different placenta and umbilical cord. The resulting
cord blood will have an increased concentration/population of human
stem cells and thereby is more useful for transplantation e.g. for
bone marrow transplantations.
5.1.2. Exsanguination of Placenta and Removal of Residual Cells
[0056] The invention provides a method for recovery of stem or
progenitor cells, including, but not limited to embryonic-like stem
cells, from a placenta that is exsanguinated, i.e., completely
drained of the cord blood remaining after birth and/or a
conventional cord blood recovery procedure. According to the
methods of the invention, the placenta is exsanguinated and
perfused with a suitable aqueous perfusion fluid, such as an
aqueous isotonic fluid in which an anticoagulant (e.g., heparin,
warfarin sodium) is dissolved. Such aqueous isotonic fluids for
perfusion are well known in the art, and include, e.g., a 0.9 N
sodium chloride solution. The perfusion fluid preferably comprises
the anticoagulant, such as heparin or warfarin sodium, at a
concentration that is sufficient to prevent the formation of clots
of any residual cord blood. In a specific embodiment, a
concentration of from 1 to 100 units of heparin is employed,
preferably a concentration of 1 to 10 units of heparin per ml is
employed. In one embodiment, apoptosis inhibitors, such as free
radical scavengers, in particular oxygen free radical scavengers,
can be used during and immediately after exsanguination and then
these agents can be washed from the placenta. In accordance with
this embodiment of the invention, the isolated placenta may be
stored under hypothermic conditions in order to prevent or inhibit
apoptosis.
[0057] According to the methods of the invention, the placenta is
exsanguinated by passage of the perfusion fluid through either or
both of the umbilical artery and umbilical vein, using a gravity
flow into the placenta. 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. In a
preferred embodiment, the umbilical artery and the umbilical vein
are connected simultaneously, as shown in FIG. 1, to a pipette that
is connected via a flexible connector to a reservoir of the
perfusion fluid. The perfusion fluid is passed into the umbilical
vein and artery and 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 fluid may also be introduced
through the umbilical cord opening and allowed to flow or perculate
out of openings in the wall of the placenta which interfaced with
the maternal uterine wall.
[0058] In a preferred 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.
[0059] In one embodiment, a sufficient amount of perfusion fluid is
used that will result in removal of all residual cord blood and
subsequent collection or recovery of placental cells, including but
not limited to embryonic-like stem cells and progenitor cells, that
remain in the placenta after removal of the cord blood.
[0060] It has been observed that when perfusion fluid is first
collected from a placenta during the exsanguination process, the
fluid is colored with residual red blood cells of the cord blood.
The perfusion fluid tends to become clearer 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 exsanguinate the placenta and to recover an initial
population of embryonic-like cells from the placenta, but more or
less perfusion fluid may be used depending on the observed
results.
5.1.3. Culturing Placenta
[0061] After exsanguination and a sufficient time of perfusion of
the placenta, the embryonic-like stem cells are observed to migrate
into the exsanguinated and perfused microcirculation of the
placenta where, according to the methods of the invention, they are
collected, preferably by washing into a collecting vessel by
perfusion. 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.
[0062] In certain embodiments of the invention, the drained,
exsanguinated placenta is cultured as a bioreactor, i.e., an ex
vivo system for propagating cells or producing biological
materials. The number of propagated cells or level of biological
material produced in the placental bioreactor is maintained in a
continuous state of balanced growth by periodically or continuously
removing a portion of a culture medium or perfusion fluid that is
introduced into the placental bioreactor, and from which the
propagated cells or the produced biological materials may be
recovered. Fresh medium or perfusion fluid is introduced at the
same rate or in the same amount.
[0063] The number and type of cells propagated may easily be
monitored 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, or by measuring changes in gene expression
using techniques well known in the art, such as PCR and gene
expression profiling.
[0064] In one embodiment, the cells 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.
[0065] 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
which specifically recognizes a cell-solid phase surface molecule
or hapten. A magnetic field is then applied, to physically
manipulate the selected beads. The beads are then mixed with the
cells to allow binding. Cells are then passed through a magnetic
field to separate out cells having cell surface markers. 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.
[0066] In preferred embodiments, the placenta to be used as a
bioreactor is exsanguinated and washed under sterile conditions so
that any adherent coagulated and non-adherent cellular contaminants
are removed. The placenta is then cultured or cultivated under
aseptic conditions in a container or other suitable vessel, and
perfused with perfusate 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).
[0067] The effluent perfusate comprises both circulated perfusate,
which has flowed through the placental circulation, and
extravasated perfusate, which exudes from or passes through the
walls of the blood vessels into the surrounding tissues of the
placenta. The effluent perfusate is collected, and preferably, both
the circulated and extravasated perfusates are collected,
preferably in a sterile receptacle. Alterations in the conditions
in which the placenta is maintained and the nature of the perfusate
can be made to modulate the volume and composition of the effluent
perfusate.
[0068] Cell types are then isolated from the collected perfusate by
employing techniques known by those skilled in the art, such as for
example, but not limited to density gradient centrifugation, magnet
cell separation, flow cytometry, affinity cell separation or
differential adhesion techniques.
[0069] In one embodiment, a placenta is placed in a sterile basin
and washed with 500 ml of phosphate-buffered normal saline. The
wash fluid is then discarded. The umbilical vein is then 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, as shown in FIG. 3. The container containing
the placenta is then covered and the placenta is maintained at room
temperature (20-25.degree. C.) for a desired period of time,
preferably from 2 to 24 hours, and preferably, no longer than 48
hours. The placenta may be perfused continually, with equal volumes
of perfusate introduced and effluent perfusate removed or
collected. Alternatively, the placenta may be perfused
periodically, e.g., at every 2 hours or at 4, 8, 12, and 24 hours,
with a volume of perfusate, e.g., 100 ml of perfusate (sterile
normal saline supplemented with or without 1000 u/l heparin and/or
EDTA and/or CPDA (creatine phosphate dextrose)). In the case of
periodic perfusion, preferably equal volumes of perfusate are
introduced and removed from the culture environment of the
placenta, so that a stable volume of perfusate bathes the placenta
at all times.
[0070] The effluent perfusate that escapes the placenta, e.g., at
the opposite surface of the placenta, is collected and processed to
isolate embryonic-like stem cells, progenitor cells or other cells
of interest.
[0071] Various media may be used as perfusion fluid for culturing
or cultivating the placenta, such as DMEM, F-12, M199, RPMI,
Fisher's, Iscore's, McCoy's and combinations thereof, supplemented
with fetal bovine serum (FBS), whole human serum (WHS), or human
umbilical cord serum collected at the time of delivery of the
placenta. The same perfusion fluid used to exsanguinate the
placenta of residual cord blood may be used to culture or cultivate
the placenta, without the addition of anticoagulant agents.
[0072] In certain embodiments, the embryonic-like stem cells are
induced to propagate in the placenta bioreactor by introduction of
nutrients, hormones, vitamins, growth factors, or any combination
thereof, into the perfusion solution. Serum and other growth
factors may be added to the propagation perfusion solution or
medium. Growth factors are usually proteins and include, but are
not limited to: cytokines, lymphokines, interferons, colony
stimulating factors (CSF's), interferons, chemokines, and
interleukins. Other growth factors that may be used include
recombinant human hematopoietic growth factors including ligands,
stem cell factors, thrombopoeitin (Tpo), granulocyte
colony-stimulating factor (G-CSF), leukemia inhibitory factor,
basic fibroblast growth factor, placenta derived growth factor and
epidermal growth factor.
[0073] The growth factors introduced into the perfusion solution
can stimulate the propagation of undifferentiated embryonic-like
stem cells, committed progenitor cells, or differentiated cells
(e.g., differentiated hematopoietic cells). The growth factors can
stimulate the production of biological materials and bioactive
molecules including, but not limited to, immunoglobulins, hormones,
enzymes or growth factors as previously described.
[0074] In one embodiment of the invention, the placenta is used as
a bioreactor for propagating endogenous cells (i.e., cells that
originate from the placenta), including but not limited to, various
kinds of pluripotent and/or totipotent embryonic-like stem cells
and lymphocytes. To use the placenta as a bioreactor, it may be
cultured for varying periods of time under sterile conditions by
perfusion with perfusate solution as disclosed herein. In specific
embodiments, the placenta is cultured for at least 12, 24, 36, or
48 hours, or for 3-5 days, 6-10 days, or for one to two weeks. In a
preferred embodiment, the placenta is cultured for 48 hours. The
cultured placenta should be "fed" periodically to remove the spent
media, depopulate released cells, and add fresh media. The cultured
placenta should be stored under sterile conditions to reduce the
possibility of contamination, and maintained under intermittent and
periodic pressurization to create conditions that maintain an
adequate supply of nutrients to the cells of the placenta. It
should be recognized that the perfusing and culturing of the
placenta can be both automated and computerized for efficiency and
increased capacity.
[0075] In another embodiment, the placenta is processed to remove
all endogenous proliferating cells, such as embryonic-like stem
cells, and to allow foreign (i.e., exogenous) cells to be
introduced and propagated in the environment of the perfused
placenta. The invention contemplates a large variety of stem or
progenitor cells that can be cultured in the placental bioreactor,
including, but not limited to, embryonic-like stem cells,
mesenchymal stem cells, stromal cells, endothelial cells,
hepatocytes, keratinocytes, and stem or progenitor cells for a
particular cell type, tissue or organ, including but not limited to
neurons, myelin, muscle, blood, bone marrow, skin, heart,
connective tissue, lung, kidney, liver, and pancreas (e.g.,
pancreatic islet cells).
[0076] Sources of mesenchymal stem cells include bone marrow,
embryonic yolk sac, placenta, umbilical cord, fetal and adolescent
skin, and blood. Bone marrow cells may be obtained from iliac
crest, femora, tibiae, spine, rib or other medullary spaces.
[0077] Methods for the selective destruction, ablation or removal
of proliferating or rapidly dividing cells from a tissue or organ
are well-known in the art, e.g., methods of cancer or tumor
treatment. For example, the perfused placenta may be irradiated
with electromagnetic, UV, X-ray, gamma- or beta-radiation to
eradicate all remaining viable, endogenous cells. The foreign cells
to be propagated are introduced into the irradiated placental
bioreactor, for example, by perfusion.
5.2. Collection of Cells from the Placenta
[0078] As disclosed above, after exsanguination and perfusion of
the placenta, embryonic-like stem cells migrate into the drained,
empty microcirculation where, according to the methods of the
invention, they are collected, preferably by collecting the
effluent perfusate in a collecting vessel.
[0079] In preferred embodiments, cells cultured in the placenta are
isolated from the effluent perfusate using techniques known by
those skilled in the art, such as, for example, density gradient
centrifugation, magnet cell separation, flow cytometry, or other
cell separation or sorting methods well known in the art, and
sorted, for example, according to the scheme shown in FIG. 4.
[0080] In a specific embodiment, cells collected from the placenta
are recovered from the effluent perfusate by centrifugation at
5000.times.g for 15 minutes at room temperature, which separates
cells from contaminating debris and platelets. The cell pellets are
resuspended in IMDM serum-free medium containing 2 U/ml heparin and
2 mM EDTA (GibcoBRL, NY). The total mononuclear cell fraction was
isolated using Lymphoprep (Nycomed Pharma, Oslo, Norway) according
to the manufacturer's recommended procedure and the mononuclear
cell fraction was resuspended. Cells were counted using a
hemocytometer. Viability was evaluated by trypan blue exclusion.
Isolation of cells is achieved by "differential trypsinization,"
using a solution of 0.05% trypsin with 0.2% EDTA (Sigma, St. Louis
Mo.). Differential trypsinization was possible because
fibroblastoid cells detached from plastic surfaces within about
five minutes whereas the other adherent populations required more
than 20-30 minutes incubation. The detached fibroblastoid cells
were harvested following trypsinization and trypsin neutralization,
using Trypsin Neutralizing Solution (TNS, BioWhittaker). The cells
were washed in H.DMEM and resuspended in MSCGM.
[0081] In another embodiment, the isolated placenta is perfused for
a period of time without collecting the perfusate, such that the
placenta may be perfused for 2, 4, 6, 8, 10, 12, 20 and 24 hours or
even days before the perfusate is collected.
[0082] In another embodiment, cells cultured in the placenta
bioreactor are isolated from the placenta by physically dissecting
the cells away from the placenta.
[0083] In another embodiment, cells cultured in the placenta
bioreactor are isolated from the placenta by dissociating the
tissues of the placenta or a portion thereof, and recovering the
cultured cells by art-known cell separation or sorting methods such
as density gradient centrifugation, magnet cell separation, flow
cytometry, etc.
[0084] In a preferred embodiment, perfusion of the placenta and
collection of effluent perfusate is repeated once or twice during
the culturing of the placenta, until the number of recovered
nucleated cells falls below 100 cells/ml. The perfusates are pooled
and subjected to light centrifugation to remove platelets, debris
and de-nucleated cell membranes. The nucleated cells are then
isolated by Ficoll-Hypaque density gradient centrifugation and
after washing, resuspended in H.DMEM. For isolation of the adherent
cells, aliquots of 5-10.times.10.sup.6 cells are placed in each of
several T-75 flasks and cultured with commercially available
Mesenchymal Stem Cell Growth Medium (MSCGM) obtained from
BioWhittaker, and placed in a tissue culture incubator (37.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.
[0085] In other embodiments, the cells collected from the placenta
are cryopreserved for use at a later time. Methods for
cryopreservation of cells, such as stem cells, are well known in
the art, for example, cryopreservation using the methods of Boyse
et al. (U.S. Pat. No. 5,192,553, issued Mar. 9, 1993) or Hu et al.
(WO 00/73421, published Dec. 7, 2000).
5.3. Cell Populations Obtained from of Cultured in Placenta
[0086] Embryonic-like stem cells obtained in accordance with the
methods of the invention may include pluripotent cells, i.e., cells
that have complete differentiation versatility, that are
self-renewing, and can remain dormant or quiescent within tissue.
The stem cells which may be obtained from the placenta include
embryonic-like stem cells, multipotent cells, committed progenitor
cells, and fibroblastoid cells.
[0087] The first collection of blood from the placenta is referred
to as cord blood which contains predominantly CD34+ and CD38+
hematopoietic progenitor cells. Within the first twenty-four hours
of post-partum perfusion, high concentrations of CD34+ and CD38-
hematopoietic progenitor cells may be isolated from the placenta,
along with high concentrations of CD34- and CD38+ hematopoietic
progenitor cells. After about twenty-four hours of perfusion, high
concentrations of CD34- and CD38- cells can be isolated from the
placenta along with the aforementioned cells. The isolated perfused
placenta of the invention provides a source of large quantities of
stem cells enriched for CD34+ and CD38- stem cells and CD34- and
CD38+ stem cells. The isolated placenta which has been perfused for
twenty-four hours or more provides a source of large quantities of
stem cells enriched for CD34- and CD38- stem cells.
[0088] In a preferred embodiment, embryonic-like stem cells
obtained by the methods of the invention are viable, quiescent,
pluripotent stem cells that exist within a full-term human placenta
and that can be recovered following successful birth and placental
expulsion, resulting in the recovery of as many as one billion
nucleated cells, which yield 50-100 million multipotent and
pluripotent stem cells.
[0089] The human placental stern cells provided by the placenta are
surprisingly embryonic-like, for example, the presence of the
following cell surface markers have been identified for these
cells: SSEA3-, SSEA4-, OCT-4+ and ABC-p.sup.+. Preferably, the
embryonic-like stem cells of the invention are characterized by the
presence of OCT-4+ and ABC-p+ cell surface markers. Thus, the
invention encompasses stem cells which have not been isolated or
otherwise obtained from an embryonic source but which can be
identified by the following markers: SSAE3-, SSAE4-, OCT-4+ and
ABC-p+. In one embodiment, the human placental stem cells do not
express MHC Class 2 antigens.
[0090] The stem cells isolated from the placenta are homogenous,
and sterile. Further, the stem cells are readily obtained in a form
suitable for administration to humans, i.e., they are of
pharmaceutical grade.
[0091] Preferred embryonic-like stem cells obtained by the methods
of the invention may be identified by the presence of the following
cell surface markers: OCT-4+ and ABC-pt. Further, the invention
encompasses embryonic stem cells having the following markers:
CD10+, CD38-, CD29+, CD34-, CD44+, CD45-, CD54+, CD90+, SH2+, SH3+,
SH4+, SSEA3-, SSEA4-, OCT-4+, and ABC-p+. Such cell surface markers
are routinely determined according to methods well known in the
art, e.g. by flow cytometry, followed by washing and staining with
an anti-cell surface marker antibody. For example, to determine the
presence of CD-34 or CD-38, cells may be washed in PBS and then
double-stained with anti-CD34 phycoerythrin and anti-CD38
fluorescein isothiocyanate (Becton Dickinson, Mountain View,
Calif.).
[0092] In another embodiment, cells cultured in the placenta
bioreactor are identified and characterized by a colony forming
unit assay, which is commonly known in the art, such as Mesen
Cult.TM. medium (stem cell Technologies, Inc., Vancouver British
Columbia)
[0093] The embryonic-like stem cells obtained by the methods of the
invention may be induced to differentiate along specific cell
lineages, including adipogenic, chondrogenic, osteogenic,
hematopoietic, myogenic, vasogenic, neurogenic, and hepatogenic. In
certain embodiments, embryonic-like stem cells obtained according
to the methods of the invention are induced to differentiate for
use in transplantation and ex vivo treatment protocols. In certain
embodiments, embryonic-like stem cells obtained by the methods of
the invention are induced to differentiate into a particular cell
type and genetically engineered to provide a therapeutic gene
product. In a specific embodiment, embryonic-like stem cells
obtained by the methods of the invention are incubated with a
compound in vitro that induces it to differentiate, followed by
direct transplantation of the differentiated cells to a subject.
Thus, the invention encompasses methods of differentiating the
human placental stem cells using standard culturing media. Further,
the invention encompasses hematopoietic cells, neuron cells,
fibroblast cells, strand cells, mesenchymal cells and hepatic
cells.
[0094] Embryonic-like stem cells may also be further cultured after
collection from the placenta using methods well known in the art,
for example, by culturing on feeder cells, such as irradiated
fibroblasts, obtained from the same placenta as the embryonic-like
stem cells or from other human or nonhuman sources, or in
conditioned media obtained from cultures of such feeder cells, in
order to obtain continued long-term cultures of embryonic-like stem
cells. The embryonic-like stem cells may also be expanded, either
within the placenta before collection from the placental bioreactor
or in vitro after recovery from the placenta. In certain
embodiments, the embryonic-like stem cells to be expanded are
exposed to, or cultured in the presence of, an agent that
suppresses cellular differentiation. Such agents are well-known in
the art and include, but are not limited to, human Delta-1 and
human Serrate-1 polypeptides (see, Sakano et al., U.S. Pat. No.
6,337,387 entitled "Differentiation-suppressive polypeptide",
issued Jan. 8, 2002), leukemia inhibitory factor (LIF) and stem
cell factor. Methods for the expansion of cell populations are also
known in the art (see e.g., Emerson et al., U.S. Pat. No. 6,326,198
entitled "Methods and compositions for the ex vivo replication of
stem cells, for the optimization of hematopoietic progenitor cell
cultures, and for increasing the metabolism, GM-CSF secretion
and/or IL-6 secretion of human stromal cells", issued Dec. 4, 2001;
Kraus et al., U.S. Pat. No. 6,338,942, entitled "Selective
expansion of target cell populations", issued Jan. 15, 2002).
[0095] The embryonic-like stem cells may 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.
[0096] In certain embodiments, the differentiation of stem cells or
progenitor cells that are cultivated in the exsanguinated, perfused
and/or cultured placenta is modulated using an agent or
pharmaceutical compositions comprising a dose and/or doses
effective upon single or multiple administration, to exert an
effect sufficient to inhibit, modulate and/or regulate the
differentiation of a cell collected from the placenta.
[0097] Agents that can induce stem or progenitor cell
differentiation are well known in the art and include, but are not
limited to, Ca.sup.2+, EGF, aFGF, bFGF, PDGF, keratinocyte growth
factor (KGF), TGF-.beta., cytokines (e.g., IL-1.alpha., IL-1.beta.,
IFN-.gamma., TFN), retinoic acid, transferrin, hormones (e.g.,
androgen, estrogen, insulin, prolactin, triiodothyronine,
hydrocortisone, dexamethasone), sodium butyrate, TPA, DMSO, NMF,
DMF, matrix elements (e.g., collagen, laminin, heparan sulfate,
Matrigel.TM.), or combinations thereof.
[0098] Agents that suppress cellular differentiation are also
well-known in the art and include, but are not limited to, human
Delta-1 and human Serrate-1 polypeptides (see, Sakano et al., U.S.
Pat. No. 6,337,387 entitled "Differentiation-suppressive
polypeptide", issued Jan. 8, 2002), leukemia inhibitory factor
(LIF), and stem cell factor.
[0099] The agent used to modulate differentiation can be introduced
into the placental bioreactor to induce differentiation of the
cells being cultured in the placenta. Alternatively, the agent can
be used to modulate differentiation in vitro after the cells have
been collected or removed from the placenta.
[0100] Determination that a stem cell has differentiated into a
particular cell type may be accomplished by methods well-known in
the art, e.g., measuring changes in morphology and cell surface
markers using techniques such as flow cytometry or
immunocytochemistry (e.g., staining cells with tissue-specific or
cell-marker specific antibodies), by examination of the morphology
of cells using light or confocal microscopy, or by measuring
changes in gene expression using techniques well known in the art,
such as PCR and gene-expression profiling.
[0101] In another embodiment, the cells cultured in the placenta
are stimulated to produce bioactive molecules, such as
immunoglobulins, hormones, enzymes.
[0102] In another embodiment, the cells cultured in the placenta
are stimulated to proliferate, for example, by administration of
erythropoietin, cytokines, lymphokines, interferons, colony
stimulating factors (CSF's), interferons, chemokines, interleukins,
recombinant human hematopoietic growth factors including ligands,
stem cell factors, thrombopoeitin (Tpo), interleukins, and
granulocyte colony-stimulating factor (G-CSF) or other growth
factors.
[0103] In another embodiment, cells cultured in the placenta are
genetically engineered either prior to, or after collection from,
the placenta, using, for example, a viral vector such as an
adenoviral or retroviral vector, or by using mechanical means such
as liposomal or chemical mediated uptake of the DNA.
[0104] A vector containing a transgene can be introduced into a
cell of interest by methods well known in the art, e.g.,
transfection, transformation, transduction, electroporation,
infection, microinjection, cell fusion, DEAE dextran, calcium
phosphate precipitation, liposomes, LIPOFECTIN.TM., lysosome
fusion, synthetic cationic lipids, use of a gene gun or a DNA
vector transporter, such that the transgene is transmitted to
daughter cells, e.g., the daughter embryonic-like stem cells or
progenitor cells produced by the division of an embryonic-like stem
cell. For various techniques for transformation or transfection of
mammalian cells, see Keown et al., 1990, Methods Enzymol. 185:
527-37; Sambrook et al., 2001, Molecular Cloning, A Laboratory
Manual, Third Edition, Cold Spring Harbor Laboratory Press,
N.Y.
[0105] Preferably, the transgene is introduced using any technique,
so long as it is not destructive to the cell's nuclear membrane or
other existing cellular or genetic structures. In certain
embodiments, the transgene is inserted into the nucleic genetic
material by microinjection. Microinjection of cells and cellular
structures is commonly known and practiced in the art.
[0106] For stable transfection of cultured mammalian cells, such as
cells culture in a placenta, only a small fraction of cells may
integrate the foreign DNA into their genome. The efficiency of
integration depends upon the vector and transfection technique
used. In order to identify and select integrants, a gene that
encodes a selectable marker (e.g., for resistance to antibiotics)
is generally introduced into the host embryonic-like stem cell
along with the gene sequence of interest. Preferred selectable
markers include those that confer resistance to drugs, such as
G418, hygromycin and methotrexate. Cells stably transfected with
the introduced nucleic acid can be identified by drug selection
(e.g., cells that have incorporated the selectable marker gene will
survive, while the other cells die). Such methods are particularly
useful in methods involving homologous recombination in mammalian
cells (e.g., in embryonic-like stem cells) prior to introduction or
transplantation of the recombinant cells into a subject or
patient.
[0107] A number of selection systems may be used to select
transformed host embryonic-like cells. In particular, the vector
may contain certain detectable or selectable markers. Other methods
of selection include but are not limited to selecting for another
marker such as: the herpes simplex virus thymidine kinase (Wigler
et al., 1977, Cell 11: 223), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska and Szybalski, 1962, Proc.
Natl. Acad. Sci. USA 48: 2026), and adenine
phosphoribosyltransferase (Lowy et al., 1980, Cell 22: 817) genes
can be employed in tk-, hgprt- or aprt-cells, respectively. Also,
antimetabolite resistance can be used as the basis of selection for
the following genes: dhfr, which confers resistance to methotrexate
(Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77: 3567; O'Hare
et al., 1981, Proc. Natl. Acad. Sci. USA 78: 1527); gpt, which
confers resistance to mycophenolic acid (Mulligan and Berg, 1981,
Proc. Natl. Acad. Sci. USA 78: 2072); neo, which confers resistance
to the aminoglycoside G-418 (Colberre-Garapin et al., 1981, J. Mol.
Biol. 150: 1); and hygro, which confers resistance to hygromycin
(Santerre et al., 1984, Gene 30: 147).
[0108] The transgene may integrate into the genome of the cell of
interest, preferably by random integration. In other embodiments
the transgene may integrate by a directed method, e.g., by directed
homologous recombination (i.e., "knock-in" or "knock-out" of a gene
of interest in the genome of cell of interest), Chappel, U.S. Pat.
No. 5,272,071; and PCT publication No. WO 91/06667, published May
16, 1991; U.S. Pat. No. 5,464,764; Capecchi et al., issued Nov. 7,
1995; U.S. Pat. No. 5,627,059, Capecchi et al. issued, May 6, 1997;
U.S. Pat. No. 5,487,992, Capecchi et al., issued Jan. 30,
1996).
[0109] Methods for generating cells having targeted gene
modifications through homologous recombination are known in the
art. The construct will comprise at least a portion of a gene of
interest with a desired genetic modification, and will include
regions of homology to the target locus, i.e., the endogenous copy
of the targeted gene in the host's genome. DNA constructs for
random integration, in contrast to those used for homologous
recombination, need not include regions of homology to mediate
recombination. Markers can be included in the targeting construct
or random construct for performing positive and negative selection
for insertion of the transgene.
[0110] To create a homologous recombinant cell, e.g., a homologous
recombinant embryonic-like stem cell, endogenous placental cell or
exogenous cell cultured in the placenta, a homologous recombination
vector is prepared in which a gene of interest is flanked at its 5'
and 3' ends by gene sequences that are endogenous to the genome of
the targeted cell, to allow for homologous recombination to occur
between the gene of interest carried by the vector and the
endogenous gene in the genome of the targeted cell. The additional
flanking nucleic acid sequences are of sufficient length for
successful homologous recombination with the endogenous gene in the
genome of the targeted cell. Typically, several kilobases of
flanking DNA (both at the 5' and 3' ends) are included in the
vector. Methods for constructing homologous recombination vectors
and homologous recombinant animals from recombinant stem cells are
commonly known in the art (see, e.g., Thomas and Capecchi, 1987,
Cell 51: 503; Bradley, 1991, Curr. Opin. Bio/Technol. 2: 823-29;
and PCT Publication Nos. WO 90/11354, WO 91/01140, and WO
93/04169.
[0111] In one embodiment, the genome of an exogenous cell cultured
in the placenta according to the methods of the invention is a
target of gene targeting via homologous recombination or via random
integration.
[0112] In a specific embodiment, the methods of Bonadio et al.
(U.S. Pat. No. 5,942,496, entitled Methods and compositions for
multiple gene transfer into bone cells, issued Aug. 24, 1999; and
PCT WO95/22611, entitled Methods and compositions for stimulating
bone cells, published Aug. 24, 1995) are used to introduce nucleic
acids into a cell of interest, such as a stem cell, progenitor cell
or exogenous cell cultured in the placenta, e.g., bone progenitor
cells.
5.4 Uses of Cultured Placenta as a Bioreactor
[0113] Exsanguinated and/or cultured placental cells can be used as
a bioreactor for the cultivation of cells, tissues, and organs. The
placental mesoderm provides an ideal stromal environment, including
an abundance of small molecules and growth factors,
lipopolysaccharides, and extracellular matrix proteins, necessary
for organogenesis and tissue neogenesis.
[0114] In one embodiment, the invention provides a method of
utilizing the isolated perfused placenta as a bioreactor for the
propagation of exogenous cells. In accordance with this embodiment,
the invention relates to an isolated placenta which contains a cell
not derived from the placenta, wherein the engraftment of said cell
into the placenta may stimulate the placenta to produce
embryonic-like stem cells, or wherein the engrafted cell produces
signals, such as cytokines and growth factors, which may stimulate
the placenta to produce stem cells. The placenta may be engrafted
with cells not placental in origin obtained from the parents,
siblings or other blood relatives of the infant associated with the
placenta. In another embodiment, the isolated placenta may be
engrafted with cells not placental in origin obtained from an
individual whom is not the infant, nor related to the infant.
Likewise, the cells, tissues, organoids and organs, which are
propagated and cultivated in the placenta may be transplanted into
the infant associated with the placenta, the parents, siblings or
other blood relatives of said infant or into an individual not
related to the infant.
[0115] In one embodiment of the invention, the placenta can be
populated with any particular cell type and used as a bioreactor
for ex vivo cultivation of cells, tissues or organs. Such cells,
tissue or organ cultures may be harvested used in transplantation
and ex vivo treatment protocols. In this embodiment, the placenta
is processed to remove all endogenous cells and to allow foreign
(i.e., exogenous) cells to be introduced and propagated in the
environment of the perfused placenta. Methods for removal of the
endogenous cells are well-known in the art. For example, the
perfused placenta is irradiated with electromagnetic, UV, X-ray,
gamma- or beta-radiation to eradicate all remaining viable,
endogenous cells. In one embodiment, sub-lethal exposure to
radiations e.g., 500 to 1500 CG.gamma. can be used to preserve the
placenta but eradicate undesired cells. For international on lethal
v. non-lethal ionizing radiation (see Chapter 5 "Biophysical and
Biological Effects of Ionizing Radiation" from the United States
Department of Defense The foreign cells of interest to be
propagated in the irradiated placental bioreactor are then
introduced, for example, by vascular perfusion or direct
intra-parenchymal injection.
[0116] In another embodiment, the bioreactor may be used to produce
and propagate novel chimeric cells, tissues, or organs. Such
chimeras may be created using placental cells and one or more
additional cell types as starting materials in a bioreactor. The
interaction, or "cross-talk" between the different cell types can
induce expression patterns distinct from either of the starting
cell types. In one embodiment, for example, an autologous chimera
is generated by propagating a patient's autologous placental cells
in a bioreactor with another cell type derived from the same
patient. In another embodiment, for example, a heterologous chimera
may be generated by addition of a patient's cells, i.e., blood
cells, to a bioreactor having heterologous placental cells. In yet
another embodiment, the placental cells may be derived from a
patient, and a second cell type from a second patient. Chimeric
cells are then recovered having a different phenotypic and/or
genetic characteristics from either of the starting cells. In a
specific embodiment, the heterologous cells are of the same
haplotype, and the chimeric cells are reintroduced into the
patient.
[0117] In other embodiments, the bioreactor may be used for
enhanced growth of a particular cell type, whether native or
synthetic in origin, or for the production of a cell-type specific
product. For example, in one embodiment, the placental bioreactor
may be used to stimulate pancreatic islet cells to produce insulin.
The bioreactor is particularly advantageous for production of
therapeutic mammalian proteins, whose therapeutic efficacy can be
dependent upon proper post-translational modification. Thus, the
bioreactor is useful for the production of therapeutic proteins,
growth factors, cytokines, and other natural or recombinant
therapeutic molecules, such as but not limited to, erythropoietin,
interleukins, and interferons.
[0118] In another embodiment, the bioreactor may be used to
propagate genetically engineered cells to provide a therapeutic
gene product, and employed for large-scale production of the
recombinant product. In one embodiment, for example, the reactor
may be used to enhance antibody production. The placenta may be
populated with antibody-producing cells, such as hybridomas, which
produce a specific monoclonal antibodies, which are homogeneous
populations of antibodies to a particular antigen. Hybridomas may
be obtained by any technique, including, but not limited to, the
hybridoma technique of Kohler and Milstein (1975, Nature 256,
495-497; and U.S. Pat. No. 4,376,110), the human B-cell hybridoma
technique (Kosbor et aL, 1983, Immunology Today 4, 72; Cole et al.,
1983, Proc. Natl. Acad. Sci. USA 80, 2026-2030), and the
EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies
And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). The
mAb-producing hybridomas may be cultivated in the bioreactor to
produce high titers of mAbs.
[0119] Alternatively, where an antigen is unknown, the bioreactor
may be used to generate antibodies specific for a particular
cell-type, which may then be used identify the unknown antigen. For
example, antibodies may be generated against an unknown
tumor-specific antigen in a cancer patient by culturing a whole
blood specimen from a cancer patient, expanding the cells in a
bioreactor, and then screened for antibodies that specifically
react against a patient's tumor cells.
[0120] In another embodiment, the bioreactor may be used to produce
viruses in culture and for screening for antiviral agents in
culture. This method is of particular interest for those viruses,
such as parvovirus and human immunodeficiency virus, which are
difficult to propagate in cell culture conditions.
[0121] The bioreactor may also be used as a support for screening
for therapeutic molecules which modulate the activity of a
particular cell type, such as the activity or expression of a gene
product of interest or the activation of a signal transduction
pathway. In this embodiment, a cell type of interest may be
cultured and expanded in the bioreactor. The cell may be naturally
occurring cell, or a cell engineered to express a recombinant gene
product. The bioreactor is then be contacted with candidate
therapeutic molecules, such as small molecules, nonpeptides,
antibodies, etc., or libraries of such candidate therapeutic
molecules. The cells are then analyzed for a change in a desired
activity in the presence or absence of the candidate therapeutic
molecule. For example, such desired activity might be an increase
or decrease in growth rate, and change in gene expression, or a
change in binding or uptake of the candidate therapeutic
molecule.
[0122] Several types of methods are likely to be particularly
convenient and/or useful for screening test agents. These include,
but are not limited to, methods which measure binding of a
compound, methods which measure a change in the ability cells to
interact with an antibody or ligand, and methods which measure the
activity or expression of "reporter" protein, that is, an enzyme or
other detectable or selectable protein, which has been placed under
the control of a control region of interest. Thus, in a preferred
embodiment, both naturally occurring and/or synthetic compounds
(e.g., libraries of small molecules or peptides), may be screened
for therapeutic activity. The screening assays can be used to
identify compounds and compositions including peptides and organic,
non-protein molecules that modulate a cell-type specific activity.
Recombinant, synthetic, and otherwise exogenous compounds may have
binding capacity and, therefore, may be candidates for
pharmaceutical agents. Alternatively, the proteins and compounds
include endogenous cellular components which interact with the
identified genes and proteins in vivo. Such endogenous components
may provide new targets for pharmaceutical and therapeutic
interventions.
[0123] In another embodiment of the invention, the placenta is used
as a bioreactor for propagating endogenous cells (i.e., cells that
originate from the placenta), including but not limited to, various
kinds of pluripotent and/or totipotent embryonic-like stem cells
and lymphocytes. In one embodiment, the placenta is incubated for
varying periods of time with perfusate solution as disclosed
herein. Such endogenous cells of placental origin may be
transformed to recombinantly express a gene of interest, to express
mutations, and/or may be engineered to delete a genetic locus,
using "knock out" technology. For example, an endogenous target
gene may be deleted by inactivating or "knocking out" the target
gene or its promoter using targeted homologous recombination (e.g.,
see Smithies, et al., 1985, Nature 317, 230-234; Thomas &
Capecchi, 1987, Cell 51, 503-512; Thompson, et al., 1989, Cell 5,
313-321; each of which is incorporated by reference herein in its
entirety). For example, a mutant, non-functional target gene (or a
completely unrelated DNA sequence) flanked by DNA homologous to the
endogenous target gene (either the coding regions or regulatory
regions of the target gene) can be used, with or without a
selectable marker and/or a negative selectable marker, to transfect
cells that express the target gene in vivo. Insertion of the DNA
construct, via targeted homologous recombination, results in
inactivation of the target gene. Such approaches may be used to
remove, replace, or alter gene expression of interest in cells,
tissue, and/or organs. This approach may be used to alter the
phenotype of a cell, tissue, or organ, which may then be introduced
into a human subject.
[0124] In other embodiments, a placenta cell may be induced to
differentiate into a particular cell type, either ex vivo or in
vivo. For example, pluripotent embryonic-like stem cells may be
injected into a damaged organ, and for organ neogenesis and repair
of injury in vivo. Such injury may be due to such conditions and
disorders including, but not limited to, myocardial infarction,
seizure disorder, multiple sclerosis, stroke, hypotension, cardiac
arrest, ischemia, inflammation, age-related loss of cognitive
function, radiation damage, cerebral palsy, neurodegenerative
disease, Alzheimer's disease, Parkinson's disease, Leigh disease,
AIDS dementia, memory loss, amyotrophic lateral sclerosis, ischemic
renal disease, brain or spinal cord trauma; heart-lung bypass,
glaucoma, retinal ischemia, or retinal trauma.
[0125] The embryonic-like stem cells isolated from the placenta may
be used, in specific embodiments, in autologous or heterologous
enzyme replacement therapy to treat specific diseases or
conditions, including, but not limited to lysosomal storage
diseases, such as Tay-Sachs, Niemann-Pick, Fabry's, Gaucher's,
Hunter's, and Hurler's syndromes, as well as other gangliosidoses,
mucopolysaccharidoses, and glycogenoses.
[0126] In other embodiments, the cells may be used as autologous or
heterologous transgene carriers in gene therapy to correct inborn
errors of metabolism, adrenoleukodystrophy, cystic fibrosis,
glycogen storage disease, hypothyroidism, sickle cell anemia,
Pearson syndrome, Pompe's disease, phenylketonuria (PKU),
porphyrias, maple syrup urine disease, homocystinuria,
mucoplysaccharide nosis, chronic granulomatous disease and
tyrosinemia and Tay-Sachs disease or to treat cancer, tumors or
other pathological conditions.
[0127] In other embodiments, the cells may be used in autologous or
heterologous tissue regeneration or replacement therapies or
protocols, including, but not limited to treatment of corneal
epithelial defects, cartilage repair, facial dermabrasion, mucosal
membranes, tympanic membranes, intestinal linings, neurological
structures (e.g., retina, auditory neurons in basilar membrane,
olfactory neurons in olfactory epithelium), burn and wound repair
for traumatic injuries of the skin, or for reconstruction of other
damaged or diseased organs or tissues.
[0128] The large numbers of embryonic-like stem cells and/or
progenitor obtained using the methods of the invention would, in
certain embodiments, reduce the need for large bone marrow
donations. Approximately 1.times.10.sup.8 to 2.times.10.sup.8 bone
marrow mononuclear cells per kilogram of patient weight must be
infused for engraftment in a bone marrow transplantation (i.e.,
about 70 ml of marrow for a 70 kg donor). To obtain 70 ml requires
an intensive donation and significant loss of blood in the donation
process. In a specific embodiment, cells from a small bone marrow
donation (e.g., 7-10 ml) could be expanded by propagation in a
placental bioreactor before infusion into a recipient.
[0129] Furthermore, a small number of stem cells and progenitor
cells normally circulate in the blood stream. In another
embodiment, such exogenous stem cells or exogenous progenitor cells
are collected by pheresis, a procedure in which blood is withdrawn,
one or more components are selectively removed, and the remainder
of the blood is reinfused into the donor. The exogenous cells
recovered by pheresis are expanded by propagation in a placental
bioreactor, thus eliminating the need for bone marrow donation
entirely.
[0130] In another embodiment, expansion of exogenous cells in a
placental bioreactor is used as a supplemental treatment in
addition to chemotherapy. Most chemotherapy agents used to target
and destroy cancer cells act by killing all proliferating cells,
i.e., cells going through cell division. Since bone marrow is one
of the most actively proliferating tissues in the body,
hematopoietic stem cells are frequently damaged or destroyed by
chemotherapy agents and in consequence, blood cell production is
diminishes or ceases. Chemotherapy must be terminated at intervals
to allow the patient's hematopoietic system to replenish the blood
cell supply before resuming chemotherapy. It may take a month or
more for the formerly quiescent stem cells to proliferate and
increase the white blood cell count to acceptable levels so that
chemotherapy may resume (when again, the bone marrow stem cells are
destroyed).
[0131] While the blood cells regenerate between chemotherapy
treatments, however, the cancer has time to grow and possibly
become more resistant to the chemotherapy drugs due to natural
selection. Therefore, the longer chemotherapy is given and the
shorter the duration between treatments, the greater the odds of
successfully killing the cancer. To shorten the time between
chemotherapy treatments, embryonic-like stem cells or progenitor
cells collected according to the methods of the invention could be
introduced into the patient. Such treatment would reduce the time
the patient would exhibit a low blood cell count, and would
therefore permit earlier resumption of the chemotherapy
treatment.
[0132] The embryonic-like stem cells, progenitor cells, foreign
cells, or engineered cells obtained from a placenta according to
the methods of the invention can be used in the manufacture of a
tissue or organ in vivo. The methods of the invention encompass
using cells obtained from the placenta, e.g., embryonic-like stem
cells, progenitor cells, or foreign stem or progenitor cells, to
seed a matrix and to be cultured under the appropriate conditions
to allow the cells to differentiate and populate the matrix. The
tissues and organs obtained by the methods of the invention may be
used for a variety of purposes, including research and therapeutic
purposes.
5.5 Use of Embryonic-Like Stem Cells
[0133] The embryonic-like stem cells of the invention can be used
for a wide variety of therapeutic protocols in which a tissue or
organ of the body is augmented, repaired or replaced by the
engraftment, transplantation or infusion of a desired cell
population, such as a stem cell or progenitor cell population. The
embryonic-like stem cells of the invention can be used to replace
or augment existing tissues, to introduce new or altered tissues,
or to join together biological tissues or structures. The
embryonic-like stem cells of the invention can also be substituted
for embryonic stem cells in therapeutic protocols in which
embryonic stem cells would be typically be used.
[0134] In a preferred embodiment of the invention, embryonic-like
stem cells and other stem cells from the placenta may be used as
autologous and allogenic, including matched and mismatched HLA type
hematopoietic transplants. In accordance with the use of
embryonic-like stem cells as allogenic hematopoietic transplants it
may be necessary to treat the host to reduce immunological
rejection of the donor cells, such as those described in U.S. Pat.
No. 5,800,539, issued Sep. 1, 1998; and U.S. Pat. No. 5,806,529,
issued Sep. 15, 1998, both of which are incorporated herein by
reference.
[0135] For example, embryonic-like stem cells of the invention can
be used in therapeutic transplantation protocols, e.g., to augment
or replace stem or progenitor cells of the liver, pancreas, kidney,
lung, nervous system, muscular system, bone, bone marrow, thymus,
spleen, mucosal tissue, gonads, or hair.
[0136] Embryonic-like stem cells may be used instead of specific
classes of progenitor cells (e.g., chondrocytes, hepatocytes,
hematopoietic cells, pancreatic parenchymal cells, neuroblasts,
muscle progenitor cells, etc.) in therapeutic or research protocols
in which progenitor cells would typically be used.
[0137] Embryonic-like stem cells of the invention can be used for
augmentation, repair or replacement of cartilage, tendon, or
ligaments. For example, in certain embodiments, prostheses (e.g.,
hip prostheses) are coated with replacement cartilage tissue
constructs grown from embryonic-like stem cells of the invention.
In other embodiments, joints (e.g., knee) are reconstructed with
cartilage tissue constructs grown from embryonic-like stem cells.
Cartilage tissue constructs can also be employed in major
reconstructive surgery for different types of joints (for
protocols, see e.g., Resnick, D., and Niwayama, G., eds., 1988,
Diagnosis of Bone and Joint Disorders, 2d ed., W. B. Saunders
Co.).
[0138] The embryonic-like stem cells of the invention can be used
to repair damage of tissues and organs resulting from disease. In
such an embodiment, a patient can be administered embryonic-like
stem cells to regenerate or restore tissues or organs which have
been damaged as a consequence of disease, e.g., enhance immune
system following chemotherapy or radiation, repair heart tissue
following myocardial infarction.
[0139] The embryonic-like stem cells of the invention can be used
to augment or replace bone marrow cells in bone marrow
transplantation. Human autologous and allogenic bone marrow
transplantation are currently used as therapies for diseases such
as leukemia, lymphoma and other life-threatening disorders. The
drawback of these procedures, however, is that a large amount of
donor bone marrow must be removed to insure that there is enough
cells for engraftment.
[0140] The embryonic-like stem cells collected according to the
methods of the invention can provide stem cells and progenitor
cells that would reduce the need for large bone marrow donation. It
would also be, according to the methods of the invention, to obtain
a small marrow donation and then expand the number of stem cells
and progenitor cells culturing and expanding in the placenta before
infusion or transplantation into a recipient.
[0141] The embryonic-like stem cells isolated from the placenta may
be used, in specific embodiments, in autologous or heterologous
enzyme replacement therapy to treat specific diseases or
conditions, including, but not limited to lysosomal storage
diseases, such as Tay-Sachs, Niemann-Pick, Fabry's, Gaucher's,
Hunter's, Hurler's syndromes, as well as other gangliosidoses,
mucopolysaccharidoses, and glycogenoses.
[0142] In other embodiments, the cells may be used as autologous or
heterologous transgene carriers in gene therapy to correct inborn
errors of metabolism such as adrenoleukodystrophy, cystic fibrosis,
glycogen storage disease, hypothyroidism, sickle cell anemia,
Pearson syndrome, Pompe's disease, phenylketonuria (PKU), and
Tay-Sachs disease, porphyrias, maple syrup urine disease,
homocystinuria, mucopolypsaccharide nosis, chronic granulomatous
disease, and tyrosinemia. or to treat cancer, tumors or other
pathological conditions.
[0143] In other embodiments, the cells may be used in autologous or
heterologous tissue regeneration or replacement therapies or
protocols, including, but not limited to treatment of corneal
epithelial defects, cartilage repair, facial dermabrasion, mucosal
membranes, tympanic membranes, intestinal linings, neurological
structures (e.g., retina, auditory neurons in basilar membrane,
olfactory neurons in olfactory epithelium), burn and wound repair
for traumatic injuries of the skin, scalp (hair) transplantation,
or for reconstruction of other damaged or diseased organs or
tissues.
[0144] The large numbers of embryonic-like stem cells and/or
progenitor obtained using the methods of the invention would, in
certain embodiments, reduce the need for large bone marrow
donations. Approximately 1.times.10.sup.8 to 2.times.10.sup.8 bone
marrow mononuclear cells per kilogram of patient weight must be
infused for engraftment in a bone marrow transplantation (i.e.,
about 70 ml of marrow for a 70 kg donor). To obtain 70 ml requires
an intensive donation and significant loss of blood in the donation
process. In a specific embodiment, cells from a small bone marrow
donation (e.g., 7-10 ml) could be expanded by propagation in a
placental bioreactor before infusion into a recipient.
[0145] In another embodiment, the embryonic-like stem cells can be
used in a supplemental treatment in addition to chemotherapy. Most
chemotherapy agents used to target and destroy cancer cells act by
killing all proliferating cells, i.e., cells going through cell
division. Since bone marrow is one of the most actively
proliferating tissues in the body, hematopoietic stem cells are
frequently damaged or destroyed by chemotherapy agents and in
consequence, blood cell production is diminishes or ceases.
Chemotherapy must be terminated at intervals to allow the patient's
hematopoietic system to replenish the blood cell supply before
resuming chemotherapy. It may take a month or more for the formerly
quiescent stem cells to proliferate and increase the white blood
cell count to acceptable levels so that chemotherapy may resume
(when again, the bone marrow stem cells are destroyed).
[0146] While the blood cells regenerate between chemotherapy
treatments, however, the cancer has time to grow and possibly
become more resistant to the chemotherapy drugs due to natural
selection. Therefore, the longer chemotherapy is given and the
shorter the duration between treatments, the greater the odds of
successfully killing the cancer. To shorten the time between
chemotherapy treatments, embryonic-like stem cells or progenitor
cells collected according to the methods of the invention could be
introduced into the patient. Such treatment would reduce the time
the patient would exhibit a low blood cell count, and would
therefore permit earlier resumption of the chemotherapy
treatment.
[0147] In another embodiment, the human placental stem cells can be
used to treat or prevent genetic diseases such as chronic
granulomatous disease.
5.6 Pharamceutical Compositions
[0148] The present invention encompasses pharmaceutical
compositions comprising a dose and/or doses effective upon single
or multiple administration, prior to or following transplantation
of conditioned or unconditioned human progenitor stem cells,
exerting effect sufficient to inhibit, modulate and/or regulate the
differentiation of human pluripotent and multipotent progenitor
stem cells of placental origin into mesodermal and/or hematopoietic
lineage cells.
[0149] In accordance with this embodiment, the embryonic-like stem
cells of the invention may be formulated as an injectable (e.g.,
PCT WO 96/39101, incorporated herein by reference in its entirety).
In an alternative embodiment, the cells and tissues of the present
invention may be formulated using polymerizable or cross linking
hydrogels as described in U.S. Pat. Nos. 5,709,854; 5,516,532;
5,654,381; each of which is incorporated by reference in their
entirety.
6. EXAMPLE
6.1. Example 1
Anaylsis of Cell Types Recovered from Perfusate of Drained
Placenta
[0150] This example describes the analysis of the cell types
recovered from the effluent perfusate of a placenta cultured
according to the methods of the invention.
[0151] Twenty ml of phosphate buffered saline solution (PBS) was
added to the perfusion liquid and a 10 ml portion was collected and
centrifuged for 25 minutes at 3000 rpm (revolutions per minute).
The effluent was divided into four tubes and placed in an ice bath.
2.5 ml of a 1% fetal calf serum (FCS) solution in PBS was added and
the tubes were centrifuged (140 minutes.times.10 g (acceleration
due to gravity)). The pellet was resuspended in 5 ml of 1% FCS and
two tubes were combined. The total mononucleocytes were calculated
by adding the total lymphocytes and the total monocytes, and then
multiplying the result by the total cell suspension volume.
[0152] The following table discloses the types of cells obtained by
perfusion of a cultured placenta according to the methods described
hereinabove.
TABLE-US-00001 WBC Total # of 1000/ml Lym % MID % GRA % Volume
Cells CB 10.5 43.2 8 48.8 60 ml 6.3 .times. 10.sup.8 (Cord Blood)
PP 12.0 62.9 18.2 18.9 15 ml 1.8 .times. 10.sup.8 (Placenta
perfusate, room temperature) PP.sub.2 11.7 56.0 19.2 24.8 30 ml 3.5
.times. 10.sup.8 (Placenta perfusate, 37.degree. C.) Samples of PP
were after Ficoll. Total cell number of PP after Ficoll was 5.3
.times. 10.sup.8 and number of CB before processing is 6.3 .times.
10.sup.8. Lym % indicates percent of lymphocytes; MID % indicates
percent of midrange white blood cells; and GRA % indicates percent
of granulocytes.
6.2. Example 2
Anaylsis of Cells Obtained by Perfusion and Incubation of
Placenta
[0153] The following example describes an analysis of cells
obtained by perfusion and incubation of placenta according to the
methods of the invention.
6.2.1. Materials and Methods
[0154] Placenta donors were recruited from expectant mothers that
enrolled in private umbilical cord blood banking programs and
provided informed consent permitting the use of the exsanguinated
placenta following recovery of cord blood for research purposes.
Donor data may be confidential. These donors also permitted use of
blinded data generated from the normal processing of their
umbilical cord blood specimens for cryopreservation. This allowed
comparison between the composition of the collected cord blood and
the effluent perfusate recovered using the experimental method
described below.
[0155] Following exsanguination of cord blood from the umbilical
cord and placenta is stored at room temperature and delivered to
the laboratory within four to twenty-four hour, according to the
methods described hereinabove, the placenta was placed in a
sterile, insulated container at room temperature and delivered to
the laboratory within 4 hours of birth. Placentas were discarded
if, on inspection, they had evidence of physical damage such as
fragmentation of the organ or avulsion of umbilical vessels.
Placentas were maintained at room temperature (23.+-.2.degree. C.)
or refrigerated (4.degree. C.) in sterile containers for 2 to 20
hours. Periodically, the placentas were immersed and washed in
sterile saline at 25.+-.3.degree. C. to remove any visible surface
blood or debris.
[0156] The umbilical cord was transected approximately 5 cm from
its insertion into the placenta and the umbilical vessels were
cannulated with TEFLON.RTM. or polypropylene catheters connected to
a sterile fluid path allowing bi-directional perfusion of the
placenta and recovery of the effluent fluid. The methods described
hereinabove enabled all aspects of placental conditioning,
perfusion and effluent collection to be performed under controlled
ambient atmospheric conditions as well as real-time monitoring of
intravascular pressure and flow rates, core and perfusate
temperatures and recovered effluent volumes. A range of
conditioning protocols were evaluated over a 24-hour postpartum
period, and the cellular composition of the effluent fluid was
analyzed by flow cytometry, light microscopy and colony forming
unit assays.
6.2.2. Placental Conditioning
[0157] The donor placentas were processed at room temperature
within 12 to 24 hours after delivery. Before processing, the
membranes were removed and the maternal site washed clean of
residual blood. The umbilical vessels were cannulated with
catheters made from 20 gauge Butterfly needles use for blood sample
collection.
[0158] The donor placentas were maintained under varying conditions
such as maintenance at 5-37.degree. 5% CO.sub.2, pH 7.2 to 7.5,
preferably pH 7.45, in an attempt to simulate and sustain a
physiologically compatible environment for the proliferation and
recruitment of residual embryonic-like stem cells. The cannula was
flushed with IMDM serum-free medium (GibcoBRL, NY) containing 2
U/ml heparin (Elkins-Sinn, NJ). Perfusion of the placenta continued
at a rate of 50 ml per minute until approximately 150 ml of
perfusate was collected. This volume of perfusate was labeled
"early fraction." Continued perfusion of the placenta at the same
rate resulted in the collection of a second fraction of
approximately 150 ml and was labeled "late fraction." During the
course of the procedure, the placenta was gently massaged to aid in
the perfusion process and assist in the recovery of cellular
material. Effluent fluid was collected from the perfusion circuit
by both gravity drainage and aspiration through the arterial
cannula.
[0159] Placentas were then perfused with heparinized (2 U/ml)
Dulbecco's modified Eagle Medium (H.DMEM) at the rate of 15
ml/minute for 10 minutes and the perfusates were collected from the
maternal sites within one hour and the nucleated cells counted. The
perfusion and collection procedures were repeated once or twice
until the number of recovered nucleated cells fell below 100/ml.
The perfusates were pooled and subjected to light centrifugation to
remove platelets, debris and de-nucleated cell membranes. The
nucleated cells were then isolated by Ficoll-Hypaque density
gradient centrifugation and after washing, resuspended in H.DMEM.
For isolation of the adherent cells, aliquots of
5-10.times.10.sup.6 cells were placed in each of several T-75
flasks and cultured with commercially available Mesenchymal Stem
Cell Growth Medium (MSCGM) obtained from BioWhittaker, and placed
in a tissue culture incubator (37.degree. C., 5% CO.sub.2). After
10 to 15 days, the non-adherent cells were removed by washing with
PBS, which was then replaced by MSCGM. The flasks were examined
daily for the presence of various adherent cell types and in
particular, for identification and expansion of clusters of
fibroblastoid cells.
6.2.3. Cell Recovery and Isolation
[0160] Cells were recovered from the perfusates by centrifugation
at 5000.times.g for 15 minutes at room temperature. This procedure
served to separate cells from contaminating debris and platelets.
The cell pellets were resuspended in IMDM serum-free medium
containing 2 U/ml heparin and 2mM EDTA (GibcoBRL, NY). The total
mononuclear cell fraction was isolated using Lymphoprep (Nycomed
Pharma, Oslo, Norway) according to the manufacturer's recommended
procedure and the mononuclear cell fraction was resuspended. Cells
were counted using a hemocytometer. Viability was evaluated by
trypan blue exclusion. Isolation of mesenchymal cells was achieved
by "differential trypsinization," using a solution of 0.05% trypsin
with 0.2% EDTA (Sigma, St. Louis Mo.). Differential trypsinization
was possible because fibroblastoid cells detached from plastic
surfaces within about five minutes whereas the other adherent
populations required more than 20-30 minutes incubation. The
detached fibroblastoid cells were harvested following
trypsinization and trypsin neutralization, using Trypsin
Neutralizing Solution (TNS, BioWhittaker). The cells were washed in
H.DMEM and resuspended in MSCGM.
[0161] Flow cytometry was carried out using a Becton-Dickinson
FACSCalibur instrument and FITC and PE labeled monoclonal
antibodies (mAbs), selected on. the basis of known markers for bone
marrow-derived MSC (mesenchymal stem cells), were purchased from
B.D. and Caltag laboratories (South San Francisco, Calif.), and
SH2, SH3 and SH4 antibody producing hybridomas were obtained from
and reactivities of the mAbs in their cultured supernatants were
detected by FITC or PE labeled F(ab)'2 goat anti-mouse antibodies.
Lineage differentiation was carried out using commercially
available induction and 20 maintenance culture media
(BioWhittaker), used as per manufacturer's instructions.
6.2.4. Isolation of Placental Embryonic-Like Stem Cells
[0162] Microscopic examination of the adherent cells in the culture
flasks revealed morphologically different cell types.
Spindle-shaped cells, round cells with large nuclei and numerous
perinuclear small vacuoles, and star-shaped cells with several
projections (through one of which star-shaped cells were attached
to the flask) were observed adhering to the culture flasks.
Although no attempts were made to further characterize these
adherent cells, similar cells were observed in the culture of bone
marrow, cord and peripheral blood, and therefore considered to be
non-stem cell-like in nature. The fibroblastoid cells, appearing
last as clusters, were candidates for being MSC (mesenchymal stem
cells) and were isolated by differential trypsinization and
subcultured in secondary flasks. Phase microscopy of the rounded
cells, after trypsinization, revealed that the cells were highly
granulated; indistinguishable from the bone marrow-derived MSC
produced in the laboratory or purchased from BioWhittaker. When
subcultured, the placenta-derived embryonic-like stem cells, in
contrast to their earlier phase, adhered within hours, assumed
characteristic fibroblastoid shape, and formed a growth pattern
identical to the reference bone marrow-derived MSC. During
subculturing and refeeding, moreover, the loosely bound mononuclear
cells were washed out and the cultures remained homogeneous and
devoid of any visible non-fibroblastoid cell contaminants.
6.2.5. Results
[0163] The expression of CD-34, CD-38, and other stem
cell-associated surface markers on early and late fraction purified
mononuclear cells was assessed by flow cytometry. Recovered, sorted
cells were washed in PBS and then double-stained with antiCD34
phycoerythrin and anti-CD38 fluorescein isothiocyanate (Becton
Dickinson, Mountain View, Calif.).
[0164] Cell isolation was achieved by using magnetic cell
separation, such as for example, Auto Macs (Miltenyi). Preferably,
CD 34+ cell isolation is performed first.
6.3 Example 3
Perfusion Medium
[0165] The following example provides a formula of the preferred
perfusate solution for the cultivation of isolated placentas
TABLE-US-00002 Stock Final Chemical Source Concentration
Concentration 500 ml DMEM-LG GibcoBRL11885-084 300 ml MCDB201 Sigma
M-6770 dissolved in H2O pH to 7.2. 200 ml filter FCS Hyclone 100%
2% 10 ml ITS Sigma I-3146 or 100.times. 1.times. 5 ml
GibcoBRL41400-045 Pen&Strep GibcoBRL15140-122 100.times.
1.times. 5 ml LA + BSA Sigma + GibcoBRL 100 .times. (1 .mu.g/ml of
LA 10 ng/ml of 5 ml BSA LA Dexamethasone Sigma D-2915 0.25 mM in
H2O 0.05 .mu.M 100 .mu.l L-Ascorbic Acid Sigma A-8960 1000 .times.
(100 mM) 1 .times. (0.1 mM) 500 .mu.l PDGF (50 .mu.g) R&D 220BD
10 .mu.g/ml in 4 mM 10 ng/ml 500 .mu.l HCl + 0.1% BSA EGF (20
.mu.g) Sigma E-9644 10 .mu.g/ml in 10 mM 10 ng/ml 500 .mu.l HAc +
0.1% BSA
[0166] The above-composition is a perfusate that may be used at a
variety of temperatures to perfuse placenta. It should be noted
that additional components such as antibiotics, anticoagulant and
other growth factors may be used in the perfusate or culture
media.
[0167] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description. Such modifications are intended to fall
within the scope of the appended claims.
[0168] All references cited herein are incorporated herein by
reference in their entirety and for all purposes to the same extent
as if each individual publication, patent or patent application was
specifically and individually indicated to be incorporated by
reference in its entirety for all purposes.
[0169] The citation of any publication is for its disclosure prior
to the filing date and should not be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention.
* * * * *