U.S. patent application number 14/792067 was filed with the patent office on 2015-10-29 for treatment of bone-related cancers using placental stem cells.
This patent application is currently assigned to ANTHROGENESIS CORPORATION. The applicant listed for this patent is ANTHROGENESIS CORPORATION. Invention is credited to Sascha Abramson, Robert J. Hariri, Shmuel Yaccoby, Xiaokui Zhang.
Application Number | 20150306150 14/792067 |
Document ID | / |
Family ID | 43798336 |
Filed Date | 2015-10-29 |
United States Patent
Application |
20150306150 |
Kind Code |
A1 |
Zhang; Xiaokui ; et
al. |
October 29, 2015 |
TREATMENT OF BONE-RELATED CANCERS USING PLACENTAL STEM CELLS
Abstract
Provided herein are methods of suppression of proliferation and
growth of cells of bone-related cancers, e.g., multiple myeloma or
chondrosarcoma cells, using placental cells, e.g., the placental
stem cells described herein, and populations of such placental
cells. Also provided herein are methods of treating individuals
having cells of a bone-related cancer.
Inventors: |
Zhang; Xiaokui; (Livingston,
NJ) ; Yaccoby; Shmuel; (Little Rock, AR) ;
Abramson; Sascha; (Holland Township, NJ) ; Hariri;
Robert J.; (Bernardsville, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ANTHROGENESIS CORPORATION |
Warren |
NJ |
US |
|
|
Assignee: |
ANTHROGENESIS CORPORATION
Warren
NJ
|
Family ID: |
43798336 |
Appl. No.: |
14/792067 |
Filed: |
July 6, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13013721 |
Jan 25, 2011 |
9121007 |
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14792067 |
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61298517 |
Jan 26, 2010 |
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61307821 |
Feb 24, 2010 |
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61352768 |
Jun 8, 2010 |
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Current U.S.
Class: |
424/93.7 ;
435/375 |
Current CPC
Class: |
A61K 35/12 20130101;
C12N 2502/025 20130101; A61K 31/4545 20130101; A61P 35/00 20180101;
C12N 2502/30 20130101; C12N 2502/1142 20130101; A61K 9/0019
20130101; A61P 19/08 20180101; C12N 5/0605 20130101; A61K 35/50
20130101; A61K 31/4545 20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 35/50 20060101
A61K035/50; A61K 9/00 20060101 A61K009/00; C12N 5/073 20060101
C12N005/073 |
Claims
1. A method of suppressing the proliferation of cells of a
bone-related cancer, comprising contacting said cells of a
bone-related cancer with a plurality of placental stem cells for a
time sufficient for said placental stem cells to suppress
proliferation of said cells of a bone-related cancer, as compared
to a plurality of said cells of a bone-related cancer not contacted
with placental stem cells, wherein said placental stem cells are
adherent to tissue culture plastic, are CD34.sup.-, CD10.sup.+,
CD105.sup.+ and CD200.sup.+ as detectable by flow cytometry, and
are not trophoblasts, cytotrophoblasts or bone marrow-derived
mesenchymal stem cells.
2. The method of claim 1, wherein said cells of a bone-related
cancer are multiple myeloma cells.
3. The method of claim 1, wherein said cells of a bone-related
cancer are chondrosarcoma cells.
4. The method of claim 1, wherein said cells of a bone-related
cancer are bone cancer cells, neuroblastoma cells, osteosarcoma
cells, Ewing sarcoma cells, chordoma cells, cells of a malignant
fibrous histiocytoma of bone, prostate cancer cells, or cells of a
fibrosarcoma of bone.
5. The method of claim 1, wherein said cells of a bone-related
cancer are not prostate cancer cells.
6. The method of claim 1, wherein said placental cells are
CD34.sup.-, CD45.sup.-, CD10.sup.+, CD90.sup.+, CD105.sup.+ and
CD200.sup.+, as detectable by flow cytometry.
7. The method of claim 1, wherein said placental cells are
CD34.sup.-, CD45.sup.-, CD10.sup.+, CD80.sup.-, CD86.sup.-,
CD90.sup.+, CD105.sup.+ and CD200.sup.+, as detectable by flow
cytometry.
8. The method of claim 1, wherein said contacting is performed in
vitro.
9. The method of claim 1, wherein said contacting is performed in
vivo.
10. The method of claim 9, wherein said contacting comprises
administering said placental stem cells to a human individual
comprising said cells of a bone related cancer.
11. The method of claim 10, wherein said contacting comprises
administering said placental stem cells to said individual at or
adjacent to a bone lesion caused by said bone-related cancer.
12. The method of claim 10, wherein said contacting comprises
administering at least 1.times.10.sup.8 of said placental stem
cells to said individual.
13. The method of claim 1, wherein said placental cells suppress
proliferation of said cells of a bone-related cancer by at least
50% compared to proliferation of an equivalent number of cells of a
bone-related cancer in the absence of said placental cells.
14. A method of treating a human individual having a bone-related
cancer, comprising administering to said individual a
therapeutically effective amount of placental stem cells for a time
sufficient for said placental stem cells to improve one or more
symptoms of, or reduce the progression of, said bone-related
cancer, wherein said placental stem cells are adherent to tissue
culture plastic, are CD34.sup.-, CD10.sup.+, CD105.sup.+ and
CD200.sup.+ as detectable by flow cytometry; are not trophoblasts,
cytotrophoblasts or bone marrow-derived mesenchymal stem cells, and
have the capacity to differentiate into osteogenic or chondrogenic
cells.
15. The method of claim 14, wherein said bone-related cancer is
multiple myeloma.
16. The method of claim 14, wherein said bone-related cancer is
chondrosarcoma.
17. The method of claim 14, wherein said bone-related cancer is
bone cancer, neuroblastoma, osteosarcoma, Ewing sarcoma, chordoma,
malignant fibrous histiocytoma of bone, prostate cancer, or
fibrosarcoma of bone.
18. The method of claim 14, wherein said cells of a bone-related
cancer are not prostate cancer cells.
19. The method of claim 14, wherein said placental stem cells are
CD34.sup.-, CD45.sup.-, CD10.sup.+, CD90.sup.+, CD105.sup.+ and
CD200.sup.+, as detectable by flow cytometry.
20. The method of claim 14, wherein said placental stem cells are
CD34.sup.-, CD45.sup.-, CD10.sup.+, CD80.sup.-, CD86.sup.-,
CD90.sup.+, CD105.sup.+ and CD200.sup.+, as detectable by flow
cytometry.
21. The method of claim 14, wherein said placental stem cells are
administered to said individual intravenously.
22. The method of claim 14, wherein said placental stem cells are
administered to said individual at or adjacent to a bone lesion
caused by said bone-related cancer.
23. The method of claim 14, comprising administering at least
1.times.10.sup.8 placental cells to said individual.
24. The method of claim 14, wherein said placental cells suppress
proliferation of cells of said bone-related cancer by at least 50%
compared to proliferation of an equivalent number of cells of said
bone-related cancer in the absence of said placental cells.
Description
[0001] This application claims the benefit of U.S. Provisional
Patent Application Ser. No. 61/298,517, filed Jan. 26, 2010; U.S.
Provisional Patent Application Ser. No. 61/307,821, filed Feb. 24,
2010; and U.S. Provisional Patent Application Ser. No. 61/352,768,
filed Jun. 8, 2010, each of which is incorporated by reference
herein in its entirety.
1. FIELD
[0002] Provided herein are methods of using tissue culture
plastic-adherent placental stem cells (referred to herein as
PDACs), and/or bone marrow-derived mesenchymal stem cells (BM-MSCs)
to treat bone related cancers, e.g., multiple myeloma, to suppress
the proliferation of cells of bone-related cancers, e.g., multiple
myeloma cells or chondrosarcoma cells, and to suppress the growth
of bone-related cancers, e.g., multiple myeloma, chondrosarcoma,
and other bone-related cancers, e.g., tumors.
2. BACKGROUND
[0003] Multiple myeloma (also known as MM, myeloma, plasma cell
myeloma, or Kahler's disease) is a type of cancer of plasma cells,
which are antibody-producing immune system cells. Symptoms of
multiple myeloma include bone pain, infection, renal failure,
anemia, and bone lesions. The disease is considered incurable, and
only a few treatments, such as lenalidomide (REVLIMID.RTM.) are
available and show promise. As such, a need exists for new
treatments for multiple myeloma. To date, no one has described the
ability of non-hematopoietic, tissue culture plastic-adherent
placental stem cells to suppress the growth of bone-related
cancers, e.g., multiple myeloma, or to suppress the proliferation
of cells of bone-related cancers.
3. SUMMARY
[0004] In one aspect, provided herein are methods of treating an
individual having a bone-related cancer, comprising administering
to the individual a therapeutically effective amount of isolated
tissue culture plastic-adherent placental stem cells, also referred
to herein as PDACs (placenta derived adherent cells), isolated
populations of such placental stem cells, or isolated populations
of cells comprising the placental stem cells; and/or isolated bone
marrow-derived mesenchymal stem cells (BM-MSCs) or bone marrow
comprising BM-MSCs.
[0005] In one embodiment, provided herein is a method of treating
an individual having a bone-related cancer, comprising
administering to said individual a therapeutically effective amount
of placental stem cells and/or BM-MSCs, wherein said
therapeutically effective amount of placental stem cells and/or
BM-MSCs improves, e.g., detectably improves, one or more symptoms
of, or reduces, e.g., detectably reduces, the progression of, said
bone-related cancer. In a specific embodiment, said bone-related
cancer is multiple myeloma. In a specific embodiment, said
bone-related cancer is chondrosarcoma. In other embodiments, said
bone-related cancer is bone cancer, neuroblastoma, osteosarcoma,
Ewing's sarcoma, chordoma, malignant fibrous histiocytoma of bone,
prostate cancer, or fibrosarcoma of bone. In a specific embodiment,
the bone-related cancer is not prostate cancer. In other
embodiments, said bone-related cancer comprises a solid tumor. In
another embodiment, said individual is a mammal. In another
embodiment, said individual is a human. In another embodiment, said
administering said placental stem cells results in a greater, e.g.,
detectably greater, improvement of said one or more symptoms than
administering an equivalent number of bone marrow-derived
mesenchymal stem cells. In certain embodiments, said bone
marrow-derived mesenchymal stem cells are one or more of
CD34.sup.-, CD45.sup.-, CD73.sup.+ and/or CD105.sup.+.
[0006] In certain embodiments, said individual exhibits a bone
lesion, e.g., a bone lesion caused by said bone-related cancer,
e.g., a bone lesion visible on an X-ray radiogram. In other
embodiments, said individual does not exhibit a bone lesion, e.g.,
a bone lesion caused by said bone-related cancer, e.g., a bone
lesion visible on an X-ray radiogram. In other embodiments, said
administering results in a delay in the appearance of, or onset of,
bone lesions, e.g., bone lesions caused by said bone-related
cancer, e.g., as visible on an X-ray radiogram, or bone lesions
caused by treatment of a cancer.
[0007] In certain embodiments, said placental stem cells, and/or
said BM-MSCs, are administered to said individual intravenously. In
other embodiments, the method of treatment comprises administering
at least about 1.times.10.sup.7, 5.times.10.sup.7,
1.times.10.sup.8, 5.times.10.sup.8, 1.times.10.sup.9,
5.times.10.sup.9, 1.times.10.sup.10, 5.times.10.sup.10 or
1.times.10.sup.11 placental stem cells, and/or BM-MSCs, to said
individual, in terms of total number of cells. In another specific
embodiment, said placental stem cells, said BM-MSCs, or both have
been proliferated in vitro for no more than 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29 or 30 population doublings prior to said administering.
In another embodiment, said placental stem cells, and/or BM-MSCs,
are administered to said individual at or adjacent to a bone
lesion, e.g., a bone lesion caused by said bone-related cancer. In
another embodiment, the method of treatment additionally comprises
administering to said individual one or more anticancer compounds.
In another embodiment of any of the embodiments herein, said
placental stem cells and/or BM-MSCs have been cryopreserved and
thawed prior to said administering.
[0008] In one embodiment, the methods of treatment can comprise
determining, once or a plurality of times before said
administering, and/or once or a plurality of times after said
administering, one or more of (1) a number or degree of bone
lesions in said individual; (2) a number of osteoclast precursors
in said individual; or (3) a number of multiple myeloma cells in
said individual, e.g., at least once before and at least once after
said administration. In certain embodiments, said therapeutically
effective amount of placental stem cells, and/or BM-MSCs, reduces,
e.g., detectably reduces, the number of, or degree of severity of,
or reduces the rate of increase in the number of, or degree of
severity, said bone lesions in said individual, e.g., as
determinable by bone densitometry or X-rays. In other embodiments,
said therapeutically effective amount of placental stem cells,
and/or BM-MSCs, reduces, e.g., detectably reduces, the number of
osteoclast precursors in said individual, e.g., as determined using
an antibody specific for osteoclast precursors to detect osteoclast
precursors in, e.g., the individual's peripheral blood or bone
marrow. In other embodiments, said therapeutically effective amount
of placental stem cells, and/or BM-MSCs, reduces the number of
bone-related cancer cells, e.g., multiple myeloma cells, in said
individual, e.g., as determinable by cell counting (e.g., by flow
cytometry), or antibody staining, of nucleated blood cells from
said individual using an antibody specific for such cells, e.g.,
multiple myeloma cells or plasma cells, e.g., an antibody specific
for cellular markers CD28 or CD138, or as determinable by assessing
the level of M proteins in blood from the individual. In other
embodiments, said placental stem cells, and/or BM-MSCs, reduce the
number of cells of said bone-related cancer, or said osteoclast
precursors, by at least, e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%,
compared to the number of said cells prior to administration of
said placental stem cells.
[0009] In another embodiment, the individual has chondrosarcoma;
e.g., the bone-related cancer is chondrosarcoma. In certain
embodiments, the method of treatment comprises determining, once or
a plurality of times before said administering, and/or once or a
plurality of times after said administering, one or more of a
number of chondrosarcoma cells in the individual or the number of
bone lesions (e.g., chondrosarcoma-caused masses) in the
individual.
[0010] In another aspect, provided herein is a method of
suppressing proliferation of cells of a bone-related cancer,
comprising contacting said cells of a bone-related cancer with a
plurality of placental stem cells, and/or BM-MSCs, for a time
sufficient for said placental stem cells and/or BM-MSCs to
suppress, e.g., detectably suppress, proliferation of said cells of
a bone-related cancer, as compared to a plurality of said cells of
a bone-related cancer not contacted with placental stem cells
and/or BM-MSCs, e.g., as determinable by a reduction, e.g., a
detectable reduction, in the number of said bone-related cancer
cells, or a detectable reduction in the increase in number of said
bone-related cancer cells. In certain embodiments, said cells of a
bone-related cancer are multiple myeloma cells. In another
embodiment of the method, said cells of a bone-related cancer are
chondrosarcoma cells. In other embodiments, said cells of a
bone-related cancer are bone cancer cells, neuroblastoma cells,
osteosarcoma cells, Ewing sarcoma cells, chordoma cells, cells of a
malignant fibrous histiocytoma of bone, or cells of a fibrosarcoma
of bone. In another specific embodiment, said cells of a
bone-related cancer are part of a solid tumor.
[0011] In certain embodiments of the method, said contacting is
performed in vitro. In certain other embodiments, said contacting
is performed in vivo. In certain embodiments, said contacting is
performed in an individual who comprises said cells of a
bone-related cancer, e.g., in an individual having a disease caused
by said cells. In other embodiments, said contacting is performed
in an individual who comprises multiple myeloma cells, e.g., in an
individual having multiple myeloma. In certain embodiments, said
individual is a mammal, e.g. a human. In another specific
embodiment, said contacting comprises administering said placental
stem cells to said individual intravenously. In another specific
embodiment, said contacting comprises administering said placental
stem cells, and/or BM-MSCs, to said individual at or adjacent to a
bone lesion in the individual.
[0012] In another embodiment, the methods of suppressing
proliferation of cells of a bone-related cancer, e.g., multiple
myeloma cells, additionally comprises contacting said cells of a
bone-related cancer with one or more anticancer compounds, e.g., a
therapeutically effective amount of one or more anticancer
compounds. In another embodiment, the method comprises
administering at least about 1.times.10.sup.7, 5.times.10.sup.7,
1.times.10.sup.8, 5.times.10.sup.8, 1.times.10.sup.9,
5.times.10.sup.9, or 1.times.10.sup.10 placental stem cells, and/or
BM-MSCs, to said individual. In certain embodiments, said placental
stem cells and/or BM-MSCs, and/or BM-MSCs, have been proliferated
in vitro for no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or
30 population doublings. In other embodiments, said placental stem
cells, and/or BM-MSCs, suppress proliferation of cells of said
bone-related cancer by at least, e.g., 10%, 15%, 20%, 25%, 30%,
35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
98% or 99%, compared to proliferation of an equivalent number of
cells of said bone-related cancer in the absence of said placental
stem cells and/or BM-MSCs, e.g., as determinable by a detectable
reduction in the number of said bone-related cancer cells, or a
detectable reduction in the increase in number of said bone-related
cancer cells, or as determinable by a detectable decrease in the
number and/or severity of bone lesions in an individual having said
cancer cells.
[0013] In other embodiments, said placental stem cells or BM-MSCs,
or both, have been cryopreserved and thawed prior to said
contacting. In another embodiment, the method comprises determining
that said placental stem cells suppress, e.g., detectably suppress,
the proliferation of a sample of said cells of a bone-related
cancer prior to said contacting.
[0014] In another embodiment, provided herein is a method of
reducing maturation of osteoclast precursors, into osteoclasts,
comprising contacting said osteoclast precursors with a plurality
of placental stem cells and/or BM-MSCs, wherein said plurality of
placental stem cells and/or BM-MSCs is a number of cells sufficient
to reduce, e.g., detectably reduce, osteoclast maturation from said
osteoclast precursors, e.g., as determinable by a detectable
reduction of, or lack of increase in, the number of osteoclasts as
a result of said contacting. In another embodiment, provided herein
is a method of increasing apoptosis of osteoclast precursors,
comprising contacting said osteoclast precursors with a plurality
of placental stem cells and/or BM-MSCs wherein said plurality of
placental stem cells and/or BM-MSCs is a number of cells sufficient
to increase, e.g., detectably increase, osteoclast precursor
apoptosis. In certain embodiments, said increase in osteoclast
precursor apoptosis is detected by a detectable increase in annexin
V and/or propidium iodide staining of osteoclast precursors from
said individual. In certain embodiments, the osteoclast precursors
are in an individual, e.g., an individual having a bone-related
cancer, e.g., multiple myeloma, chondrosarcoma, or one of the other
bone-related cancers described herein. In certain other
embodiments, the method comprises contacting said osteoclast
precursors with lenalidomide, e.g., administering lenalidomide to
an individual having said osteoclast precursors.
[0015] In certain embodiments of the above methods, said contacting
takes place in vitro. In other embodiments, said contacting takes
place in vivo. In another embodiment, said contacting takes place
in a human. In another embodiment, said contacting takes place in
an individual having a bone-related cancer, e.g., an individual
having cells of a bone related cancer, or a disease caused by such
cells. In another embodiment, said individual is an individual
having multiple myeloma or multiple myeloma cells. In another
embodiment, said individual has at least one symptom of multiple
myeloma. In another embodiment, said individual has at least one
bone lesion caused by multiple myeloma.
[0016] In certain embodiments of any of the above methods, said
placental stem cells are one or more of: (1) adherent to tissue
culture plastic; (2) CD34.sup.-, CD10.sup.+, CD105.sup.+ and
CD200.sup.+ as detectable by flow cytometry; and/or (3) have the
capacity to differentiate into osteogenic or chondrogenic cells,
e.g., either in vitro or in vivo, or both. In another embodiment,
said placental stem cells are adherent to tissue culture plastic;
CD34.sup.-, CD10.sup.+, CD105.sup.+ and CD200.sup.+ as detectable
by flow cytometry; and have the capacity to differentiate into
cells having one or more characteristics of osteogenic or
chondrogenic cells, e.g., characteristics of osteocytes or
chondrocytes, e.g., either in vitro or in vivo, or both. In other
embodiments, the placental stem cells additionally have the ability
to differentiate into cells having one or more characteristics of
neural cells or neurogenic cells, e.g., characteristics of neurons;
one or more characteristics of glial cells, e.g., characteristics
of glia or astrocytes; one or more characteristics of adipocytic
cells, e.g., characteristics of adipocytes; one or more
characteristics of pancreatic cells; and/or one or more
characteristics of cardiac cells. In a specific embodiment of each
of the embodiments of placental stem cells herein, the placental
stem cells are isolated placental stem cells.
[0017] In another embodiment, said placental stem cells are
CD34.sup.-, CD10.sup.+, CD105.sup.+ and CD200.sup.+, and one or
more of CD44.sup.+, CD45.sup.-, CD90.sup.+, CD166.sup.+, KDR.sup.-,
or CD133.sup.-. In a more specific embodiment, said placental stem
cells are CD34.sup.-, CD10.sup.+, CD105.sup.+ and CD200.sup.+,
CD44.sup.+, CD45.sup.-, CD90.sup.+, CD166.sup.+, KDR.sup.-, and
CD133.sup.-. In another embodiment, the placental stem cells are
CD34.sup.-, CD10.sup.+, CD105.sup.+ and CD200.sup.+, and one or
more of HLA ABC.sup.+, HLA DR,DQ,DP.sup.-, CD80.sup.-, CD86.sup.-,
CD98.sup.-, or PD-L1.sup.+. In a more specific embodiment, said
placental stem cells are CD34.sup.-, CD10.sup.+, CD105.sup.+ and
CD200.sup.+, HLA ABC.sup.+, HLA DR,DQ,DP.sup.-, CD80.sup.-,
CD86.sup.-, CD98.sup.-, and PD-L1.sup.+. In another embodiment,
said placental stem cells are CD34.sup.-, CD10.sup.+, CD105.sup.+
and CD200.sup.+, and one or more of CD38.sup.-, CD45.sup.-,
CD80.sup.-, CD86.sup.-, CD133.sup.-, HLA-DR,DP,DQ.sup.-,
SSEA3.sup.-, SSEA4.sup.-, CD29.sup.+, CD44.sup.+, CD73.sup.+,
CD90.sup.+, CD105.sup.+, HLA-A,B,C.sup.+, PDL1.sup.+, ABC-p.sup.+,
and/or OCT-4.sup.+, as detectable by flow cytometry and/or RT-PCR.
In another embodiment, the placental stem cells are CD34.sup.-,
CD45.sup.-, CD10.sup.+, CD90.sup.+, CD105.sup.+ and CD200.sup.+, as
detectable by flow cytometry. In another embodiment, said placental
stem cells CD34.sup.-, CD45.sup.-, CD10.sup.+, CD80.sup.-,
CD86.sup.-, CD90.sup.+, CD105.sup.+ and CD200.sup.+, as detectable
by flow cytometry. In another embodiment, said placental stem cells
are CD34.sup.-, CD45.sup.-, CD10.sup.+, CD80.sup.-, CD86.sup.-,
CD90.sup.+, CD105.sup.+ and CD200.sup.+, and additionally one or
more of CD29.sup.+, CD38.sup.-, CD44.sup.+, CD54+, SH3.sup.+ or
SH4.sup.+, as detectable by flow cytometry. In another embodiment,
said placental stem cells are CD34.sup.-, CD38.sup.-, CD45.sup.-,
CD10.sup.+, CD29.sup.+, CD44.sup.+, CD54.sup.+, CD73.sup.+,
CD80.sup.-, CD86.sup.-, CD90.sup.+, CD105.sup.+, and CD200.sup.+ as
detectable by flow cytometry.
[0018] In another embodiment, said CD34.sup.-, CD10.sup.+,
CD105.sup.+ and CD200.sup.+ placental stem cells are additionally
one or more of CD3.sup.-, CD9.sup.-, CD117.sup.-, CD133.sup.-,
CD146.sup.+, CD166.sup.+, KDR.sup.- (VEGFR2.sup.-),
HLA-A,B,C.sup.+, HLA-DP,DQ,DR.sup.-, or Programmed Death-1 Ligand
(PDL1).sup.+, or any combination thereof. In another specific
embodiment, said placental stem cells are CD3.sup.-, CD9.sup.-,
CD34.sup.-, CD38.sup.-, CD45.sup.-, CD10.sup.+, CD29.sup.+,
CD44.sup.+, CD54.sup.+, CD73.sup.+, CD80.sup.-, CD86.sup.-,
CD90.sup.+, CD105.sup.+, CD117.sup.-, CD133.sup.-, CD146.sup.+,
CD166.sup.+, CD200.sup.+, KDR.sup.- (VEGFR2.sup.-),
HLA-A,B,C.sup.+, HLA-DP,DQ,DR.sup.-, or Programmed Death-1 Ligand
(PDL1).sup.+, as detectable by flow cytometry.
[0019] In another embodiment, any of the placental stem cells
described herein are additionally ABC-p.sup.+, as detectable by
flow cytometry, or OCT-4.sup.+ (POU5F1.sup.+), e.g., as
determinable by RT-PCR, wherein ABC-p is a placenta-specific ABC
transporter protein (also known as breast cancer resistance protein
(BCRP) and as mitoxantrone resistance protein (MXR)). In another
embodiment, any of the placental stem cells described herein are
additionally SSEA3.sup.- or SSEA4.sup.-, e.g., as determinable by
flow cytometry, wherein SSEA3 is Stage Specific Embryonic Antigen
3, and SSEA4 is Stage Specific Embryonic Antigen 4. In another
embodiment, any of the placental stem cells described herein are
additionally SSEA3.sup.- and SSEA4.sup.-.
[0020] In another embodiment of the methods described herein, any
of the placental stem cells populations of isolated placental stem
cells described herein are additionally one or more of MHC-I.sup.+
(e.g., HLA-A,B,C.sup.+), MHC-II.sup.- (e.g., HLA-DP,DQ,DR.sup.-) or
HLA-G.sup.-. In another embodiment, any of the placental stem cells
described herein are additionally each of MHC-I.sup.+ (e.g.,
HLA-A,B,C.sup.+), MHC-II.sup.- (e.g., HLA-DP,DQ,DR.sup.-) and
HLA-G.sup.-.
[0021] In another embodiment, the CD34.sup.-, CD10.sup.+,
CD105.sup.+, CD200.sup.+ placental stem cells are additionally one
or more of CD3.sup.-, CD9.sup.-, CD29.sup.+, CD38.sup.-,
CD44.sup.+, CD54.sup.+, CD80.sup.-, CD86.sup.-, CD146.sup.+,
CD166.sup.+, SH3.sup.+ or SH4.sup.+. In another embodiment, the
CD34.sup.-, CD10.sup.+, CD105.sup.+, CD200.sup.+ placental stem
cells are additionally CD44.sup.+. In another embodiment, the
CD34.sup.-, CD10.sup.+, CD105.sup.+, CD200.sup.+ placental stem
cells are additionally one or more of CD3.sup.-, CD9.sup.-,
CD13.sup.+, CD29.sup.+, CD33.sup.+, CD38.sup.-, CD44.sup.+,
CD45.sup.-, CD54.sup.+, CD62E.sup.-, CD62L.sup.-, CD62P.sup.-,
SH3.sup.+ (CD73.sup.+), SH4.sup.+ (CD73.sup.+), CD80.sup.-,
CD86.sup.-, CD90.sup.+, SH2.sup.+ (CD105+), CD106/VCAM.sup.+,
CD117.sup.-, CD144/VE-cadherin.sup.low, CD184/CXCR4.sup.-,
CD133.sup.-, OCT-4.sup.+, SSEA3.sup.-, SSEA4.sup.-, ABC-p.sup.+,
KDR.sup.- (VEGFR2.sup.-), HLA-A,B,C.sup.+, HLA-DP,DQ,DR.sup.-,
HLA-G.sup.-, or Programmed Death-1 Ligand (PDL1).sup.+, or any
combination thereof. In another embodiment, the CD34.sup.-,
CD10.sup.+, CD105.sup.+, CD200.sup.+ placental stem cells are
additionally CD13.sup.+, CD29.sup.+, CD33.sup.+, CD38.sup.-,
CD44.sup.+, CD45.sup.-, CD54/ICAM.sup.+, CD62E.sup.-, CD62L.sup.-,
CD62P.sup.-, SH3.sup.+ (CD73.sup.+), SH4.sup.+ (CD73+), CD80.sup.-,
CD86.sup.-, CD90.sup.+, SH2.sup.+ (CD105.sup.+), CD106/VCAM.sup.+,
CD117.sup.-, CD144/VE-cadherin.sup.dim, CD146.sup.+, CD166.sup.+,
CD184/CXCR4.sup.-, CD133.sup.-, OCT-4.sup.+, SSEA3.sup.-,
SSEA4.sup.-, ABC-p.sup.+, KDR.sup.- (VEGFR2.sup.-),
HLA-A,B,C.sup.+, HLA-DP,DQ,DR.sup.-, HLA-G.sup.-, and Programmed
Death-1 Ligand (PDL1).sup.+.
[0022] In other embodiments of the methods disclosed herein, the
isolated placental stem cells are CD200.sup.+ and HLA-G.sup.-;
CD73.sup.+, CD105.sup.+, and CD200.sup.+; CD200.sup.+ and
OCT-4.sup.+; CD73.sup.+, CD105.sup.+ and HLA-G.sup.-; CD73.sup.+
and CD105.sup.+; or OCT-4.sup.+; or any combination thereof.
[0023] In certain embodiments of the methods disclosed herein, the
placental stem cells are one or more of CD10.sup.+, CD29.sup.+,
CD34.sup.-, CD38.sup.-, CD44.sup.+, CD45.sup.-, CD54.sup.+,
CD90.sup.+, SH2.sup.+, SH3.sup.+, SH4.sup.+, SSEA3.sup.-,
SSEA4.sup.-, OCT-4.sup.+, MHC-I.sup.+ or ABC-p.sup.+, where ABC-p
is a placenta-specific ABC transporter protein (also known as
breast cancer resistance protein (BCRP) and as mitoxantrone
resistance protein (MXR)). In another embodiment, the placental
stem cells are CD10.sup.+, CD29.sup.+, CD34.sup.-, CD38.sup.-,
CD44.sup.+, CD45.sup.-, CD54.sup.+, CD90.sup.+, SH2.sup.+,
SH3.sup.+, SH4.sup.+, SSEA3.sup.-, SSEA4.sup.-, and OCT-4.sup.+. In
another embodiment, the placental stem cells are CD10.sup.+,
CD29.sup.+, CD34.sup.-, CD38.sup.-, CD45.sup.-, CD54.sup.+,
SH2.sup.+, SH3.sup.+, and SH4.sup.+. In another embodiment, the
placental stem cells are CD10.sup.+, CD29.sup.+, CD34.sup.-,
CD38.sup.-, CD45.sup.-, CD54.sup.+, SH2.sup.+, SH3.sup.+, SH4.sup.+
and OCT-4.sup.+. In another embodiment, the placental stem cells
are CD10.sup.+, CD29.sup.+, CD34.sup.-, CD38.sup.-, CD44.sup.+,
CD45.sup.-, CD54.sup.+, CD90.sup.+, MHC-1.sup.+, SH2.sup.+,
SH3.sup.+, SH4.sup.+. In another embodiment, the placental stem
cells are OCT-4.sup.+ and ABC-p.sup.+. In another embodiment, the
placental stem cells are SH2.sup.+, SH3.sup.+, SH4.sup.+ and
OCT-4.sup.+. In another embodiment, the placental stem cells are
OCT-4.sup.+, CD34.sup.-, SSEA3.sup.-, and SSEA4.sup.-. In a
specific embodiment, said OCT-4.sup.+, CD34.sup.-, SSEA3.sup.-, and
SSEA4.sup.- placental stem cells are additionally CD10.sup.+,
CD29.sup.+, CD34.sup.-, CD44.sup.+, CD45.sup.-, CD54.sup.+,
CD90.sup.+, SH2.sup.+, SH3.sup.+, and SH4.sup.+. In another
embodiment, the placental stem cells are OCT-4.sup.+ and
CD34.sup.-, and either SH3.sup.+ or SH4.sup.+. In another
embodiment, the placental stem cells are CD34.sup.- and either
CD10.sup.+, CD29.sup.+, CD44.sup.+, CD54.sup.+, CD90.sup.+, or
OCT-4.sup.+. In certain embodiments, the placental stem cells are
CD10.sup.+, CD34.sup.-, CD105.sup.+ and CD200.sup.+.
[0024] In another embodiment, the placental stem cells are one or
more of CD10.sup.+, CD29.sup.+, CD44.sup.+, CD45.sup.-,
CD54/ICAM.sup.-, CD62-E.sup.-, CD62-L.sup.-, CD62-P.sup.-,
CD80.sup.-, CD86.sup.-, CD103.sup.-, CD104.sup.-, CD105.sup.+,
CD106/VCAM.sup.+, CD144/VE-cadherin.sup.dim, CD184/CXCR4.sup.-,
.beta.2-microglobulin.sup.dim, MHC-I.sup.dim, MHC-II.sup.-,
HLA-G.sup.dim, and/or PDL1.sup.dim. In certain embodiments, such
placental stem cells or population of isolated placental stem cells
are at least CD29.sup.+ and CD54.sup.-. In another embodiment, such
placental stem cells are at least CD44.sup.+ and CD106.sup.+. In
another embodiment, such placental stem cells are at least
CD29.sup.+.
[0025] In certain embodiments of any of the above characteristics,
expression of the cellular marker (e.g., cluster of differentiation
or immunogenic marker) is determined by flow cytometry. In certain
other embodiments, expression of the cellular marker is determined
by RT-PCR.
[0026] In another embodiment, the placental stem cells, e.g., said
CD10.sup.+, CD34.sup.-, CD105.sup.+, CD200.sup.+ cells, e.g., the
cells in the aggregate, express one or more genes at a higher leve,
e.g., a detectably higher level, than an equivalent number of bone
marrow-derived mesenchymal stem cells, wherein said one or more
genes are one or more of, or all of, ACTG2, ADARB1, AMIGO2, ARTS-1,
B4GALT6, BCHE, C11orf9, CD200, COL4A1, COL4A2, CPA4, DMD, DSC3,
DSG2, ELOVL2, F2RL1, FLJ10781, GATA6, GPR126, GPRC5B, ICAM1, IER3,
IGFBP7, IL1A, IL6, IL18, KRT18, KRT8, LIPG, LRAP, MATN2, MEST,
NFE2L3, NUAK1, PCDH7, PDLIM3, PKP2, RTN1, SERPINB9, ST3GAL6,
ST6GALNAC5, SLC12A8, TCF21, TGFB2, VTN, and ZC3H12A, and wherein
said bone marrow-derived mesenchymal stem cells have undergone a
number of passages in culture equivalent to the number of passages
said isolated placental stem cells have undergone. In certain
embodiments, said expression of said one or more genes is
determined, e.g., by RT-PCR or microarray analysis, e.g., using a
U133-A microarray (Affymetrix). In another embodiment, said
placental stem cells express, e.g., differentially express, said
one or more genes when cultured for, e.g., anywhere from about 3 to
about 35 population doublings, in a medium comprising 60% DMEM-LG
(e.g., from Gibco) and 40% MCDB-201 (e.g., from Sigma); 2% fetal
calf serum (e.g., from Hyclone Labs.); 1.times.
insulin-transferrin-selenium (ITS); 1.times. linoleic acid-bovine
serum albumin (LA-BSA); 10.sup.-9 M dexamethasone (e.g., from
Sigma); 10.sup.-4 M ascorbic acid 2-phosphate (e.g., from Sigma);
epidermal growth factor 10 ng/mL (e.g., from R&D Systems); and
platelet-derived growth factor (PDGF-BB) 10 ng/mL (e.g., from
R&D Systems). In another embodiment, said placental stem cells
express, e.g., differentially express, said one or more genes when
cultured for from about 3 to about 35 population doublings in a
medium comprising 60% DMEM-LG (e.g., from Gibco) and 40% MCDB-201
(e.g., from Sigma); 2% fetal calf serum (e.g., from Hyclone Labs.);
1.times. insulin-transferrin-selenium (ITS); 1.times. linoleic
acid-bovine serum albumin (LA-BSA); 10.sup.-9 M dexamethasone
(e.g., from Sigma); 10.sup.-4 M ascorbic acid 2-phosphate (Sigma);
epidermal growth factor 10 ng/mL (e.g., from R&D Systems); and
platelet-derived growth factor (PDGF-BB) 10 ng/mL (e.g., from
R&D Systems).
[0027] In certain embodiments, the placental stem cells express
CD200 and ARTS1 (aminopeptidase regulator of type 1 tumor necrosis
factor); ARTS-1 and LRAP (leukocyte-derived arginine
aminopeptidase); IL6 (interleukin-6) and TGFB2 (transforming growth
factor, beta 2); IL6 and KRT18 (keratin 18); IER3 (immediate early
response 3), MEST (mesoderm specific transcript homolog) and TGFB2;
CD200 and IER3; CD200 and IL6; CD200 and KRT18; CD200 and LRAP;
CD200 and MEST; CD200 and NFE2L3 (nuclear factor (erythroid-derived
2)-like 3); or CD200 and TGFB2 at a higher level, e.g., a
detectably higher level, than an equivalent number of bone
marrow-derived mesenchymal stem cells (BM-MSCs) wherein said bone
marrow-derived mesenchymal stem cells have undergone a number of
passages in culture equivalent to the number of passages said
placental stem cells have undergone. In other embodiments, the
placental stem cells express ARTS-1, CD200, IL6 and LRAP; ARTS-1,
IL6, TGFB2, IER3, KRT18 and MEST; CD200, IER3, IL6, KRT18, LRAP,
MEST, NFE2L3, and TGFB2; ARTS-1, CD200, IER3, IL6, KRT18, LRAP,
MEST, NFE2L3, and TGFB2; or IER3, MEST and TGFB2 at a higher level,
e.g., a detectably higher level, than an equivalent number of bone
marrow-derived mesenchymal stem cells BM-MSCs, wherein said bone
marrow-derived mesenchymal stem cells have undergone a number of
passages in culture equivalent to the number of passages said
placental stem cells have undergone.
[0028] In various embodiments, said placental stem cells useful in
the methods disclosed herein are contained within a population of
cells, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the cells of
which are said placental stem cells. In certain other embodiments,
the placental stem cells in said population of cells are
substantially free of cells having a maternal genotype; e.g., at
least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
98% or 99% of the placental stem cells in said population have a
fetal genotype, i.e., are fetal in origin. In certain other
embodiments, the population of cells comprising said placental stem
cells are substantially free of cells having a maternal genotype;
e.g., at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 98% or 99% of the cells in said population have a fetal
genotype, i.e., are fetal in origin. In certain other embodiments,
the population of cells comprising said placental stem cells
comprise cells having a maternal genotype; e.g., at least 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 98% or 99% of the cells in said population have a
maternal genotype, i.e., are maternal in origin.
[0029] In an embodiment of any of the embodiments of placental stem
cells herein, the placental stem cells facilitate the formation of
one or more embryoid-like bodies in a population of placental cells
comprising said isolated placental stem cells when said population
is cultured under conditions that allow the formation of an
embryoid-like body e.g., culture under proliferation
conditions).
[0030] In certain embodiments of any of the placental stem cells or
BM-MSCs disclosed herein, the cells are mammalian, e.g., human.
[0031] In certain embodiments, any of the placental stem cells
and/or BM-MSCs described herein are autologous to a recipient,
e.g., an individual who has a bone-related cancer, e.g., an
individual who has multiple myeloma, or has a symptom of a
bone-related cancer, e.g., a symptom of multiple myeloma. In
certain other embodiments, the placental stem cells and/or BM-MSCs
are allogeneic to a recipient, e.g., an individual who has a
bone-related cancer, e.g., an individual who has multiple myeloma,
or has a symptom of a bone-related cancer, e.g., a symptom of
multiple myeloma.
[0032] In certain embodiments of the methods of treatment or
methods of suppressing bone-related cancer cell proliferation
disclosed herein, the placental stem cells and/or BM-MSCs are
cryopreserved prior to said administering. In another embodiment,
said placental stem cells are obtained from a cell bank, e.g., a
placental stem cell bank. In another embodiment, said BM-MSCs are
obtained from a bank of bone marrow-derived mesenchymal stem
cells.
[0033] In any of the embodiments of placental stem cells herein,
the placental stem cells generally do not differentiate during
culturing in growth medium, i.e., medium formulated to promote
proliferation, e.g., during proliferation in growth medium. In
another embodiment, said placental stem cells do not require a
feeder layer in order to proliferate, e.g., do not require a feeder
layer to proliferate when cultured in growth medium. In another
embodiment, said placental stem cells do not differentiate in
culture solely as the result of culture in the absence of a feeder
cell layer.
[0034] In any of the embodiments of isolated BM-MSCs herein, the
cells generally do not differentiate during culturing in growth
medium, i.e., medium formulated to promote proliferation, e.g.,
during proliferation in growth medium. In another embodiment, said
isolated BM-MSCs do not require a feeder layer in order to
proliferate, e.g., do not require a feeder layer to proliferate
when cultured in growth medium. In another embodiment, said
isolated BM-MSCs do not differentiate in culture solely as the
result of culture in the absence of a feeder cell layer.
[0035] In certain embodiments, said placental stem cells are
obtained by perfusion of a post-partum placenta that has been
drained of blood and perfused to remove residual blood; drained of
blood but not perfused to remove residual blood; or neither drained
of blood nor perfused to remove residual blood. In another specific
embodiment, said placental stem cells are obtained by physical
and/or enzymatic disruption of placental tissue. In another
specific embodiment, said placental stem cells are obtained by
culturing a portion of a placenta and allowing the placental stem
cells to proliferate out of said portion of a placenta.
[0036] Cell surface, molecular and genetic markers characteristic
of placental stem cells useful in the methods provided herein are
described in detail in Section 5.2, below.
[0037] In another specific embodiment of the method, a
therapeutically effective amount of said placental stem cells
and/or BM-MSCs is a number of cells that results in elimination of,
a detectable improvement in, lessening of the severity of, or
slowing of the progression of one or more symptoms of, a
bone-related cancer, e.g., multiple myeloma. In a specific
embodiment, said symptom of a bone-related cancer, e.g., said
symptom of multiple myeloma, is a bone lesion. In another specific
embodiment, said therapeutically effective amount of placental stem
cells and/or BM-MSCs increases, e.g., detectably increases, bone
mineral density (BMD) in at least one bone of an individual
receiving the cells, e.g., as measured by densitometry, or bone
mineral content (BMC), e.g., as measured by densitometry. In
another specific embodiment, said therapeutically effective amount
of placental stem cells and/or BM-MSCs reduces, e.g., detectably
reduces, a bone lesion, e.g., at least one bone lesion, caused by
said bone-related cancer, e.g., as visible by X-ray, MRI, or CAT
scan, or the like.
[0038] In another specific embodiment, said placental stem cells
and/or BM-MSCs are administered to an individual having a
bone-related cancer, e.g., multiple myeloma, at or adjacent to a
bone lesion caused by said bone-related cancer, i.e.,
intralesionally. In another specific embodiment of the methods
described above, said isolated placental stem cells and/or BM-MSCs
are administered by bolus injection. In another specific
embodiment, said placental stem cells and/or BM-MSCs are
administered by intravenous infusion. In a specific embodiment,
said intravenous infusion is intravenous infusion over about 1 to
about 8 hours. In another specific embodiment, said placental stem
cells and/or BM-MSCs are administered intracranially. In another
specific embodiment, said isolated placental stem cells are
administered intraperitoneally. In another specific embodiment,
said placental stem cells and/or BM-MSCs are administered
intra-arterially. In another specific embodiment of the method of
treatment, said placental stem cells and/or BM-MSCs are
administered intramuscularly, intradermally, subcutaneously, or
intraocularly.
[0039] In another embodiment of the methods described above, said
placental stem cells and/or BM-MSCs are administered by surgical
implantation into said individual of a composition of matter
comprising said cells, e.g., at or adjacent to a bone lesion caused
by a bone-related cancer. In a specific embodiment, said
composition of matter is a matrix or scaffold. In another specific
embodiment, said matrix or scaffold is a hydrogel. In another
specific embodiment, said matrix or scaffold is a decellularized
tissue. In another specific embodiment, said matrix or scaffold is
a synthetic biodegradable composition. In another specific
embodiment, said matrix or scaffold is a foam. In another specific
embodiment, said matrix or scaffold is a physiologically-acceptable
ceramic material, e.g., mono-, di-, tri-, alpha-tri-, beta-tri-,
and tetra-calcium phosphate, hydroxyapatite, a fluoroapatite, a
calcium sulfate, a calcium fluoride, a calcium oxide, a calcium
carbonate, a magnesium calcium phosphate, a biologically active
glass (e.g., BIOGLASS.RTM.), or a mixture of any thereof. In
another specific embodiment, said matrix or scaffold is a porous
biocompatible ceramic material (e.g., SURGIBONE.RTM., ENDOBON.RTM.,
CEROS.RTM. or the like), or a mineralized collagen bone grafting
product (e.g., HEALOS.TM., VITOSS.RTM., RHAKOSS.TM., and
CORTOSS.RTM., or the like).
[0040] In another specific embodiment of the methods described
above, said placental stem cells and/or BM-MSCs are administered
once to said individual. In another specific embodiment, said
placental stem cells and/or BM-MSCs are administered to said
individual in two or more separate administrations. In another
specific embodiment, said administering comprises administering
between about 1.times.10.sup.4 and 1.times.10.sup.5 placental stem
cells and/or BM-MSCs, e.g., per kilogram of said individual. In
another specific embodiment, said administering comprises
administering between about 1.times.10.sup.5 and 1.times.10.sup.6
placental stem cells and/or BM-MSCs per kilogram of said
individual. In another specific embodiment, said administering
comprises administering between about 1.times.10.sup.6 and
1.times.10.sup.7 placental stem cells and/or BM-MSCs per kilogram
of said individual. In another specific embodiment, said
administering comprises administering between about
1.times.10.sup.7 and 1.times.10.sup.8 placental stem cells and/or
BM-MSCs per kilogram of said individual. In other specific
embodiments, said administering comprises administering between
about 1.times.10.sup.6 and about 2.times.10.sup.6 placental stem
cells and/or BM-MSCs per kilogram of said individual; between about
2.times.10.sup.6 and about 3.times.10.sup.6 placental stem cells
and/or BM-MSCs per kilogram of said individual; between about
3.times.10.sup.6 and about 4.times.10.sup.6 placental stem cells
and/or BM-MSCs per kilogram of said individual; between about
4.times.10.sup.6 and about 5.times.10.sup.6 placental stem cells
and/or BM-MSCs per kilogram of said individual; between about
5.times.10.sup.6 and about 6.times.10.sup.6 placental stem cells
and/or BM-MSCs per kilogram of said individual; between about
6.times.10.sup.6 and about 7.times.10.sup.6 placental stem cells
and/or BM-MSCs per kilogram of said individual; between about
7.times.10.sup.6 and about 8.times.10.sup.6 placental stem cells
and/or BM-MSCs per kilogram of said individual; between about
8.times.10.sup.6 and about 9.times.10.sup.6 placental stem cells
and/or BM-MSCs per kilogram of said individual; or between about
9.times.10.sup.6 and about 1.times.10.sup.7 placental stem cells
and/or BM-MSCs per kilogram of said individual. In another specific
embodiment, said administering comprises administering between
about 1.times.10.sup.7 and about 2.times.10.sup.7 placental stem
cells and/or BM-MSCs per kilogram of said individual to said
individual. In another specific embodiment, said administering
comprises administering between about 1.3.times.10.sup.7 and about
1.5.times.10.sup.7 placental stem cells and/or BM-MSCs per kilogram
of said individual to said individual. In another specific
embodiment, said administering comprises administering up to about
3.times.10.sup.7 placental stem cells and/or BM-MSCs per kilogram
of said individual to said individual. In a specific embodiment,
said administering comprises administering between about
5.times.10.sup.6 and about 2.times.10.sup.7 placental stem cells
and/or BM-MSCs to said individual. In another specific embodiment,
said administering comprises administering about 150.times.10.sup.6
placental stem cells and/or BM-MSCs in about 20 milliliters of
solution to said individual.
[0041] In certain embodiments of the methods described above, said
BM-MSCs are used in an amount, by numbers of cells, generally at
least 50% greater than for said placental stem cells.
[0042] In a specific embodiment, said administering comprises
administering between about 5.times.10.sup.6 and about
2.times.10.sup.7 placental stem cells and/or BM-MSCs to said
individual, wherein said cells are contained in a solution
comprising 10% dextran, e.g., dextran-40, 5% human serum albumin,
and optionally an immunosuppressant.
[0043] In another specific embodiment, said administering comprises
administering between about 5.times.10.sup.7 and 3.times.10.sup.9
placental stem cells and/or BM-MSCs intravenously. In specific
embodiments, said administering comprises administering about
9.times.10.sup.8 placental stem cells and/or BM-MSCs or about
1.8.times.10.sup.9 placental stem cells and/or BM-MSCs
intravenously. In another specific embodiment, said administering
comprises administering between about 5.times.10.sup.7 and
1.times.10.sup.8 placental stem cells and/or BM-MSCs
intralesionally. In another specific embodiment, said administering
comprises administering about 9.times.10.sup.7 placental stem cells
and/or BM-MSCs intralesionally.
[0044] In another specific embodiment of the method of treatment,
said placental stem cells and/or BM-MSCs are administered to said
individual within 21-30, e.g., 21 days; within 7 days; within 48
hours; or within 24 hours of diagnosis of a bone-related cancer,
e.g., multiple myeloma, or development of one or more symptoms of a
bone-related cancer.
[0045] The placental stem cells and/or BM-MSCs used in the methods
provided herein can, in certain embodiments, be genetically
engineered to produce one or more proteins that suppress the growth
or proliferation of cells of a bone-related cancer, e.g., multiple
myeloma cells. For example, in certain embodiments, said one or
more proteins can comprise osteoprotegerin, one or more bone
morphogenetic proteins (BMPs); one or more connexins, e.g.,
connexin 26 (Cx26) and/or connexin 43 (Cx43); osteocontin; or
activin A. In other embodiments, the placental stem cells and/or
BM-MSCs have been engineered to express exogenous IFN-.beta. or
IL-2, e.g., in an amount that results in greater, e.g., detectably
greater, suppression of tumor cell proliferation, when said tumor
cells are contacted with said placental stem cells and/or BM-MSCs
compared to such cells not expressing exogenous IFN-.beta. or IL-2.
Also provided herein are pharmaceutical compositions comprising
such genetically-engineered placental stem cells and/or BM-MSCs for
use in suppressing the growth or proliferation of bone-related
cancer cells, e.g., multiple myeloma cells, or for treating an
individual having bone-related cancer cells, e.g., multiple myeloma
cells.
3.1 DEFINITIONS
[0046] As used herein, the term "about," when referring to a stated
numeric value, indicates a value within plus or minus 10% of the
stated numeric value.
[0047] As used herein, "bone lesion," in the context of a
bone-related cancer, means an anomaly in the growth or structure of
a bone, which is caused by, or is a symptom of, the bone-related
cancer. In a non-limiting example, multiple myeloma generally
causes lytic bone lesions, which are hollowed-out areas of a bone
caused by demineralization of the bone. In another non-limiting
example, chondrosarcoma generally causes lesions characterized by a
growth on one or more bones, usually comprising a cartilaginous
growth that may be calcified.
[0048] As used herein, "bone marrow-derived mesenchymal stem
cells," also referred to as BM-MSCs, refers to mesenchymal stem
cells obtained from bone marrow, or cultured from mesenchymal stem
cells obtained from bone marrow, e.g., the cells disclosed in U.S.
Pat. No. 5,486,359, the disclosure of which is incorporated by
reference herein.
[0049] As used herein, the term "bone-related cancer" refers to a
cancer that, in any phase of the disease, affects or metastasizes
to one or more bones in an individual having the cancer. For
example, multiple myeloma is a bone-related cancer because the
cancer affects bones; one aspect of multiple myeloma is the
development of bone lesions due at least in part to upregulation of
osteoclast activity resulting from cytokines secreted by multiple
myeloma cells.
[0050] As used herein, "contacting," in the context of contacting
placental stem cells with cells of a bone-related cancer,
encompasses, but does not require, placing the cells in such
proximity such that they actually physically contact each other
(e.g., in co-culture in a multiwell plate or the like), and placing
the cells in the same space, without actual physical contact, but
in the same space, e.g., in a TRANSWELL.RTM. culture system or
administration to an individual, e.g., a human. "Contacting" as
used herein encompasses bringing the placental stem cells and
bone-related cancer cells together in vitro, e.g., in a single
container (e.g., culture dish, flask, vial, etc.). "Contacting"
also encompasses bringing placental stem cells and tumor cells
together or in vivo, for example, in the same individual (e.g.,
mammal, for example, mouse, rat, dog, cat, sheep, goat, horse,
human, etc.). For example, placental stem cells can be contacted
with bone-related cancer cells by administering the placental stem
cells intravenously to an individual having said bone-related
cancer, or by direct injection into the site of a tumor, e.g., a
bone lesion caused by a bone-related cancer, or the like. Placental
stem cells can be contacted with bone-related cancer cells by
administering the placental stem cells and bone-related cancer
cells intravenously to, for example, an experimental animal.
[0051] As used herein, the term "SH2" refers to an antibody that
binds an epitope on the cellular marker CD105. Thus, cells that are
referred to as SH2.sup.+ are CD105.sup.+.
[0052] As used herein, the terms "SH3" and SH4" refer to antibodies
that bind epitopes present on the cellular marker CD73. Thus, cells
that are referred to as SH3.sup.+ and/or SH4.sup.+ are
CD73.sup.+.
[0053] A placenta has the genotype of the fetus that develops
within it, but is also in close physical contact with maternal
tissues during gestation. As such, as used herein, the term "fetal
genotype" means the genotype of the fetus, e.g., the genotype of
the fetus associated with the placenta from which particular
isolated placental stem cells, as described herein, are obtained,
as opposed to the genotype of the mother that carried the fetus. As
used herein, the term "maternal genotype" means the genotype of the
mother that carried the fetus, e.g., the fetus associated with the
placenta from which particular isolated placental stem cells, as
described herein, are obtained.
[0054] As used herein, stem cells, e.g., placental stem cells, are
"isolated" if at least 50%, 60%, 70%, 80%, 90%, 95%, or at least
99% of the other cells with which the stem cells are naturally
associated are removed from the stem cells, e.g., during collection
and/or culture of the stem cells.
[0055] As used herein, "multipotent," when referring to a cell,
means that the cell has the ability to differentiate into some, but
not necessarily all, types of cells of the body, or into cells
having characteristics of some, but not all, types of cells of the
body, or into cells of one or more of the three germ layers. In
certain embodiments, for example, isolated placental stem cells
(PDAC), as described in Section 5.2, below, that have the capacity
to differentiate into cells having characteristics of neurogenic,
chondrogenic and/or osteogenic cells are multipotent cells.
[0056] As used herein, the term "population of isolated cells"
means a population of cells that is substantially separated from
other cells of the tissue, e.g., placenta, from which the
population of cells is derived or isolated. In certain embodiments,
the population of cells is separated from at least 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of other cells of the
tissue, e.g., placenta, from which the population of cells is
derived or isolated.
[0057] As used herein, the term "placental stem cell" refers to a
stem cell or progenitor cell that is derived from a mammalian
placenta, e.g., as described below, either as a primary isolate or
a cultured cell regardless of whether the cell is a primary cell,
part of a primary cell culture, or has been passaged after a
primary culture. A cell e.g., a "placental stem cell," is
considered a "stem cell" if the cell displays one, two, or all
three of a marker or gene expression profile associated with one or
more types of stem cells; the ability to replicate at least 10-40
times in culture; and the ability to differentiate into cells
displaying characteristics of differentiated cells of one or more
of the three germ layers. Unless otherwise noted herein, the term
"placental" includes the umbilical cord. The isolated placental
cells, e.g., placental stem cells, disclosed herein, in certain
embodiments, differentiate in vitro under differentiating
conditions), differentiate in vivo, or both.
[0058] As used herein, a cell or population of cells is "positive"
for a particular marker when that marker is detectable above
background, via, for example, antibody-mediated or nucleic
acid-mediated detection. Detection of a particular marker can, for
example, be accomplished either by use of antibodies, or by
oligonucleotide probes or primers based on the sequence of the gene
or mRNA encoding the marker. For example, a placental stem cell is
positive for, e.g., CD73 because CD73 is detectable on placental
stem cells in an amount greater, e.g., detectably greater, than
background (in comparison to, e.g., an antibody isotype control). A
cell is also positive for a marker when that marker can be used to
distinguish the cell from at least one other cell type, or can be
used to select or isolate the cell when present or expressed by the
cell. In the context of, e.g., antibody-mediated detection,
"positive," as an indication a particular cell surface marker is
present, means that the marker is detectable using an antibody,
e.g., a fluorescently-labeled antibody, specific for that marker;
"positive" also refers to a cell exhibiting the marker in an amount
that produces a signal, e.g., in a cytometer, that is above, e.g.,
detectably above, background. For example, a cell is "CD200.sup.+"
where the cell is labeled, e.g., detectably labeled, with an
antibody specific to CD200, and the signal from the antibody is
higher, e.g., detectably higher than that of a control (e.g.,
background or an isotype control). For example, a cell or
population of cells can be determined to be OCT-4.sup.+ if the
amount of OCT-4 RNA detected in RNA from the cell or population of
cells is detectably greater than background as determined, e.g., by
a method of detecting RNA such as RT-PCR, slot blots, etc. In
certain embodiments, OCT-4 is determined to be present, and a cell
is "OCT-4.sup.+" if OCT-4 is detectable using RT-PCR. Unless
otherwise noted herein, cluster of differentiation ("CD") markers
are detected using antibodies. With respect to HLA-G, a population
of placental stem cell is, in certain embodiments, positive for
HLA-G if more than 5% the cells in the population are positive for
HLA-G, e.g., detectably stain with an antibody against HLA-G.
[0059] As used herein for all markers except HLA-G, a placental
stem cell is "negative" for a particular cellular marker if the
cellular marker is not detectable, e.g., using an antibody specific
for that marker compared to a control (e.g., background or an
isotype control), or is not detectable using a nucleic acid-based
detection method, e.g., RT-PCR. For example, a cell is "CD34.sup.-"
where the cell is not reproducibly detectably labeled with an
antibody specific to CD34 to a greater degree than a control (e.g.,
background or an isotype control). Markers, e.g., markers not
detected, or not detectable, using antibodies, can be determined to
be positive or negative in a similar manner, using an appropriate
control, using other, for example, nucleic acid-mediated detection
methods. Unless otherwise noted herein, cluster of differentiation
("CD") markers are detected using antibodies. With respect to
HLA-G, a population of placental stem cell is, in certain
embodiments, negative for HLA-G if 5% or fewer of the cells in the
population are positive for HLA-G, e.g., detectably stain with an
antibody against HLA-G.
[0060] As used herein, the designation "low" or "dim," when
referring to the expression of a marker detectable in flow
cytometry, means that the marker is expressed by fewer than 10% of
cells tested, or that fluorescence attributable to the marker in,
e.g., flow cytometry, is less than 1 log above background.
[0061] As used herein, "treat" encompasses the cure of, remediation
of, improvement of, lessening of the severity of, or reduction in
the time course of, a disease, disorder or condition, or any
parameter or symptom thereof.
4. BRIEF DESCRIPTION OF THE FIGURES
[0062] FIG. 1: Placental stem cells improve repair of bone defects
in experimental rats. Y axis: degree of skull bone defect closure,
as assessed by area. X axis conditions: HEALOS.RTM. alone;
HEALOS.RTM. in combination with bone morphogenctic protein-2
(BMP-2); HEALOS.RTM. and placental stem cells; HEALOS.RTM. and bone
marrow-derived mesenchymal stem cells (BM-MSCs); or no repair
(empty). Asterisk indicates significant (p<0.05) improvement in
repair of the defect in HEALOS.RTM. in combination with bone
morphogenetic protein-2 (BMP-2); HEALOS.RTM. in combination with
placental stem cells; and HEALOS.RTM. in combination with BM-MSCs,
versus controls.
[0063] FIG. 2: Placental stem cells suppress the differentiation of
osteoclast precursors. X axis: osteoclasts (OC); osteoclast
precursors not co-cultured with placental stem cells, or bone
marrow-derived mesenchymal stem cells (BM-MSCs). Y axis: numbers of
mature osteoclasts formed.
[0064] FIG. 3: Placental stem cells reduce the growth of tumor
cells from different individuals. Multiple myeloma cells (cell
lines BN, JB, ARP1, U266, Dn and Hale) were transfected with a gene
encoding luciferase and co-cultured with (from left to right in
each condition) fetal mesenchymal stem cells (FB-MSC), patient bone
marrow-derived mesenchymal stem cells (Pt-MSC), or placental stem
cells. Reduction of multiple myeloma cell growth after several
weeks was expressed as fold-value of luciferase expression in
multiple myeloma cells co-cultured with placental stem cells or
Pt-MSC compared to luciferase expression by multiple myeloma cells
co-cultured with FB-MSCs.
[0065] FIG. 4: Diagram of a TRANSWELL.RTM. experiment, in which
placental stem cells or mesenchymal stem cells are cultured on the
underside of a membrane, and multiple myeloma cells are cultured on
the upper side of the membrane.
[0066] FIG. 5: Suppression of multiple myeloma cells from six
different patients (X-axis) by placental stem cells (PDACs) and
fetal bone marrow-derived mesenchymal stem cells (Fetal MSCs).
Y-axis: percent viability compared to myeloma cells cultured in the
absence of placental stem cells.
[0067] FIG. 6: Placental stem cells reduce multiple myeloma cell
growth in SCID-rab/SCID-hu mice. The number of multiple myeloma
cells was assessed by the titer of human antibody present in sera
from the mice, as assessed by ELISA. Pre-Rx: titer of human
antibody before administration of myeloma cells. 2WK, 4WK: titer of
human antibody two weeks and four weeks post-administration of
multiple myeloma cells, either alone (control) or with placental
stem cells.
[0068] FIG. 7: Change in bone mass density, as assessed by X-rays,
of bone implants in SCID-rab/SCID-hu mice. BMD: bone mineral
density.
[0069] FIGS. 8-9: Placental stem cells effects on myeloma bone
disease and tumor growth is dose dependent and comparable with
fetal MSCs. SCID-rab mice engrafted with patient's 2 myeloma cells.
(A-C) Upon establishment of high tumor burden hosts were
intralesionally injected with vehicle, with 0.1, 0.5 and
1.times.10.sup.6 placental stem cells, or subcutaneously engrafted
with 5.times.10.sup.6 placental stem cells using HyStem-C hydrogel
carrier (see Methods for details) (6-7 mice/group).
[0070] FIG. 8: Changes in human immunoglobulin (Ig) prior to
treatment (Prc-Rx), 2 and 4 weeks after treatment with placental
stem cells. IL=intralesional administration of 0.1, 0.5 or
1.times.10.sup.6 cells. SC=subcutaneous administration of cells.
CONT=control (no cells).
[0071] FIG. 9: Changes in bone mineral density from pretreatment
levels of the implanted myelomatous bone. IL=intralesional
administration of 0.1, 0.5 or 1.times.10.sup.6 cells.
SC=subcutaneous administration of cells. CONT=control (no
cells).
[0072] FIG. 10: Hosts were intralesionally injected with vehicle,
or with 1.times.10.sup.6 placental stem cells or MSCs. FIG. 10
depicts changes in bone mineral density from pretreatment levels of
the implanted myelomatous bone. Left condition: control (no cells);
middle condition: placental stem cells; right condition: bone
marrow-derived mesenchymal stem cells.
[0073] FIG. 11: Hosts were intralesionally injected with vehicle,
or with 1.times.10.sup.6 placental stem cells or MSCs. FIG. 11
depicts changes in human Ig prior to treatment (Pre-Rx) and
experiment's end. Left condition: control (no cells); middle
condition: placental stem cells; right condition: bone
marrow-derived mesenchymal stem cells.
[0074] FIG. 12: Comparison of the average number (n=3) of multiple
myeloma cell line cells (U-266, RPMI-8226, L363 and OMP-2) per well
in the presence or absence of placental stem cells on day 5 of
culture or co-culture. P-values are provided for control and
experimental conditions for each multiple myeloma cell line.
[0075] FIGS. 13A-13C: Reduction in the phosphorylation of
retinoblastoma (Rb) protein at Serine 780 (S780), or at serines 807
and 811 (S807/S811) for multiple myeloma cell lines H929 (FIG.
13A), OPM-2 (FIG. 13B) and LP1 (FIG. 13C). D2: Day 2 of co-culture.
D4: Day 4 of co-culture. GM: geometric mean of increase/decrease in
Rb phosphorylation. .DELTA.: Change in geometric mean.
[0076] FIG. 14: Number of osteoclasts formed when cultured in the
presence of 1 .mu.M lenalidomide. Thick horizontal line represents
the number of osteoclasts formed in the control wells.
[0077] FIG. 15: Number of osteoclasts formed when co-cultured with
placental stem cells (PDACs) alone, bone marrow-derived mesenchymal
stem cells (BM-MCS), or the combination of PDACs and
lenalidomide.
5. DETAILED DESCRIPTION
5.1 Treatment of Bone-Related Cancers Using Isolated Placental Stem
Cells and/or Mesenchymal Stem Cells
[0078] Provided herein are methods of treating an individual having
a bone-related cancer comprising administering to the individual
isolated placental stem cells, in particular, the isolated
placental stem cells described in detail in Section 5.2, below,
also referred to herein as PDACs (Placenta Derived Adherent Cells),
and/or bone marrow-derived mesenchymal stem cells (BM-MSCs), e.g.,
a therapeutically effective amount of either or both of said cells.
Bone-related cancers include, without limitation, multiple myeloma,
bone cancer, neuroblastoma, osteosarcoma, Ewing's sarcoma,
chondrosarcoma, chordoma, malignant fibrous histiocytoma of bone,
fibrosarcoma of bone, prostate cancer, and any form of metastatic
cancer characterized by bone metastases. In certain embodiments,
the bone-related cancer does not include prostate cancer. In
certain embodiments, administration of isolated placental stem
cells and/or BM-MSCs is therapeutically effective to reduce,
ameliorate or reverse one or more symptoms associated with the
bone-related cancer, e.g., a symptom caused by, associated with, or
related to an effect of the cancer on one or more bones in the
individual, e.g., a bone defect attributable to the bone-related
cancer. Treatment of bone-related cancers with placental stem cells
and/or BM-MSCs as provided herein, can occur before, after, or
concurrently with a second anti-cancer therapy, as discussed below.
Accordingly, in one embodiment, bone defects that are a symptom of
a bone-related cancer are treated before the cancer is treated with
a second anti-cancer therapy. In another embodiment, bone defects
that are a symptom of a bone-related cancer are treated at the same
time, or near the same time, that the cancer is treated with a
second anti-cancer therapy. In another embodiment, bone defects
that are a symptom of a bone-related cancer are treated after the
cancer is treated with a second anti-cancer therapy.
[0079] In one aspect, provided herein are methods of treating an
individual having a bone-related cancer, comprising administering
to the individual a therapeutically effective amount of placental
stem cells and/or BM-MSCs. In one embodiment, provided herein is a
method of treating an individual having a bone-related cancer,
comprising administering to said individual a therapeutically
effective amount of placental stem cells and/or BM-MSCs for a time
sufficient for said placental stem cells and/or BM-MSCs to improve,
e.g., detectably improve, one or more symptoms of, or reduce, e.g.,
detectably reduce, the progression of, said bone-related cancer. In
a specific embodiment, said bone-related cancer is multiple
myeloma. In another specific embodiment, the bone-related cancer is
sarcoma. In other specific embodiments, said bone-related cancer is
bone cancer, neuroblastoma, osteosarcoma, Ewing's sarcoma,
chordoma, malignant fibrous histiocytoma of bone, or fibrosarcoma
of bone. In another specific embodiment, said bone-related cancer
comprises a solid tumor. In another specific embodiment, said
bone-related cancer is not prostate cancer.
[0080] As used herein, "administering for a time sufficient," and
the like, encompasses, for example, administration of cells, e.g.,
a unit of cells, followed by evaluation of the one or more symptoms
of bone-related cancer, e.g., multiple myeloma, over a time
sufficient to determine any change in the one or more symptoms,
e.g., over the course of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours, or 1, 2, 3, 4, 5,
or 6 days, or over 1, 2, 3, or 4 weeks, or the like. If no change
is detected, one or more subsequent administrations of cells can
take place.
[0081] In certain embodiments, treatment of bone-related cancers,
e.g., multiple myeloma, comprises administering an amount, e.g., a
therapeutically effective amount, of isolated placental stem cells
and/or BM-MSCs, to an individual having bone-related cancer cells,
e.g., multiple myeloma cells, wherein at least some of said
placental stem cells and/or BM-MSCs directly contact at least some
of the bone-related cancer cells, e.g., there is direct cell-cell
contact between at least some of said placental stem cells and/or
BM-MSCs, and at least some of said bone-related cancer cells. In
certain other embodiments, treatment of bone-related cancers, e.g.,
multiple myeloma, comprises administering an amount, e.g., a
therapeutically effective amount, of placental stem cells and/or
BM-MSCs to an individual having bone-related cancer cells, e.g.,
multiple myeloma cells, wherein none, or substantially none, of
said placental stem cells and/or BM-MSCs directly contact said
bone-related cancer cells, e.g., there is no, or substantially no,
direct cell-cell contact between most, or any, of said placental
stem cells and/or BM-MSCs, and said bone-related cancer cells.
[0082] In certain embodiments, the placental stem cells and/or
BM-MSCs, are administered intralesionally, e.g., directly into, or
adjacent to (e.g., within about 1-5 cm of) one or more bone lesions
caused by the bone-related cancer. In certain embodiments, the
placental stem cells and/or BM-MSCs, are administered in
combination with a matrix, e.g., an injectable matrix. In certain
other embodiments, the placental stem cells and/or BM-MSCs, are
administered to an individual having a bone-related cancer in
combination with alginate, or with platelet-rich plasma. In certain
other embodiments, the placental stem cells and/or BM-MSCs are
administered to an individual having a bone-related cancer in
combination with a solid matrix, e.g., a bone substitute, a matrix
or bone substitute described in Section 5.7.4, below.
[0083] In certain other embodiments, the placental stem cells
and/or BM-MSCs are administered intravenously to the individual.
The placental stem cells and/or BM-MSCs can be administered from
any container, and by any delivery system, medically suitable for
the delivery of fluids, e.g., fluids comprising cells, to an
individual. Such containers can be, for example, a sterile plastic
bag, flask, jar, or other container from which the placental stem
cells or BM-MSCs can be easily dispensed. For example, the
container can be a blood bag or other plastic, medically-acceptable
bag suitable for the intravenous administration of a liquid to a
recipient.
[0084] Intralesional or intravenous administration can comprise,
e.g., about, at least, or no more than 1.times.10.sup.5,
5.times.10.sup.5, 1.times.10.sup.6, 5.times.10.sup.6,
1.times.10.sup.7, 5.times.10.sup.7, 1.times.10.sup.8,
5.times.10.sup.8, 1.times.10.sup.9, 5.times.10.sup.9,
1.times.10.sup.10, 5.times.10.sup.10, 1.times.10.sup.11 or more
isolated placental stem cells and/or BM-MSCs in a single dose. In
certain embodiments, a dose of BM-MSCs comprises approximately 50%
more cells than a dose of placental stem cells, e.g., PDACs.
[0085] In one embodiment, intralesional or intravenous
administration can comprise about 2.times.10.sup.8 placental stem
cells in a single dose. In another embodiment, intralesional or
intravenous administration can comprise about 8.times.10.sup.8
placental stem cells in a single dose. The isolated placental stem
cells may be administered once, or more than once, during a course
of therapy. Preferably, the administered placental stem cells
comprise at least 50% viable cells or more (that is, at least about
50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% of the placental stem
cells in a population of placental stem cells are functional or
living). Preferably, at least about 60% of the cells in the
population are viable. More preferably, at least about 70%, 80%,
90%, 95%, or 99% of the cells in the population in the
pharmaceutical composition are viable.
[0086] Administration of isolated placental stem cells and/or
BM-MSCs, in addition to treating symptoms of bone-related cancers,
e.g., symptoms of multiple myeloma, such as bone lesions, can
suppress the proliferation or growth of cells of the bone-related
cancer, e.g., multiple myeloma cells. Suppression of proliferation
can encompass, e.g., reducing the growth or proliferation rate of
tumor cells, or killing some or all of the tumor cells. Thus, in
another aspect, provided herein is a method of suppressing
proliferation of cells of a bone-related cancer, comprising
contacting said plurality of tumor cells with a plurality of
placental stem cells and/or BM-MSCs for a time sufficient for said
placental stem cells and/or BM-MSCs to suppress, e.g., detectably
suppress, proliferation of said cells of a bone-related cancer, as
compared to a plurality of said cells of a bone-related cancer not
contacted with placental stem cells and/or BM-MSCs, e.g., as
determinable by a detectable reduction in the number of such cells
after treatment, a detectable reduction in the increase in the
number of such cells after treatment, or the like. In specific
embodiments, said cells of a bone-related cancer are multiple
myeloma cells, bone cancer cells, neuroblastoma cells, osteosarcoma
cells, Ewing's sarcoma cells, chondrosarcoma cells, chordoma cells,
cells of a malignant fibrous histiocytoma of bone, cells of a
cancer that metastasizes to the bone, prostate cancer cells, or
cells of a fibrosarcoma of bone. In another specific embodiment,
said cells of a bone-related cancer are cells of or within a solid
tumor. In another specific embodiment, said cells of a bone-related
cancer are not prostate cancer cells.
[0087] In another specific embodiment, said contacting is performed
in vitro. In another specific embodiment, said contacting is
performed in vivo, e.g., by administration of the cells to an
individual having cells of a bone-related cancer, e.g., multiple
myeloma or a chondrosarcoma. In another specific embodiment, said
individual is a mammal. In another specific embodiment, said mammal
is a human. In another specific embodiment, said contacting
comprises administering said placental stem cells and/or BM-MSCs to
said individual intravenously. In another specific embodiment, said
contacting comprises administering said placental stem cells and/or
BM-MSCs to said individual at or adjacent to a bone lesion caused
by said bone-related cancer, e.g., intralesionally or
intraosseously.
[0088] In another embodiment, the method of suppressing
proliferation of cells of a bone-related cancer, e.g., multiple
myeloma cells, by contacting said cells of a bone-related cancer
with placental stem cells and/or BM-MSCs additionally comprises
contacting said cells of a bone-related cancer with one or more
anticancer compounds, e.g., one or more of the anticancer compounds
in Section 5.1.3, e.g., administering one or more of said
anticancer compounds to said individual.
[0089] In another embodiment, the method comprises administering at
least 1.times.10.sup.7 placental stem cells and/or BM-MSCs to said
individual, by total numbers of cells. In another specific
embodiment, the method comprises administering at least
1.times.10.sup.8 placental stem cells and/or BM-MSCs to said
individual, by total numbers of cells. In another specific
embodiment, said placental stem cells and/or BM-MSCs have been
proliferated in vitro prior to administration for no more than 30
population doublings. In another specific embodiment, said
placental stem cells and/or BM-MSCs have been proliferated in vitro
prior to administration for no more than 10 population doublings.
In another specific embodiment, said placental stem cells and/or
BM-MSCs have been cryopreserved and thawed prior to said
contacting.
[0090] In another specific embodiment of the method, said placental
stem cells and/or BM-MSCs suppress proliferation of said cells of a
bone-related cancer, e.g., by at least 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80% or 90%, e.g., as compared to proliferation of an
equivalent number of cells of a bone-related cancer in the absence
of said placental stem cells and/or BM-MSCs. In certain
embodiments, the percent reduction in proliferation can be assessed
by, for example, comparing a number of bone-related cancer cells in
a tissue (e.g., blood) sample from an individual having the
bone-related cancer before and after administration of the
placental stem cells and/or BM-MSCs. In another specific
embodiment, the method comprises determining, prior to said
contacting, that said placental stem cells and/or BM-MSCs suppress,
e.g., detectably suppress, the proliferation of a sample of said
cells of a bone-related cancer. In such an embodiment, for example,
the placental stem cells and/or BM-MSCs could be determined to
suppress the proliferation of a sample of said cells of a
bone-related cancer by, e.g., taking a sample of a population of
placental stem cells and/or BM-MSCs (for example, a sample from a
unit or lot from a stem cell bank, or the like).
[0091] In any of the methods disclosed herein, e.g., methods of
treatment, methods of suppressing tumor growth, or methods of
suppressing osteoclast maturation, disclosed herein, placental stem
cells, e.g., PDACs, or BM-MSCs may be used alone, or the placental
stem cells and BM-MSCs may be used in combination. When used in
combination, the cells may be combined so as to be administrable at
the same time, e.g., in the same unit of cells; or may be
administrable separately, e.g., maintained in separate cell units,
for example, in separate blood-type bags. When used in combination,
administration of the placental stem cells and BM-MSCs can take
place together, at the same time, or can take place at separate
times.
[0092] Further provided herein is a method of reducing the
maturation of osteoclast precursors into osteoclasts, comprising
contacting said osteoclast precursors with a plurality of isolated
placental stem cells and/or BM-MSCs (e.g., isolated BM-MSCs or
BM-MSCs in bone marrow), wherein said plurality of placental stem
cells and/or BM-MSCs is a number of placental stem cells and/or
BM-MSCs, sufficient to reduce, e.g., detectably reduce, osteoclast
maturation from said osteoclast precursors. In a specific
embodiment, said contacting takes place in vitro. In another
specific embodiment, said contacting takes place in vivo. In
another specific embodiment, said contacting takes place in a
mammal, e.g., in a human. In another specific embodiment, said
contacting takes place in an individual having multiple myeloma, or
comprising multiple myeloma cells, or who has one or more symptoms
of multiple myeloma. In another specific embodiment, said one or
more symptoms of multiple myeloma comprise one or more bone
lesions, or bone pain resulting from osteoclast activity.
[0093] In another embodiment, provided herein is a method of
increasing apoptosis of osteoclast precursors, comprising
contacting said osteoclast precursors with a plurality of placental
stem cells and/or BM-MSCs (e.g., isolated BM-MSCs or BM-MSCs in
bone marrow), wherein said plurality of placental stem cells and/or
BM-MSCs, is a number of placental stem cells and/or BM-MSCs,
sufficient to increase, e.g., detectably increase, osteoclast
precursor apoptosis. In a specific embodiment, said contacting
takes place in vitro. In another specific embodiment, said
contacting takes place in vivo. In another specific embodiment,
said contacting takes place in a human. In another specific
embodiment, said contacting takes place in an individual having
multiple myeloma, or comprising multiple myeloma cells, or who has
one or more symptoms of multiple myeloma. In another specific
embodiment, said one or more symptoms of multiple myeloma comprise
one or more bone lesions, or bone pain resulting from osteoclast
activity. In another specific embodiment, said increase in
osteoclast precursor apoptosis is detected by a detectable increase
in annexin V and propidium iodide staining of osteoclast precursors
from said individual.
[0094] In any of the above embodiments, the placental stem cells
and/or BM-MSCs can be genetically engineered placental stem cells,
e.g., the genetically engineered cells described in Section 5.7.2,
below.
[0095] In any of the methods disclosed herein, said individual is a
mammal. In a specific embodiment, said mammal is a human.
[0096] In any of the methods disclosed herein, the BM-MSCs can be
isolated bone marrow-derived mesenchymal stem cells, e.g., BM-MSCs
that have been cultured or purchased from a commercial source, or
can be BM-MSCs contained within bone marrow, e.g., bone marrow
aspirate, crude bone marrow, or the like.
[0097] 5.1.1 Treatment of Multiple Myeloma
[0098] Provided herein are methods of treating an individual having
multiple myeloma, comprising administering to said individual
placental stem cells and/or BM-MSCs, wherein said isolated
placental stem cells have any combination of, or all of, the
characteristics described in Section 5.2, below.
[0099] Multiple myeloma is a cancer of plasma cells, which are
antibody-producing cells of the immune system. The disease
typically presents with three main characteristics: bone lesions,
the development of which can result in bone pain and elevated blood
calcium; anemia; and renal failure.
[0100] In certain embodiments, provided herein are methods of
treating individuals having one or more multiple myeloma-related
diseases or conditions, or symptoms thereof. In specific
embodiments, said multiple myeloma-related diseases or conditions
are monoclonal gammopathy of unknown significance (MGUS),
smoldering myeloma (e.g., smoldering multiple myeloma), solitary
plasmacytoma, benign monoclonal gammopathy, asymptomatic monoclonal
gammopathy, non-myelomatous monoclonal gammopathy, discrete
monoclonal gammopathy, cryptogenic monoclonal gammopathy, lanthanic
monoclonal gammopathy, rudimentary monoclonal gammopathy,
dysimmunoglobulinemia, asymptomatic paraimmunoglobulinemia, or
idiopathic paraproteincmia. In certain embodiments, a person having
smoldering myeloma exhibits blood paraprotein, but no other
symptoms of multiple myeloma.
[0101] In certain embodiment, the symptoms are as follows.
[0102] Bone Lesions and Bone Pain--
[0103] Myeloma cells secrete osteoclast activating factor, which is
a cytokine that activates osteoclasts to break down bone, creating
painful bone lesions. These bone lesions, visible, e.g., in X-ray
radiographs, are lytic in nature and typically appear as one or
more regions in which the bone appears absent or "punched out."
Myeloma bone pain usually involves the spine and ribs, and worsens
with activity. Persistent localized pain may be present, and can
indicate a pathological bone fracture. Involvement of the vertebrae
may lead to spinal cord compression. The breakdown of bone also
leads to release of calcium into the blood, leading to
hypercalcemia and its associated symptoms.
[0104] Anemia--
[0105] The anemia found in myeloma is usually normocytic and
normochromic, and results from the replacement of normal bone
marrow by infiltrating tumor cells and inhibition of normal red
blood cell production (hematopoiesis) by cytokines.
[0106] Renal Failure--
[0107] Multiple myeloma also tends to result in renal failure,
which may develop both acutely and chronically. Renal failure in
multiple myeloma is largely attributable to hypercalcemia, which
develops as osteoclasts dismantle existing bone. Renal failure is
also caused by tubular damage from excretion of light chains, also
called Bence Jones proteins, which can manifest as the Fanconi
syndrome (type II renal tubular acidosis). Other causes include
glomerular deposition of amyloid, hyperuricemia, recurrent
infections (e.g., pyelonephritis), and local infiltration of tumor
cells. Renal failure can be associated with elevated levels of
serum creatinin.
[0108] Multiple myeloma can present with other symptoms, as well,
as follows.
[0109] Infection--
[0110] Another common symptom of multiple myeloma is infection, as
the immune system is disrupted. The increased risk of infection is
due to immune deficiency resulting from diffuse
hypogammaglobulinemia, which is due to decreased production and
increased destruction of normal antibodies. The most common
infections are pneumonias and pyelonephritis. Common pneumonia
pathogens causing disease in multiple myeloma patients include
Streptococcus pneumoniae, Staphylococcus aureus, and Klebsiella
pneumoniae, while common pathogens causing pyelonephritis include
Escherichia coli. Typically, infection occurs in the initial few
months after the start of chemotherapy.
[0111] Neurological Symptoms--
[0112] Symptoms of multiple myeloma include a spectrum of
neurological conditions, including weakness, confusion and fatigue
due to hypercalcemial headache, visual changes and retinopathy,
which can be the result of hyperviscosity of the blood depending on
the properties of paraprotein (see below). Other neurological
symptoms include radicular pain, loss of bowel or bladder control
(for example, due to involvement of spinal cord leading to cord
compression), and carpal tunnel syndrome and other neuropathics
(for example, due to infiltration of peripheral nerves by amyloid).
Multiple myeloma may give rise to paraplegia in late presenting
cases.
[0113] Presence of Paraprotein--
[0114] A diagnostic symptom of multiple myeloma is the presence in
the blood and/or urine of paraprotein, which is a monoclonal
protein (M protein), e.g., an immunoglobulin light-chain that is
produced by the clonal proliferation of plasma cells, or
immunoglobulin fragments. Presence of paraprotein can be determined
by analyzing protein from urine and/or serum from an individual by
agarose gel electrophoresis, or by immunofixation using one or more
antibodies to an immunoglobulin light or heavy chain.
[0115] Symptomatic multiple myeloma, in certain embodiments, is
diagnosed when the following symptoms or signs are present: clonal
plasma cells constituting greater than 10% of cells on bone marrow
biopsy or, in any quantity in a biopsy from other tissues (e.g.,
plasmacytoma); paraprotein in either serum or urine; evidence of
end-organ damage (related organ or tissue impairment), for example,
hypercalcemia (e.g., corrected calcium greater than about 12 mg per
deciliter of blood, or greater than about 2.75 mmol in the blood),
renal insufficiency attributable to myeloma, anemia defined as
hemoglobin <10 g/dL blood, bone lesions (e.g., lytic lesions or
osteoporosis with compression fractures, frequent severe infections
(>2 a year), amyloidosis (the deposition of amyloid protein) of
other organs, and hyperviscosity syndrome (increase in the
viscosity of blood), e.g., a blood viscosity of above 1.8
centipoises, e.g., a blood viscosity of at least 2, 3, 4, or 5
centipoises.
[0116] Individuals having multiple myeloma, in certain embodiments,
fall into one of the following groups. In one embodiment, the
individual having multiple myeloma has never been treated for the
disease. In another embodiment, the individual has responsive
myeloma; that is, multiple myeloma that is responding to therapy.
In a specific embodiment, such an individual exhibits a decrease in
M protein (paraprotein) of at least 50% as a result of treatment.
In another specific embodiment, the individual exhibits a decrease
in M protein of between 25% and 50% as a result of treatment. In
another embodiment, the individual has stable multiple myeloma,
which refers to myeloma that has not responded to treatment (for
example, the decrease in M protein has not reached 50%), but has
not progressed or gotten worse. In another embodiment, the
individual has progressive multiple myeloma, which refers to active
myeloma that is worsening (for example, increasing M protein and
worsening organ or tissue impairment or end organ damage). In
another embodiment, the individual has relapsed multiple myeloma,
which refers to myeloma disease that initially responded to therapy
but has then begun to progress again. In specific embodiments, the
individual has relapsed after initial therapy or has relapsed after
subsequent therapy. In another embodiment, the individual has
refractory multiple myeloma. In a specific embodiment, the
refractory multiple myeloma is multiple myeloma that has not
responded to initial therapy. In another specific embodiment, the
refractory multiple myeloma is relapsed multiple myeloma that has
not responded to subsequent treatment. In another specific
embodiment, the refractory multiple myeloma is non-responding
progressing refractory disease, which refers to refractory disease
that is progressing. In another specific embodiment, the refractory
multiple myeloma is non-responding non-progressing refractory
disease, which refers to refractory disease that is not
worsening.
[0117] Thus, in one embodiment, provided herein is a method of
treating an individual having multiple myeloma, comprising
administering to the individual isolated placental stem cells
and/or BM-MSCs (e.g., isolated BM-MSCs or BM-MSCs in bone marrow),
wherein said administration results in the detectable reduction of
progression, detectable lessening of worsening, and/or detectable
improvement, of one or more symptoms of multiple myeloma, e.g., any
one or more of the symptoms of multiple myeloma described herein,
without limitation. In specific embodiments, said one or more
symptoms comprise elevated blood or urine calcium compared to
normal, the presence of bone lesions, anemia, or renal failure. In
another specific embodiment, said one or more symptoms comprise
plasma cells, e.g., clonal plasma cells constituting greater than
10% of cells on bone marrow biopsy or, in any quantity in a biopsy
from other tissues (e.g., plasmacytoma); paraprotein in either
serum or urine; and/or evidence of end-organ damage. In another
specific embodiment, said one or more symptoms is a concentration
of calcium in the blood of greater than about 2.75 mmol/L, renal
insufficiency, less than about 10 g hemoglobin per deciliter of
blood, the presence of bone lesions, or amyloidosis of one or more
organs other than bone marrow.
[0118] In another specific embodiment, said symptom is infection,
e.g., infection caused by hypergammaglobulinemia. In certain
embodiments, the infection is pneumonia or pyelonephritis. In
certain embodiments, said infection occurs within 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11 or 12 months following the start of chemotherapy,
e.g., chemotherapy to treat said multiple myeloma.
[0119] In another specific embodiment, said symptom is a
neurological symptom. In other specific embodiments, said
neurological symptoms are weakness, confusion, fatigue, headache,
visual changes, retinopathy, radicular pain, loss of bowel or
bladder control, carpal tunnel syndrome, and/or paraplegia.
[0120] In a specific embodiment, provided herein is a method of
treating an individual having multiple myeloma, comprising
administering to the individual placental stem cells and/or BM-MSCs
(e.g., isolated BM-MSCs or BM-MSCs in bone marrow), wherein said
administration results in the detectable reduction in number of
multiple myeloma cells, e.g., clonal multiple myeloma cells, in one
or more organs or tissues of the individual.
[0121] In a specific embodiment, provided herein is a method of
treating an individual having multiple myeloma, comprising
administering to the individual isolated placental stem cells
and/or BM-MSCs (e.g., isolated BM-MSCs or BM-MSCs in bone marrow),
wherein said administration results in the detectable increase in
hemoglobin in the blood of the individual, e.g., an increase to
within normal limits. Normal hemoglobin levels vary by the age and
sex of the individual, as shown in Table 1A, below:
TABLE-US-00001 TABLE 1A Newborns 17-22 gm/dl One (1) week of age
15-20 gm/dl One (1) month of age 11-15 gm/dl Children 11-13 gm/dl
Adult males 14-18 gm/dl Adult women 12-16 gm/dl Men after middle
age 12.4-14.9 gm/dl Women after middle age 11.7-13.8 gm/dl
[0122] Thus, in another specific embodiment, provided herein is a
method of treating an individual having multiple myeloma,
comprising administering to the individual isolated placental stem
cells and/or BM-MSCs (e.g., isolated BM-MSCs or BM-MSCs in bone
marrow), wherein said administration results in the increase of
blood hemoglobin levels in said individual to between 11 g/dL blood
and 20 g/dL blood. In another specific embodiment, said
administering results in the increase of blood hemoglobin levels in
said individual to between 11 g/dL blood and 13 g/dL blood. In
another specific embodiment, said administering results in the
increase of blood hemoglobin levels in said individual to between
12 g/dL blood and 16 g/dL blood. In another specific embodiment,
said administering results in the increase of blood hemoglobin
levels in said individual to between 14 g/dL blood and 18 g/dL
blood. In another embodiment, provided herein is a method of
treating an individual having anemia, e.g., having less than about
10 g hemoglobin per deciliter of blood, comprising administering to
the individual a therapeutically effective amount of placental stem
cells, wherein said anemia is caused by multiple myeloma, and
wherein said therapeutically effective amount is an amount
sufficient to cause a rise in hemoglobin in blood from the
individual to about 10 grams per deciliter or more.
[0123] In another embodiment, provided herein is a method of
treating an individual having multiple myeloma, comprising
administering to the individual isolated placental stem cells, a
population of isolated placental stem cells or a population of
cells comprising isolated placental stem cells, wherein said
administration results in detectable reduction in the level of
paraprotein in blood or urine from said individual. In a specific
embodiment, said administering results in the reduction of
paraprotein in blood or urine of said individual to an undetectable
level. In another embodiment, provided herein is a method of
treating an individual having paraprotein in the individual's
blood, comprising administering to the individual a therapeutically
effective amount of placental stem cells and/or BM-MSCs, wherein
the presence of paraprotein is caused by multiple myeloma, and
wherein said therapeutically effective amount is an amount
sufficient to cause a detectable drop in paraprotein in the
individual's blood.
[0124] In another embodiment, provided herein is a method of
treating an individual having a number of clonal plasma cells
greater than 10%, out of all nucleated cells, in a bone marrow
biopsy or blood sample from said individual, comprising
administering to the individual a therapeutically effective amount
of placental stem cells and/or BM-MSCs, wherein said number of
clonal plasma cells is caused by multiple myeloma, and wherein said
therapeutically effective amount is an amount sufficient to cause a
detectable drop in said number of clonal plasma cells in a bone
marrow biopsy or blood sample to below 10%.
[0125] In another embodiment, provided herein is a method of
treating an individual having hypercalcemia comprising
administering to the individual a therapeutically effective amount
of placental stem cells and/or BM-MSCs, wherein said hypercalcemia
is caused by multiple myeloma, and wherein said therapeutically
effective amount is an amount sufficient to cause a detectable drop
in calcium in blood from the individual. In another embodiment,
provided herein is a method of treating an individual having high
blood calcium levels (e.g., corrected calcium greater than about 12
mg per deciliter of blood, or greater than about 2.75 mmol),
comprising administering to the individual a therapeutically
effective amount of placental stem cells and/or BM-MSCs, wherein
the high blood calcium levels are caused by multiple myeloma, and
wherein said therapeutically effective amount is an amount
sufficient to cause a detectable drop in said blood calcium levels,
e.g., a drop in said blood calcium levels to below about 12 mg per
deciliter of blood, or below about 2.75 mmol.
[0126] In another embodiment, provided herein is a method of
treating an individual having anemia, wherein said anemia is caused
by multiple myeloma, wherein said anemia is defined as blood
hemoglobin of less than 10 g/dL blood, comprising administering to
the individual a therapeutically effective amount of placental stem
cells and/or BM-MSCs, wherein said therapeutically effective amount
is an amount sufficient to cause a detectable increase in
hemoglobin in blood from the individual. In a specific embodiment,
said therapeutically effective amount is an amount that results in
an increased of hemoglobin in blood from the individual to 10 g/dL
or greater.
[0127] In another embodiment, provided herein is a method of
treating an individual having blood hyperviscosity syndrome,
wherein said blood has a viscosity of above 1.8 centipoises,
wherein said blood hyperviscosity syndrome is caused by multiple
myeloma, comprising administering to the individual a
therapeutically effective amount of placental stem cells and/or
BM-MSCs, wherein said therapeutically effective amount is an amount
sufficient to cause a detectable decrease in viscosity of blood
from the individual. In specific embodiments, said therapeutically
effective amount is an amount that results in a decrease in
viscosity of blood in the individual to below 5, 4, 3, 2, or 1.8
centipoises.
[0128] In another embodiment, provided herein is a method of
treating an individual having greater than, e.g., 6%, 8%, 10%, 12%,
14%, 16%, 18% or 20% plasma cells in bone marrow of said
individual, comprising administering to the individual a
therapeutically effective amount of placental stem cells and/or
BM-MSCs, wherein said therapeutically effective amount is an amount
sufficient to cause a detectable decrease in the percentage of
plasma cells in bone marrow from the individual.
[0129] In another embodiment, provided herein is a method of
treating an individual having multiple myeloma, comprising
administering to the individual isolated placental stem cells
and/or BM-MSCs (e.g., isolated BM-MSCs or BM-MSCs in bone marrow),
wherein said administration results in detectable reduction in the
severity and/or number of bone lesions caused by multiple myeloma
in said individual, as determinable by, e.g., bone scan or
radiography. In another embodiment, provided herein is a method of
treating an individual having multiple myeloma, comprising
administering to the individual isolated placental stem cells
and/or BM-MSCs, wherein said administration results in detectable
reduction in loss of bone mass or bone mineral content, cessation
of loss of bone mass or bone mineral content, or increase in bone
mass or bone mineral content, in said individual.
[0130] In another specific embodiment of the method of treatment,
said one or more symptoms of multiple myeloma are bone pain,
osteocytic lesions (e.g., visible by X-ray or magnetic resonance
imaging (MRI)), osteoporosis, anemia, hypercalcemia or a symptom
due to hypercalcemia, or renal failure. In other specific
embodiments, said individual has never been treated for multiple
myeloma; said individual has been treated for multiple myeloma and
responds to non-placental stem cell and/or BM-MSC therapy; said
individual has been treated for multiple myeloma and has not
responded to non-placental stem cell and/or BM-MSC therapy, but the
course of multiple myeloma in said individual has not progressed;
or said individual has progressive multiple myeloma.
[0131] In another embodiment, administration of the placental stem
cells and/or BM-MSCs (e.g., isolated BM-MSCs or BM-MSCs in bone
marrow), are sufficient to cause a detectable increase in one or
more markers of bone formation in said individual. For instance,
bone formation may be assessed by analysis of levels of bone
specific alkaline phosphatase (BSAP) and/or serum intact
procollagen type I N-terminal peptide (PINP) in, e.g., a serum
sample from said individual. A detectable increase in serum BSAP
and/or PINP after administration of placental stem cells and/or
BM-MSCs to an individual having multiple myeloma is an indication
of an increase in bone formation. Thus, in another embodiment,
provided herein is a method of treating an individual having
multiple myeloma, comprising administering to the individual
isolated placental stem cells and/or BM-MSCs, wherein said
administering results in a detectable increase in either BSAP or
PINP in serum from the individual.
[0132] In another embodiment, administration of isolated placental
stem cells and/or BM-MSCs, is sufficient to cause a detectable
decrease in one or more markers of bone resorption. For instance,
bone resorption may be assessed by analysis of levels of serum
C-terminal type I collagen telopeptide (CTX) and/or serum
tartrate-resistant acid phosphatase isoform-5b (TRACP-5b). A
detectable decrease in CTX or TRACP-5b after administration of
placental stem cells and/or BM-MSCs, to an individual having
multiple myeloma is an indication of a decrease in bone resorption.
Thus, in another embodiment, provided herein is a method of
treating an individual having multiple myeloma, comprising
administering to the individual isolated placental stem cells
and/or BM-MSCs (e.g., isolated BM-MSCs or BM-MSCs in bone marrow),
wherein said administering results in a detectable decrease in
either CTX or TRACP-5b in serum from the individual.
[0133] In another embodiment, provided herein is a method of
treating an individual having Stage I multiple myeloma, comprising
administering to the individual a therapeutically effective amount
of isolated placental stem cells and/or BM-MSCs (e.g., isolated
BM-MSCs or BM-MSCs in bone marrow), wherein said Stage I multiple
myeloma is characterized by: (i) hemoglobin level of 10 g/dL or
more; (ii) normal bone, or only 1-2 lesions, as seen on a
radiogram; (iii) less than 12 mg/dL blood calcium; and detectable
levels of paraprotein; wherein said therapeutically effective
amount of said placental stem cells and/or BM-MSCs is an amount
sufficient to result in improvement of one or more of said
symptoms, and/or a detectable reduction in the number of plasma
cells in blood from the individual.
[0134] In another embodiment, provided herein is a method of
treating an individual having Stage II multiple myeloma, comprising
administering to the individual a therapeutically effective amount
of isolated placental stem cells and/or BM-MSCs (e.g., isolated
BM-MSCs or BM-MSCs in bone marrow), wherein said Stage II multiple
myeloma is characterized by the symptoms: (i) blood hemoglobin
below 8.5 g/dL; (ii) blood calcium level above 12 mg/dL; (iii) 3 or
more areas of bone lesions as seen on a radiogram; and (iv) high
levels of paraprotein; wherein said therapeutically effective
amount of said isolated placental stem cells and/or BM-MSCs is an
amount sufficient to result in improvement of one or more of said
symptoms, and/or a detectable reduction in the number of plasma
cells in blood from the individual.
[0135] In another embodiment, provided herein is a method of
treating an individual having Stage I multiple myeloma, comprising
administering to the individual a therapeutically effective amount
of isolated placental stem cells and/or BM-MSCs (e.g., isolated
BM-MSCs or BM-MSCs in bone marrow), wherein said Stage I multiple
myeloma is characterized by serum beta-2 microglobulin less than
3.5 mg/L and a serum albumin level of 3.5 g/dL or higher, and
wherein said therapeutically effective amount of said placental
stem cells and/or BM-MSCs is an amount sufficient to reduce, e.g.,
detectably reduce, the level of serum beta-2 microglobulin, or
increase, e.g., detectably increase, the blood albumin level in
said individual.
[0136] In another embodiment, provided herein is a method of
treating an individual having Stage II multiple myeloma, comprising
administering to the individual a therapeutically effective amount
of isolated placental stem cells and/or BM-MSCs (e.g., isolated
BM-MSCs or BM-MSCs in bone marrow), wherein said Stage II multiple
myeloma is characterized by serum beta-2 microglobulin of between
about 3.3 mg/L and 5.5 mg/L with any level of serum albumin, or
serum albumin level of below about 3.5 g/dL and serum beta-2
microglobulin less than about 3.5 g/L, and wherein said
therapeutically effective amount of said placental stem cells
and/or BM-MSCs is an amount sufficient to reduce, e.g., detectably
reduce, the level of serum beta-2 microglobulin, e.g., to below
about 3.3 mg/L, or increase, e.g., detectably increase, the blood
albumin level, in said individual.
[0137] In another embodiment, provided herein is a method of
treating an individual having Stage III multiple myeloma,
comprising administering to the individual a therapeutically
effective amount of isolated placental stem cells and/or BM-MSCs
(e.g., isolated BM-MSCs or BM-MSCs in bone marrow), wherein said
Stage III multiple myeloma is characterized by serum beta-2
microglobulin of greater than 5.5 mg/L with any level of serum
albumin, wherein said therapeutically effective amount of said
placental stem cells and/or BM-MSCs, is an amount sufficient to
reduce, e.g., detectably reduce, the amount of serum beta-2
microglobulin in blood or serum of said individual, e.g., to below
about 5.5 mg/L, or to below about 3.5 mg/L.
[0138] In certain other specific embodiments of any of the above,
the individual having multiple myeloma is refractory to one or more
non-cell multiple myeloma therapies, e.g., melphalan (with or
without prednisolone), cyclophosphamide (with or without
prednisolone), alkylating agents, VAD (vincristine, adriamycin and
high-dose dexamethasone), ABCM (vincristine, adriamycin,
prednisolone and carmustine), high-dose dexamethasone, thalidomide,
biphosphonates, etc.
[0139] In another aspect, provided herein is a method of
suppressing the proliferation of multiple myeloma cells, comprising
contacting said multiple myeloma cells with isolated placental stem
cells, e.g., the isolated placental stem cells described in Section
5.2, below, a population of such isolated placental stem cells, or
a population of cells comprising the isolated placental stem cells,
and/or isolated BM-MSCs or bone marrow comprising BM-MSCs, such
that proliferation of said multiple myeloma cells is suppressed,
e.g., detectably suppressed. In certain embodiments, provided
herein is a method of suppressing the proliferation of multiple
myeloma cells in vivo, comprising administering a therapeutically
effective amount of placental stem cells and/or BM-MSCs, to an
individual comprising multiple myeloma cells, wherein said
administering reduces, e.g., detectably reduces, proliferation of
said multiple myeloma cells. In a specific embodiment, said
administering reduces, e.g., detectably reduces (e.g., improves),
one or more symptoms or signs of multiple myeloma, or lessens the
worsening of said one or more symptoms or signs of multiple
myeloma. A reduction in the proliferation of multiple myeloma cells
after administration of placental stem cells and/or BM-MSCs, can be
assessed, e.g., by detecting a reduction in the number of plasma
cells from blood or bone marrow of an individual having multiple
myeloma, e.g., using one or more antibodies specific to plasma
cells or multiple myeloma cells, for example, antibodies to CD28 or
CD138.
[0140] In another aspect, provided herein is a method of reducing a
number of multiple myeloma cells, e.g., in an individual having
multiple myeloma, comprising contacting said multiple myeloma cells
with isolated placental stem cells and/or BM-MSCs (e.g., isolated
BM-MSCs or BM-MSCs in bone marrow), such that the number of
multiple myeloma cells in said individual is suppressed, e.g.,
detectably suppressed, after said contacting. In a specific
embodiment, said contacting is performed by administering said
placental stem cells and/or BM-MSCs, to said individual. In another
specific embodiment, said administering reduces, e.g., detectably
reduces (e.g., improves), one or more symptoms or signs of multiple
myeloma, or lessens the worsening of said one or more symptoms or
signs of multiple myeloma. A reduction in the number of multiple
myeloma cells after administration of placental stem cells and/or
BM-MSCs, as compared to before administration, can be assessed,
e.g., by detection of a reduction in the number of plasma cells
from blood or bone marrow of an individual having multiple myeloma,
e.g., using one or more antibodies specific to plasma cells or
multiple myeloma cells, for example, antibodies to CD28 or
CD138.
[0141] Typically, an individual presenting with one or more
symptoms of multiple myeloma is assessed for multiple myeloma at
least once before a final diagnosis of multiple myeloma, e.g., as a
part of tests performed to arrive at a diagnosis of multiple
myeloma. Also, typically, an individual diagnosed with multiple
myeloma is assessed at least once, usually more than once, after a
diagnosis of multiple myeloma, for symptoms of multiple myeloma to
gauge progress of the disease. Such an assessment may comprise a
determination of the extent and/or number of bone lesions using,
e.g. X-ray analysis, magnetic resonance imaging (MRI), computerized
tomography (CT) scanning, positron emission tomography (PET)
scanning, or the like; a determination of the level of calcium in
the blood; a determination of the level of M proteins (antibodies
or fragments of antibodies) in the blood or urine, and the like.
Effectiveness of treatment of multiple myeloma, e.g., effectiveness
of administering placental stem cells and/or BM-MSCs, can be
assessed by any one, or more, of such symptoms of multiple myeloma,
e.g., by improvement in any one, or more, of such symptoms of
multiple myeloma. Effectiveness can also be assessed by determining
the number of multiple myeloma cells in blood or bone marrow of
said individual, before and after administration of said placental
stem cells and/or BM-MSCs.
[0142] Thus, in specific embodiments, any of the above methods
comprises determining, once or a plurality of times before said
administering, and, optionally, once or a plurality of times after
said administering, one or more of (1) a number or degree of bone
lesions in said individual; (2) a level of M proteins (paraprotein)
in blood or urine from the individual; (3) a level of calcium in
blood from the individual; and/or (4) a number of multiple myeloma
cells in blood or bone marrow from the individual. In certain
embodiments, if the level of calcium in blood from the individual,
or the level of M proteins in the blood or urine from the
individual, drops, e.g., detectably drops, after administration of
isolated placental stem cells and/or BM-MSCs, compared to the level
before administration, the placental stem cells and/or BM-MSCs, are
therapeutically effective. Similarly, in certain embodiments,
administration of the placental stem cells and/or BM-MSCs is
therapeutically effective if the number of bone lesions, or the
degree of severity of bone lesions, in the individual, is lessened
after said administration relative to the number of bone lesions,
or the degree of severity of bone lesions before said
administration. In certain other embodiments, administration of the
placental stem cells and/or BM-MSCs is also therapeutically
effective, e.g., if administration of the placental stem cells
and/or BM-MSCs results in a lessening of an increase in the level
of M protein in the blood or urine of the individual, or lessening
in an increase in the level of calcium in the blood of the
individual, or a lessening in an increase in the number or severity
of bone lesions in the individual. In certain specific embodiments,
if there is no detectable change in the number or severity of bone
lesions in the individual, the level of M protein in blood or urine
of the individual, or the level of blood calcium in the individual,
after administration of said placental stem cells and/or BM-MSCs,
administration of either the placental stem cells, BM-MSCs, or both
is repeated.
[0143] Effectiveness of administration of isolated placental stem
cells and/or BM-MSCs may also be assessed by determining that an
amount, e.g., a therapeutically effective amount, of the placental
stem cells and/or BM-MSCs reduces, e.g., detectably reduces, the
number of osteoclast precursors or multiple myeloma cells in the
individual following administration. Reduction of the number of
osteoclast precursors in said individual may be determined by any
medically-acceptable method. For example, the number of osteoclast
precursors may be determined using an antibody specific for
osteoclast precursors to detect osteoclast precursors in, e.g., a
sample of the individual's peripheral blood or bone marrow; the
number of labeled cells may be assessed, e.g., by histology,
counting cells under a microscope, sorting labeled cells by flow
cytometry, or the like. In another specific embodiment, said
therapeutically effective amount of placental stem cells and/or
BM-MSCs reduces the number of multiple myeloma cells in said
individual, e.g., as determinable by cell counting (e.g., by flow
cytometry), or antibody staining, of nucleated blood cells from
said individual using an antibody specific for multiple myeloma
cells or plasma cells, e.g., an antibody specific for cellular
markers CD28 or CD138.
[0144] In any of the methods of treating multiple myeloma, treating
a symptom of multiple myeloma, or suppressing proliferation of
multiple myeloma cells, as described herein, the multiple myeloma
cells exhibit a translocation of genetic material from chromosome 4
to chromosome 14 (e.g., a t(4:14) translocation). In other
embodiments, the multiple myeloma cells exhibit a t(14:16)
translocation, a t(11:14) translocation, and/or an illegitimate IgH
rearrangement with an unknown chromosomal partner. In certain other
embodiments, the multiple myeloma cells do not secrete detectable
amounts of immunoglobulin. In certain other embodiments, the
multiple myeloma cells secrete only, or substantially only, light
chain immunoglobulin, e.g., .kappa. (kappa) light chain, .lamda.
(lambda) light chain, or both. In certain other embodiments, the
multiple myeloma cells secrete immunoglobulin comprising a heavy
chain and a light chain. In other embodiments, the multiple myeloma
cells produce IgG immunoglobulin, IgA immunoglobulin, or both.
[0145] In any of the above embodiments, the isolated placental stem
cells can be, e.g., the genetically engineered placental stem cells
described below. In any of the above embodiments, the BM-MSCs can
be genetically engineered BM-MSCs. BM-MSCs can be genetically
engineered in any manner as described for genetic engineering of
placental stem cells, as described Section 5.7.2, below.
[0146] In certain embodiments, the individual having multiple
myeloma is additionally treated with a chemotherapeutic compound,
e.g., an anticancer compounds described in Section 5.1.3, below,
for example one or more of the anticancer compounds as well as with
placental stem cells and/or BM-MSCs. Placental stem cells and/or
BM-MSCs can be administered to said individual to treat multiple
myeloma, e.g., at the same time as, or within 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11 or 12 months following the start of chemotherapy,
e.g., chemotherapy to treat said multiple myeloma. In other
embodiments, the anticancer compound is administered within 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months following administration
of said placental stem cells and/or BM-MSCs.
[0147] 5.1.2 Treatment of Chondrosarcoma
[0148] In another respect, provided herein is a method of treating
an individual having a chondrosarcoma, comprising administering to
said individual isolated placental stem cells and/or BM-MSCs (e.g.,
isolated BM-MSCs or BM-MSCs in bone marrow). As elsewhere herein,
said isolated placental stem cells can have any combination of, or
all of, the characteristics described in Section 5.2, below.
Additionally, the BM-MSCs may be isolated or present in, e.g., bone
marrow comprising BM-MSCs.
[0149] Thus, in one embodiment, provided herein is a method of
treating an individual having a chondrosarcoma, comprising
administering to the individual isolated placental stem cells
and/or BM-MSCs, wherein said administration results in the
detectable reduction of progression, detectable lessening of
worsening, and/or detectable improvement, of one or more symptoms
of chondrosarcoma. In specific embodiments, said symptoms include,
but are not limited to, bone pain, one or more bone lesions
visible, e.g., on an X-ray, swelling of the bone, e.g., at the site
of the tumor, or enlargement of one or more bones.
[0150] In a specific embodiment, the chondrosarcoma is a clear cell
chondrosarcoma. In another specific embodiment, the chondrosarcoma
is a benign chondrosarcoma (enchondroma). In another specific
embodiment, the chondrosarcoma is a low-grade malignant
chondrosarcoma (Grade I chondrosarcoma; characterized by tumors
resembling normal cartilage; tumors may surround areas of lamellar
bone and/or show atypical cells including binucleate cells). In
another specific embodiment, the chondrosarcoma is an intermediate
grade malignant chondrosarcoma (Grade II chondrosarcoma;
characterized by significant cellularity with many atypical cells,
many of which have hyperchromasia (an abundance of darkly-staining
DNA in the nucleus) and increased nuclear size, compared to Grade
I). In another specific embodiment, the chondrosarcoma is a high
grade malignant chondrosarcoma (Grade III chondrosarcoma;
characterized by areas of marked pleomorphism, large cells with
significant hyperchromasia, occasional giant cells and abundant
necrosis. In another specific embodiment, the chondrosarcoma is a
dedifferentiated chondrosarcoma (a chondrosarcoma comprising a
well-differentiated cartilage tumor (enchondroma or Grade II or II
chondrosarcoma) adjacent to a high-grade non-cartilaginous
sarcoma). In another specific embodiment, the condrosarcoma is a
mesenchymal chondrosarcoma.
[0151] In certain embodiments, said placental stem cells and/or
BM-MSCs are administered to the individual without any further
treatment of the chondrosarcoma. In certain other embodiments, said
placental stem cells and/or BM-MSCs are administered to the
individual after surgery to remove part or all of the
chondrosarcoma tumor, or to remove part or all of a bone affected
by chondrosarcoma. In certain other embodiments, said placental
stem cells and/or BM-MSCs are administered to the individual prior
to, or at the time of, surgery to remove part or all of the
chondrosarcoma tumor, or to remove part or all of a bone affected
by chondrosarcoma. In certain other embodiments, the placental stem
cells and/or BM-MSCs are administered systemically to the
individual, e.g., at a site or by a route other than the site of
the chondrosarcoma in the individual; e.g., intravenously,
intraarterially, peritoneally, or the like. In certain other
embodiments, the placental stem cells and/or BM-MSCs are
administered at or adjacent to the site of the chondrosarcoma (if
the tumor has not been removed), e.g., the site of the
chondrosarcoma in the individual, or the site from which the
chondrosarcoma was removed, if surgical removal has taken
place.
[0152] 5.1.3 Combination Therapies
[0153] Treatment of a bone-related cancer, e.g., multiple myeloma,
chondrosarcoma, or one of the other bone-related cancers noted
herein, can comprise administration of placental stem cells and/or
BM-MSCs (e.g., isolated BM-MSCs or BM-MSCs in bone marrow) in
combination with a second therapy, to the individual having the
cancer. In various embodiments, the second therapy is administered
at the same time as said placental stem cells and/or BM-MSCs in the
same course of treatment as said placenta cells, after said
placental stem cells and/or BM-MSCs have been administered (e.g.,
after completion of a course of treatment comprising administering
placental stem cells and/or BM-MSCs), or before administration of
placental stem cells and/or BM-MSCs (e.g., before initiation of a
course of treatment comprising administering placental stem cells
and/or BM-MSCs). In certain embodiments, the placental stem cells
and/or BM-MSCs, and second therapy, are formulated together to be
administered, e.g., from the same package or container. In certain
other embodiments, the placental stem cells and/or BM-MSCs, and
second therapy, are each formulated for separate
administration.
[0154] Thus, in another aspect, provided herein is a method of
treating an individual having a bone-related cancer, e.g., multiple
myeloma or chondrosarcoma, or one of the other bone-related cancers
listed herein, comprising administering to the individual isolated
placental stem cells and/or BM-MSCs, in combination with one or
more other anticancer therapies, e.g., one or more chemotherapies
or chemotherapeutic compounds. Such other anticancer therapies can
be administered to the individual at the same time as, during the
same course of treatment as, or separately from, said
administration of placental stem cells and/or BM-MSCs. In a
specific embodiment, the one or more anticancer therapies is/are
administered sequentially with administration of said placental
stem cells and/or BM-MSCs. In another specific embodiment, said
other anticancer therapy or anticancer therapies are administered
to said individual before administration of said placental stem
cells and/or BM-MSCs; e.g., a course of such other anticancer
therapies is administered to the individual, and completed, prior
to administration to the individual of placental stem cells and/or
BM-MSCs. In another specific embodiment, said placental stem cells
and/or BM-MSCs are administered to the individual before
administration of said other anticancer therapies; e.g., a course
of placental stem cells and/or BM-MSCs is administered to said
individual before administration of said other anticancer
therapies, and completed, prior to administration to the individual
said other anticancer therapy or anticancer therapies.
[0155] As used herein, "anticancer agent" or "anticancer therapy"
is an agent or therapy that has been identified, e.g., in clinical,
pre-clinical or scientific studies (including anecdotal studies) to
have a tumoristatic or tumoricidal effect on one or more types of
tumor or cancer cells.
[0156] In a specific embodiment, the anticancer agent is melphalan
(also known as L-phenylalanine mustard or L-PAM; trade name
Albertan). Thus, in one embodiment, the method of treating an
individual having multiple myeloma comprises administering to said
individual melphalan, e.g., a therapeutically effective dose or
doses of melphalan (e.g., ALKERAN.RTM.). Administration is
typically oral or intravenous. In another specific embodiment, the
anticancer agent is thalidomide. In another specific embodiment,
the anticancer agent is an amino-substituted thalidomide analog or
an amino-substituted imidazole. In another specific embodiment, the
anticancer agent is pomalidomide (sold under the trade name
ACTIMID.RTM.); lenalidomide (sold under the trade name
REVLIMID.RTM.); or lenalidomide in combination with dexamethasone.
In another specific embodiment, the anticancer treatment is
bortezomib (e.g., VELCADE.RTM.). In another specific embodiment,
the anticancer agent comprises a combination of melphalan,
prednisone, and thalidomide (administered separately or together).
In another specific embodiment, the anticancer agent is the
combination of bortezomib, melphalan and prednisone (administered
separately or together). In other specific embodiments, the
anticancer agent is one or more of cyclophosphamide (e.g.,
CYTOXAN.RTM.), vincristine (e.g., ONCOVIN.RTM., VINCASAR PFS.RTM.),
doxorubicin (e.g., ADRIAMYCIN RDF.RTM., ADRIAMYCIN PFS.RTM.), or
liposomal doxorubicin (e.g., DOXIL.RTM.).
[0157] Other anticancer agents are well-known in the art. Thus, in
other specific embodiments, the anticancer agents include, but are
not limited to: acivicin; aclarubicin; acodazole hydrochloride;
acronine; adozelesin; aldesleukin; altretamine; ambomycin;
ametantrone acetate; amsacrine; anastrozole; anthramycin;
asparaginase; asperlin; azacitidine; azetepa; azotomycin;
batimastat; benzodepa; bicalutamide; bisantrene hydrochloride;
bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar
sodium; bropirimine; busulfan; cactinomycin; calusterone;
caracemide; carbetimer; carboplatin; carmustine; carubicin
hydrochloride; carzelesin; cedefingol; celecoxib (COX-2 inhibitor);
chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol
mesylate; cytarabine; dacarbazine; dactinomycin; daunorubicin
hydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguanine
mesylate; diaziquone; docetaxel; doxorubicin hydrochloride;
droloxifene; droloxifene citrate; dromostanolone propionate;
duazomycin; edatrexate; eflomithine hydrochloride; elsamitrucin;
enloplatin; enpromate; epipropidine; epirubicin hydrochloride;
erbulozole; esorubicin hydrochloride; estramustine; estramustine
phosphate sodium; etanidazole; etoposide; etoposide phosphate;
etoprine; fadrozole hydrochloride; fazarabine; fenretinide;
floxuridine; fludarabine phosphate; fluorouracil; flurocitabine;
fosquidone; fostriecin sodium; gemcitabine; gemcitabine
hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide;
ilmofosine; iproplatin; irinotecan; irinotecan hydrochloride;
lanreotide acetate; letrozole; leuprolide acetate; liarozole
hydrochloride; lometrexol sodium; lomustine; losoxantrone
hydrochloride; masoprocol; maytansine; mechlorethamine
hydrochloride; megestrol acetate; melengestrol acetate; melphalan;
menogaril; mercaptopurine; methotrexate; methotrexate sodium;
metoprine; meturedepa; mitindomide; mitocarcin; mitocromin;
mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone
hydrochloride; mycophenolic acid; nocodazole; nogalamycin;
ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin;
pentamustine; peplomycin sulfate; perfosfamide; pipobroman;
piposulfan; piroxantrone hydrochloride; plicamycin; plomestane;
porfimer sodium; porfiromycin; prednimustine; procarbazine
hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin;
riboprine; safingol; safingol hydrochloride; semustine; simtrazene;
sparfosate sodium; sparsomycin; spirogermanium hydrochloride;
spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur;
talisomycin; tecogalan sodium; taxotere; tegafur; teloxantrone
hydrochloride; temoporfin; teniposide; teroxirone; testolactone;
thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine;
toremifene citrate; trestolone acetate; triciribine phosphate;
trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole
hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin;
vinblastine sulfate; vincristine sulfate; vindesine; vindesine
sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine
sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine
sulfate; vorozole; zeniplatin; zinostatin; and zorubicin
hydrochloride.
[0158] Other anti-cancer drugs include, but are not limited to:
20-cpi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone;
aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin;
ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine;
aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole;
andrographolide; angiogenesis inhibitors; antagonist D; antagonist
G; antarelix; anti-dorsalizing morphogenetic protein-1;
antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston;
antisense oligonucleotides; aphidicolin glycinate; apoptosis gene
modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA;
arginine deaminase; asulacrine; atamestane; atrimustine;
axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin;
azatyrosine; baccatin III derivatives; balanol; batimastat; BCR/ABL
antagonists; benzochlorins; benzoylstaurosporine; beta lactam
derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF
inhibitor; bicalutamide; bisantrene; bisaziridinylspermine;
bisnafide; bistratene A; bizelesin; breflate; bropirimine;
budotitane; buthionine sulfoximine; calcipotriol; calphostin C;
camptothecin derivatives; capecitabine; carboxamide-amino-triazole;
carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived
inhibitor; carzelesin; casein kinase inhibitors (ICOS);
castanospermine; cecropin B; cetrorelix; chlorlns;
chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin;
cladribine; clomifene analogues; clotrimazole; collismycin A;
collismycin B; combretastatin A4; combretastatin analogue;
conagenin; crambescidin 816; crisnatol; cryptophycin 8;
cryptophycin A derivatives; curacin A; cyclopentanthraquinones;
cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;
cytostatin; dacliximab; decitabine; dehydrodidenmin B; deslorelin;
dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;
diaziquone; didemnin B; didox; diethylnorspermine;
dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin; diphenyl
spiromustine; docetaxel; docosanol; dolasetron; doxifluridine;
doxorubicin; droloxifene; dronabinol; duocarmycin SA; ebselen;
ecomustine; edelfosine; edrecolomab; eflomithine; elemene;
emitefur; epirubicin; epristeride; estramustine analogue; estrogen
agonists; estrogen antagonists; etanidazole; etoposide phosphate;
exemestane; fadrozole; fazarabine; fenretinide; filgrastim;
finasteride; flavopiridol; flezelastine; fluasterone; fludarabine;
fluorodaunorunicin hydrochloride; forfenimex; formestane;
fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate;
galocitabine; ganirelix; gelatinase inhibitors; gemcitabine;
glutathione inhibitors; hepsulfam; heregulin; hexamethylene
bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene;
idramantone; ilmofosine; ilomastat; imatinib (e.g., GLEEVEC.RTM.),
imiquimod; immunostimulant peptides; insulin-like growth factor-1
receptor inhibitor; interferon agonists; interferons; interleukins;
iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine;
isobengazolc; isohomohalicondrin B; itasetron; jasplakinolide;
kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin;
lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia
inhibiting factor; leukocyte alpha interferon;
leuprolide+estrogen+progesterone; leuprorelin; levamisole;
liarozole; linear polyamine analogue; lipophilic disaccharide
peptide; lipophilic platinum compounds; lissoclinamide 7;
lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone;
loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic
peptides; maitansine; mannostatin A; marimastat; masoprocol;
maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors;
menogaril; merbarone; meterelin; methioninase; metoclopramide; MIF
inhibitor; mifepristone; miltefosine; mirimostim; mitoguazone;
mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast
growth factor-saporin; mitoxantrone; mofarotene; molgramostim;
Erbitux, human chorionic gonadotrophin; monophosphoryl lipid
A+myobacterium cell wall sk; mopidamol; mustard anticancer agent;
mycaperoxide B; mycobacterial cell wall extract; myriaporone;
N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;
naloxone+pentazocine; napavin; naphterpin; nartograstim;
nedaplatin; nemorubicin; neridronic acid; nilutamide; nisamycin;
nitric oxide modulators; nitroxide antioxidant; nitrullyn;
oblimersen (e.g., GENASENSE.RTM.); O.sup.6-benzylguanine;
octreotide; okicenone; oligonucleotides; onapristone; ondansetron;
ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone;
oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues;
paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic
acid; panaxytriol; panomifene; parabactin; pazelliptine;
pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin;
pentrozole; perflubron; perfosfamide; perillyl alcohol;
phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil;
pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A;
placetin B; plasminogen activator inhibitor; platinum complex;
platinum compounds; platinum-triamine complex; porfimer sodium;
porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2;
protcasome inhibitors; protein A-based immune modulator; protein
kinase C inhibitor; protein kinase C inhibitors, microalgal;
protein tyrosine phosphatase inhibitors; purine nucleoside
phosphorylase inhibitors; purpurins; pyrazoloacridine;
pyridoxylated hemoglobin polyoxyethylene conjugate; raf
antagonists; raltitrexed; ramosetron; ras farnesyl protein
transferase inhibitors; ras inhibitors; ras-GAP inhibitor;
retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin;
ribozymes; RII retinamide; rohitukine; romurtide; roquinimex;
rubiginone B1; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A;
sargramostim; Sdi 1 mimetics; semustine; senescence derived
inhibitor 1; sense oligonucleotides; signal transduction
inhibitors; sizofiran; sobuzoxane; sodium borocaptate; sodium
phenylacetate; solverol; somatomedin binding protein; soncrmin;
sparfosic acid; spicamycin D; spiromustine; splenopentin;
spongistatin 1; squalamine; stipiamide; stromelysin inhibitors;
sulfinosine; superactive vasoactive intestinal peptide antagonist;
suradista; suramin; swainsonine; tallimustine; tamoxifen
methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur;
tellurapyrylium; telomerase inhibitors; temoporfin; teniposide;
tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline;
thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin
receptor agonist; thymotrinan; thyroid stimulating hormone; tin
ethyl etiopurpurin; tirapazamine; titanocene bichloride; topsentin;
toremifene; translation inhibitors; tretinoin; triacetyluridine;
triciribine; trimetrexate; triptorelin; tropisetron; turosteride;
tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex;
urogenital sinus-derived growth inhibitory factor; urokinase
receptor antagonists; vapreotide; variolin B; velaresol; veramine;
verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole;
zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer.
[0159] In other embodiments, the combination therapy comprises
administration of placental stem cells and/or BM-MSCs, to an
individual in combination with an inhibitor of osteoclasts, e.g.,
an inhibitor of osteoclast formation or differentiation of
osteoclast precursors into osteoclasts. In a specific embodiment,
the osteoclast inhibitor is an inhibitor of RANKL, e.g., Denosumab.
In another specific embodiment, the osteoclast inhibitor is an
integrin or cathepsin K inhibitor.
[0160] In another embodiment, the combination therapy comprises
administration of placental stem cells and/or BM-MSCs in
combination with bisphosphonates. In specific embodiments, the
bisphosphonates are aledronate (e.g., at a dosage of about 5 mg to
about 10 mg per day, or about 35 mg to about 70 mg once a week;
with or without supplemental vitamin D), ibandronate, risedronate,
clodronate and/or pamidronate. In another embodiment, the
combination therapy comprises administration of placental stem
cells and/or BM-MSCs in combination with one or more of calcitonin,
estrogen, parathyroid hormone (e.g., teriparatide, e.g.,
FORTEO.RTM.) or raloxifene.
[0161] In another embodiment, provided herein is a method of
treating an individual having a bone-related cancer, e.g., multiple
myeloma, chondrosarcoma, or one of the other bone-related cancers
listed herein, comprising administering to the individual isolated
placental stem cells and/or BM-MSCs, in combination with a compound
having activin antagonist or activin receptor RIIa (ActRIIa)
antagonist activity, e.g., an ActRIIa antagonist. In a specific
embodiment, said ActRIIa antagonist is a soluble activin receptor
type IIA IgC-Fc fusion protein (e.g., ACE-011.RTM.). See, e.g.,
U.S. Patent Application Publication No. 2009/0142333, which is
incorporated by reference herein in its entirety.
[0162] In another embodiment, provided herein is a method of
treating an individual having a bone-related cancer, e.g., multiple
myeloma, chondrosarcoma, or one of the other bone-related cancers
listed herein, comprising administering to the individual isolated
placental stem cells and/or BM-MSCs, in combination with
radiotherapy. In certain embodiments, the radiotherapy comprises
administering X-rays to an organ or tissue in the individual having
the bone-related cancer, which is affected by the bone-related
cancer. In specific embodiments, for example, said radiotherapy,
e.g., X-rays, is administered to a bone lesion caused by multiple
myeloma, or a bone lesion caused by chondrosarcoma. In other
specific embodiments, said radiotherapy, e.g., X-rays, is
administered to a half of the individual's body that is affected by
said bone-related cancer. In other specific embodiments, said
radiotherapy, e.g., X-rays, is administered to the whole of the
affected individual's body. In other embodiments, said radiation
therapy comprises administering a proton beam or an electron beam
to an organ or tissue in the individual having the bone-related
cancer, which is affected by the bone-related cancer. In certain
other embodiments, said radiotherapy is administered in preparation
for hematopoietic stem cell replacement therapy (e.g., radiotherapy
to kill the individual's existing hematopoietic system).
[0163] In another embodiment, isolated placental stem cells and/or
BM-MSCs are combined with a bone substitute, e.g., to treat a bone
lesion associated with a bone-related cancer, e.g., by
administration at or adjacent to a bone lesion caused by a
bone-related cancer. In a specific embodiment, said bone substitute
is a physiologically-acceptable ceramic material, e.g., mono-, di-,
tri-, alpha-tri-, beta-tri-, and tetra-calcium phosphate,
hydroxyapatite, a fluoroapatite, a calcium sulfate, a calcium
fluoride, a calcium oxide, a calcium carbonate, a magnesium calcium
phosphate, a biologically active glass (e.g., BIOGLASS.RTM.), or a
mixture of any thereof. In another specific embodiment, said bone
substitute is a porous biocompatible ceramic material (e.g.,
SURGIBONE.RTM., ENDOBON.RTM., CEROS.RTM. or the like), or a
mineralized collagen bone grafting product (e.g., HEALOS.TM.,
VITOSS.RTM., RHAKOSS.TM., and CORTOSS.RTM., or the like)
5.2 Placental Stem Cells
[0164] The isolated placental stem cells useful in the treatment of
individuals having a bone-related cancer, e.g., multiple myeloma,
or having cells of a bone-related cancer, e.g., multiple myeloma
cells, are cells, obtainable from a placenta or part thereof, that
adhere to a tissue culture substrate (e.g., uncoated tissue culture
plastic), and have characteristics of multipotent cells or stem
cells. In certain embodiments, the isolated placental stem cells
useful in the methods disclosed herein have the capacity to
differentiate into one or more non-placental cell types. Placental
stem cells useful in the methods disclosed herein are described
herein and, e.g., in U.S. Pat. No. 7,486,276, and in U.S. Patent
Application Publication No. 2007/0275362, the disclosures of which
are hereby incorporated by reference in their entireties. Placental
stem cells are not trophoblasts, cytotrophoblasts, embryonic germ
cells, or embryonic stem cells, as those cells are understood by
persons of skill in the art
[0165] The isolated placental stem cells useful in the methods
disclosed herein can be either fetal or maternal in origin (that
is, can have the genotype of either the fetus or mother,
respectively). Preferably, the isolated placental stem cells and
populations of isolated placental stem cells are fetal in origin.
Isolated placental stem cells, or populations of cells comprising
the isolated placental stem cells, can comprise isolated placental
stem cells that are solely fetal or maternal in origin, or can
comprise a mixed population of isolated placental stem cells of
both fetal and maternal origin. In certain embodiments of any of
the placental stem cells described herein, said isolated placental
stem cells are non-maternal in origin. In certain other
embodiments, said placental stem cells are substantially free of
maternal cells; e.g., at least about 40%, 45%, 5-0%, 55%, 60%, 65%,
70%, 75%, 90%, 85%, 90%, 95%, 98% or 99% of said cells are
non-maternal in origin.
[0166] The isolated placental stem cells, and populations of cells
comprising the isolated placental stem cells, can be identified and
selected by the morphological, marker, and culture characteristics
discussed below. In certain embodiments, any of the placental stem
cells described herein, are autologous to a recipient, e.g., an
individual who has a bone-related cancer, or cells of a
bone-related cancer, e.g., multiple myeloma or multiple myeloma
cells, or chondrosarcoma or chondrosarcoma cells, or another bone
related cancer of cells of another bone-related cancer. In certain
other embodiments, any of the placental stem cells described
herein, are heterologous to a recipient, e.g., an individual who
has a bone-related cancer, or cells of a bone-related cancer, e.g.,
multiple myeloma or multiple myeloma cells, or chondrosarcoma or
chondrosarcoma cells, or another bone related cancer of cells of
another bone-related cancer.
[0167] In certain embodiments, the placental stem cells useful in
the methods of the invention, e.g., the placental stem cells
described herein, are obtained from term placenta, that is, a
post-partum mammalian, e.g., human, placenta. In certain other
embodiments, the placental stem cells useful in the methods of the
invention, e.g., the placental stem cells described herein, are
obtained from preterm mammalian, e.g., human, placenta.
[0168] 5.2.1 Physical and Morphological Characteristics
[0169] The isolated placental stem cells described herein (PDACs),
when cultured in primary cultures or in cell culture, adhere to the
tissue culture substrate, e.g., tissue culture container surface
(e.g., tissue culture plastic), or to a tissue culture surface
coated with extracellular matrix or ligands such as laminin,
collagen (e.g., native or denatured), gelatin, fibronectin,
ornithine, vitronectin, and extracellular membrane protein (e.g.,
MATRIGEL.RTM. (BD Discovery Labware, Bedford, Mass.)). The isolated
placental stem cells in culture assume a generally fibroblastoid,
stellate appearance, with a number of cytoplasmic processes
extending from the central cell body. The cells are, however,
morphologically distinguishable from fibroblasts cultured under the
same conditions, as the isolated placental stem cells exhibit a
greater number of such processes than do fibroblasts.
Morphologically, isolated placental stem cells are also
distinguishable from hematopoietic stem cells, which generally
assume a more rounded, or cobblestone, morphology in culture, and
do not adhere to tissue culture plastic. Morphologically, the
placental stem cells are also distinguishable from trophoblasts or
cytotrophoblasts, which tend to appear rounded or epitheloid, and,
in the case of cytotrophoblasts, multinucleate as compared to the
uninucleate placental stem cells. The placental stem cells are
unicellular and remain unicellular during culture and over multiple
passages, and do not form, e.g., multinuclear cells in culture,
e.g., in culture in growth medium in air, or culture in growth
medium in 95% air/5% CO.sub.2.
[0170] In certain embodiments, the isolated placental stem cells
useful in the methods disclosed herein, e.g., the methods of
treatment or methods of suppressing the growth of bone-related
cancer cells or suppressing differentiation of osteoclast
precursors into osteoclasts, when cultured in a growth medium,
develop embryoid-like bodies. Embryoid-like bodies are well-known
in the art, and are noncontiguous clumps of cells that can grow on
top of an adherent layer of proliferating isolated placental stem
cells. The term "embryoid-like" is used because the clumps of cells
resemble embryoid bodies, clumps of cells that grow from cultures
of embryonic stem cells. Growth medium in which embryoid-like
bodies can develop in a proliferating culture of isolated placental
stem cells includes medium comprising, e.g., DMEM-LG (e.g., from
Gibco); 2% fetal calf serum (e.g., from Hyclone Labs.); 1.times.
insulin-transferrin-selenium (ITS); 1.times. linoleic acid-bovine
serum albumin (LA-BSA); 10.sup.-9 M dexamethasone (e.g., from
Sigma); 10.sup.-4 M ascorbic acid 2-phosphate (e.g., from Sigma);
epidermal growth factor 10 ng/mL (e.g., from R&D Systems); and
platelet-derived growth factor (PDGF-BB) 10 ng/mL (e.g., from
R&D Systems).
[0171] 5.2.2 Cell Surface, Molecular and Genetic Markers
[0172] The isolated placental stem cells are tissue culture
plastic-adherent human placental stem cells that have
characteristics of multipotent cells or stem cells, and express a
plurality of markers that can be used to identify and/or isolate
the cells, or populations of cells that comprise the stem cells.
The isolated placental stem cells include cells and placental stem
cell-containing cell populations obtained directly from the
placenta, or a part thereof. Isolated placental stem cell
populations also include populations of (that is, two or more)
isolated placental stem cells in culture, and a population in a
container, e.g., a bag. The isolated placental stem cells described
herein are not bone marrow-derived mesenchymal cells,
adipose-derived mesenchymal stem cells, or mesenchymal cells
obtained from umbilical cord blood, placental blood, or peripheral
blood. Placental stem cells useful in the methods and compositions
described herein are described, e.g., in U.S. Pat. Nos. 7,311,904;
7,311,905; and 7,468,276; and in U.S. Patent Application
Publication No. 2007/0275362, the disclosures of which are hereby
incorporated by reference in their entireties. In a specific
embodiments of any of the embodiments of the placental stem cells
described herein, the cells are mammalian, e.g., human.
[0173] In certain embodiments, the isolated placental stem cells
are isolated placental stem cells. In certain other embodiments,
the isolated placental cells are isolated placental multipotent
cells. In one embodiment, isolated placental stem cells useful in
the methods described herein are CD34.sup.-, CD10.sup.+ and
CD105.sup.+ as detectable by flow cytometry. As used herein, the
phrase "as detectable by," "as determinable by," and the like, does
not indicate that the cells need to be assessed for expression of
the recited markers in order for the cells to be "isolated," nor to
the cells need to be isolated using the markers. In another
specific embodiment, the isolated CD34.sup.-, CD10.sup.+,
CD105.sup.+ placental stem cells have the potential to
differentiate into cells of a neural phenotype, cells of an
osteogenic phenotype, and/or cells of a chondrogenic phenotype,
e.g., either in vitro or in vivo, or both. In another specific
embodiment, the isolated CD34.sup.-, CD10.sup.+, CD105.sup.+
placental stem cells are additionally CD200.sup.+. In another
specific embodiment, the isolated CD34.sup.-, CD10.sup.+,
CD105.sup.+ placental stem cells are additionally CD45.sup.- or
CD90.sup.+. In another specific embodiment, the isolated
CD34.sup.-, CD10.sup.+, CD105.sup.+ placental stem cells are
additionally CD45.sup.- and CD90.sup.+, as detectable by flow
cytometry. In another specific embodiment, the isolated CD34.sup.-,
CD10.sup.+, CD105.sup.+, CD200.sup.+ placental stem cells are
additionally CD90.sup.+ or CD45.sup.-, as detectable by flow
cytometry. In a specific embodiment, the isolated CD34.sup.-,
CD10.sup.+, CD105.sup.+, CD200.sup.+ placental stem cells are
additionally one or more of CD44.sup.+, CD45.sup.-, CD90.sup.+,
CD166.sup.+, KDR.sup.+, or CD133.sup.-. In another specific
embodiment, the isolated CD34.sup.-, CD10.sup.+, CD105.sup.+,
CD200.sup.+ placental stem cells are additionally CD44.sup.+,
CD45.sup.-, CD90.sup.+, CD166.sup.+, KDR.sup.+, and CD133.sup.-. In
another specific embodiment, the isolated CD34.sup.-, CD10.sup.+,
CD105.sup.+, CD200.sup.+ placental stem cells are additionally
CD90.sup.+ and CD45.sup.-, as detectable by flow cytometry, i.e.,
the placental stem cells are CD34.sup.-, CD10.sup.+, CD45.sup.-,
CD90.sup.+, CD105.sup.+ and CD200.sup.+. In another specific
embodiment, said CD34.sup.-, CD10.sup.+, CD45.sup.-, CD90.sup.+,
CD105.sup.+, CD200.sup.+ placental stem cells are additionally
CD44.sup.+, CD80.sup.- and/or CD86.sup.-. In another specific
embodiment, said CD34.sup.-, CD10.sup.+, CD44.sup.+, CD45.sup.-,
CD90.sup.+, CD105.sup.+, CD200.sup.+ placental stem cells are
additionally one or more of CD80.sup.-, CD86.sup.-, CD117.sup.-,
CD133.sup.-, cytokeratin.sup.+, KDR.sup.+, HLA-A,B,C.sup.+,
HLA-DR,DP,DQ.sup.-, and HLA-G.sup.-. In another specific
embodiment, the CD34.sup.-, CD10.sup.+, CD105.sup.+ placental stem
cells are additionally one or more of SSEA1.sup.-, SSEA3.sup.-
and/or SSEA4.sup.-. In another specific embodiment, the CD34.sup.-,
CD10.sup.+, CD105.sup.+ placental stem cells are additionally
SSEA1.sup.-, SSEA3.sup.- and SSEA4.sup.-.
[0174] In another embodiment, said placental stem cells are
CD34.sup.-, CD10.sup.+, CD105.sup.+ and CD200.sup.+, and one or
more of CD44.sup.+, CD45.sup.-, CD90.sup.+, CD166.sup.+, KDR.sup.-,
or CD133.sup.-. In a more specific embodiment, said placental stem
cells are CD34.sup.-, CD10.sup.+, CD105.sup.+ and CD200.sup.+,
CD44.sup.+, CD45.sup.-, CD90.sup.+, CD166.sup.+, KDR.sup.-, and
CD133.sup.-. In another embodiment, said placental stem cells are
CD34.sup.-, CD10.sup.+, CD105.sup.+ and CD200.sup.+, and one or
more of HLA ABC.sup.+, HLA DR,DQ,DP.sup.-, CD80.sup.-, CD86.sup.-,
CD98.sup.-, or PD-L1.sup.+. In a more specific embodiment, said
placental stem cells are CD34.sup.-, CD10.sup.+, CD105.sup.+ and
CD200.sup.+, HLA ABC.sup.+, HLA DR,DQ,DP.sup.-, CD80.sup.-,
CD86.sup.-, CD98.sup.-, and PD-L1.sup.+. In certain embodiments,
said placental stem cells are CD34.sup.-, CD10.sup.+, CD105.sup.+
and CD200.sup.+, and one or more of CD3.sup.-, CD9.sup.-,
CD38.sup.-, CD45.sup.-, CD80.sup.-, CD86.sup.-, CD133.sup.-,
HLA-DR,DP,DQ.sup.-, SSEA3.sup.-, SSEA4.sup.-, CD29.sup.+,
CD44.sup.+, CD73.sup.+, CD90.sup.+, CD105.sup.+, HLA-A,B,C.sup.+,
PDL1.sup.+, ABC-p.sup.+, and/or OCT-4.sup.+, as detectable by flow
cytometry. In other embodiments, any of the CD34.sup.-, CD10.sup.+,
CD105.sup.+ placental stem cells described above are additionally
one or more of CD29.sup.+, CD38.sup.-, CD44.sup.+, CD54.sup.+,
SH3.sup.+ or SH4.sup.+. In another specific embodiment, the
placental stem cells are additionally CD44.sup.+. In another
specific embodiment of any of the isolated CD34.sup.-, CD10.sup.+,
CD105.sup.+ placental stem cells above, the cells are additionally
one or more of CD117.sup.-, CD133.sup.-, KDR.sup.- (VEGFR2.sup.-),
HLA-A,B,C.sup.+, HLA-DP,DQ,DR.sup.-, or Programmed Death-1 Ligand
(PDL1)+, or any combination thereof.
[0175] In another embodiment, the isolated CD34.sup.-, CD10.sup.+,
CD105.sup.+ placental stem cells are additionally one or more of
CD3.sup.-, CD9.sup.-, CD13.sup.+, CD29.sup.+, CD33.sup.+,
CD38.sup.-, CD44.sup.+, CD45.sup.-, CD54.sup.+, CD62E.sup.-,
CD62L.sup.-, CD62P.sup.-, SH3.sup.+ (CD73.sup.+), SH4.sup.+
(CD73.sup.+), CD80.sup.-, CD86.sup.-, CD90.sup.+, SH2.sup.+
(CD105.sup.+), CD106/VCAM.sup.+, CD117.sup.-,
CD144/VE-cadherin.sup.low, CD146.sup.+, CD166.sup.+,
CD184/CXCR4.sup.-, CD200.sup.+, CD133.sup.-, OCT-4.sup.+,
SSEA3.sup.-, SSEA4.sup.-, ABC-p.sup.+, KDR.sup.- (VEGFR2.sup.-),
HLA-A,B,C.sup.+, HLA-DP,DQ,DR.sup.-, HLA-G.sup.-, or Programmed
Death-1 Ligand (PDL1).sup.+, or any combination thereof. In another
embodiment, the CD3.sup.-, CD9.sup.-, CD34.sup.-, CD10.sup.+,
CD105.sup.+ placental stem cells are additionally CD13.sup.+,
CD29.sup.+, CD33.sup.+, CD38.sup.-, CD44.sup.+, CD45.sup.-,
CD54/ICAM.sup.+, CD62E.sup.-, CD62L.sup.-, CD62P.sup.-, SH3.sup.+
(CD73.sup.+), SH4.sup.+ (CD73.sup.+), CD80.sup.-, CD86.sup.-,
CD90.sup.+, SH2.sup.+ (CD105.sup.+), CD106/VCAM.sup.+, CD117.sup.-,
CD144/VE-cadherin.sup.low, CD146.sup.+, CD166.sup.+,
CD184/CXCR4.sup.-, CD200.sup.+, CD133.sup.-, OCT-4.sup.+,
SSEA3.sup.-, SSEA4.sup.-, ABC-p.sup.+, KDR.sup.- (VEGFR2.sup.-),
HLA-A,B,C.sup.+, HLA-DP,DQ,DR.sup.-, HLA-G.sup.-, and Programmed
Death-1 Ligand (PDL1).sup.+.
[0176] In another specific embodiment, any of the isolated
placental stem cells described herein are ABC-p.sup.+, as
detectable by flow cytometry, and/or OCT-4.sup.+ (POU5F1.sup.+), as
determinable by RT-PCR, wherein ABC-p is a placenta-specific ABC
transporter protein (also known as breast cancer resistance protein
(BCRP) and as mitoxantrone resistance protein (MXR)), and OCT-4 is
the Octamer-4 protein (POU5F1). In another specific embodiment, any
of the placental stem cells described herein are additionally SSEA3
or SSEA4.sup.-, as determinable by flow cytometry, wherein SSEA3 is
Stage Specific Embryonic Antigen 3, and SSEA4 is Stage Specific
Embryonic Antigen 4. In another specific embodiment, any of the
placental stem cells described herein are additionally SSEA3.sup.-
and SSEA4.sup.-.
[0177] In another specific embodiment, any of the placental stem
cells described herein are one or more of MHC-I.sup.+ (e.g.,
HLA-A,B,C.sup.+), MHC-II.sup.- (e.g., HLA-DP,DQ,DR.sup.-) or
HLA-G.sup.-. In another specific embodiment, any of the placental
stem cells described herein are one or more of MHC-I.sup.+ (e.g.,
HLA-A,B,C.sup.+), MHC-II.sup.- (e.g., HLA-DP,DQ,DR.sup.-) and
HLA-G.sup.-.
[0178] Also provided herein are populations of cells comprising,
e.g., that are enriched for, the isolated placental stem cells,
that are useful in the methods and compositions disclosed herein.
Preferred populations of cells comprise the isolated placental stem
cells, wherein at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% of the
cells in said population of cells are isolated CD10.sup.+,
CD105.sup.+ and CD34.sup.- placental stem cells. In a specific
embodiment, the isolated CD34.sup.-, CD10.sup.+, CD105.sup.+
placental stem cells are additionally CD200.sup.+. In another
specific embodiment, the isolated CD34.sup.-, CD10.sup.+,
CD105.sup.+, CD200.sup.+ placental stem cells are additionally
CD90.sup.+ or CD45.sup.-, as detectable by flow cytometry. In
another specific embodiment, the isolated CD34.sup.-, CD10.sup.+,
CD105.sup.+, CD200.sup.+ placental stem cells are additionally
CD90.sup.+ and CD45.sup.-, as detectable by flow cytometry. In
another specific embodiment, any of the isolated CD34.sup.-,
CD10.sup.+, CD105.sup.+ placental stem cells described above are
additionally one or more of CD29.sup.+, CD38.sup.-, CD44.sup.+,
CD54.sup.+, SH3.sup.+ or SH4.sup.+. In another specific embodiment,
the isolated CD34.sup.-, CD10.sup.+, CD105.sup.+ placental stem
cells, or isolated CD34.sup.-, CD10.sup.+, CD105.sup.+, CD200.sup.+
placental stem cells, are additionally CD44.sup.+. In a specific
embodiment of any of the populations of cells comprising isolated
CD34.sup.-, CD10.sup.+, CD105.sup.+ placental stem cells above, the
isolated placental stem cells are additionally one or more of
CD13.sup.+, CD29.sup.+, CD33.sup.+, CD38.sup.-, CD44.sup.+,
CD45.sup.-, CD54.sup.+, CD62E.sup.-, CD62L.sup.-, CD62P.sup.-,
SH3.sup.+ (CD73.sup.+), SH4.sup.+ (CD73.sup.+), CD80.sup.-,
CD86.sup.-, CD90.sup.+, SH2.sup.+ (CD105.sup.+), CD106/VCAM.sup.+,
CD117.sup.-, CD144/VE-cadherin.sup.low, CD184/CXCR4.sup.-,
CD200.sup.+, CD133.sup.-, OCT-4.sup.+, SSEA3.sup.-, SSEA4.sup.-,
ABC-p.sup.+, KDR.sup.- (VEGFR2.sup.-), HLA-A,B,C.sup.+,
HLA-DP,DQ,DR.sup.-, HLA-G.sup.-, or Programmed Death-1 Ligand
(PDL1).sup.+, or any combination thereof. In another specific
embodiment, the CD34.sup.-, CD10.sup.+, CD105.sup.+ cells are
additionally CD13.sup.+, CD29.sup.+, CD33.sup.+, CD38.sup.-,
CD44.sup.+, CD45.sup.-, CD54/ICAM.sup.+, CD62E.sup.-, CD62L.sup.-,
CD62P.sup.-, SH3.sup.+ (CD73.sup.-), SH4.sup.+ (CD73.sup.+),
CD80.sup.-, CD86.sup.-, CD90.sup.+, SH2.sup.+ (CD105.sup.+),
CD106/VCAM.sup.+, CD117.sup.-, CD144/VE-cadherin.sup.low,
CD184/CXCR4.sup.-, CD200.sup.+, CD133.sup.-, OCT-4.sup.+,
SSEA3.sup.-, SSEA4.sup.-, ABC-p.sup.+, KDR.sup.- (VEGFR2.sup.-),
HLA-A,B,C.sup.+, HLA-DP,DQ,DR.sup.-, HLA-G.sup.-, and Programmed
Death-1 Ligand (PDL1).sup.+.
[0179] In certain embodiments, the isolated placental stem cells
useful in the methods and compositions described herein are one or
more, or all, of CD10.sup.+, CD29.sup.+, CD34.sup.-, CD38.sup.-,
CD44.sup.+, CD45.sup.-, CD54.sup.+, CD90.sup.+, SH2.sup.+,
SH3.sup.+, SH4.sup.+, SSEA3.sup.-, SSEA4.sup.-, OCT-4.sup.+, and
ABC-p.sup.+, wherein said isolated placental stem cells are
obtained by physical and/or enzymatic disruption of placental
tissue. In a specific embodiment, the isolated placental stem cells
are OCT-4.sup.+ and ABC-p.sup.+. In another specific embodiment,
the isolated placental stem cells are OCT-4.sup.+ and CD34.sup.-,
wherein said isolated placental stem cells have at least one, or
all, of the following characteristics: CD10.sup.+, CD29.sup.+,
CD44.sup.+, CD45.sup.-, CD54.sup.+, CD90.sup.+, SH3.sup.+,
SH4.sup.+, SSEA3.sup.-, and SSEA4.sup.-. In another specific
embodiment, the isolated placental stem cells are OCT-4.sup.+,
CD34.sup.-, CD10.sup.+, CD29.sup.+, CD44.sup.+, CD45.sup.-,
CD54.sup.+, CD90.sup.+, SH3.sup.+, SH4.sup.+, SSEA3.sup.-, and
SSEA4.sup.-. In another embodiment, the isolated placental stem
cells are OCT-4.sup.+, CD34.sup.-, SSEA3.sup.-, and SSEA4.sup.-. In
another specific embodiment, the isolated placental stem cells are
OCT-4.sup.+ and CD34.sup.-, and is either SH2.sup.+ or SH3.sup.+.
In another specific embodiment, the isolated placental stem cells
are OCT-4.sup.+, CD34.sup.-, SH2.sup.+, and SH3.sup.+. In another
specific embodiment, the isolated placental stem cells are
OCT-4.sup.+, CD34.sup.-, SSEA3.sup.-, and SSEA4.sup.-, and are
either SH2.sup.+ or SH3.sup.+. In another specific embodiment, the
isolated placental stem cells are OCT-4.sup.+ and CD34.sup.-, and
either SH2.sup.+ or SH3.sup.+, and is at least one of CD10.sup.+,
CD29.sup.+, CD44.sup.+, CD45.sup.-, CD54.sup.+, CD90.sup.+,
SSEA3.sup.-, or SSEA4.sup.-. In another specific embodiment, the
isolated placental stem cells are OCT-4.sup.+, CD34.sup.-,
CD10.sup.+, CD29.sup.+, CD44.sup.+, CD45.sup.-, CD54.sup.+,
CD90.sup.+, SSEA3.sup.-, and SSEA4.sup.-, and either SH2.sup.+ or
SH3.sup.+.
[0180] In another embodiment, the isolated placental stem cells
useful in the methods and compositions disclosed herein are
SH2.sup.+, SH3.sup.+, SH4.sup.+ and OCT-4.sup.+. In another
specific embodiment, the isolated placental stem cells are
CD10.sup.+, CD29.sup.+, CD44.sup.+, CD54.sup.+, CD90.sup.+,
CD34.sup.-, CD45.sup.-, SSEA3.sup.-, or SSEA4.sup.-. In another
embodiment, the isolated placental stem cells are SH2.sup.+,
SH3.sup.+, SH4.sup.+, SSEA3.sup.- and SSEA4.sup.-. In another
specific embodiment, the isolated placental stem cells are
SH2.sup.+, SH3.sup.+, SH4.sup.+, SSEA3.sup.- and SSEA4.sup.-,
CD10.sup.+, CD29.sup.+, CD44.sup.+, CD54.sup.+, CD90.sup.+,
OCT-4.sup.+, CD34.sup.- or CD45.sup.-.
[0181] In another embodiment, the isolated placental stem cells
useful in the methods and compositions disclosed herein are
CD10.sup.+, CD29.sup.+, CD34.sup.-, CD44.sup.+, CD45.sup.-,
CD54.sup.+, CD90.sup.+, SH2.sup.+, SH3.sup.+, and SH4.sup.+;
wherein said isolated placental stem cells are additionally one or
more of OCT-4.sup.+, SSEA3.sup.- or SSEA4.sup.-.
[0182] In certain embodiments, isolated placental stem cells useful
in the methods and compositions disclosed herein are CD200.sup.+ or
HLA-G.sup.-. In a specific embodiment, the isolated placental stem
cells are CD200.sup.+ and HLA-G.sup.-. In another specific
embodiment, the isolated placental stem cells are additionally
CD73.sup.+ and CD105.sup.+. In another specific embodiment, the
isolated placental stem cells are additionally CD34.sup.-,
CD38.sup.- or CD45.sup.-. In another specific embodiment, the
isolated placental stem cells are additionally CD34.sup.-,
CD38.sup.- and CD45.sup.-. In another specific embodiment, said
isolated placental stem cells are CD34.sup.-, CD38.sup.-,
CD45.sup.-, CD73.sup.+ and CD105.sup.+. In another specific
embodiment, said isolated CD200.sup.+ or HLA-G.sup.- placental stem
cells facilitate the formation of embryoid-like bodies in a
population of placental cells comprising the isolated placental
stem cells, under conditions that allow the formation of
embryoid-like bodies. In another specific embodiment, the isolated
placental stem cells are isolated away from placental cells that
are not stem or multipotent cells. In another specific embodiment,
said isolated placental stem cells are isolated away from placental
cells that do not display these markers.
[0183] In another embodiment, a cell population useful in the
methods and compositions described herein is a population of cells
comprising, e.g., that is enriched for, CD200.sup.+, HLA-G.sup.-
placental stem cells. In a specific embodiment, said population is
a population of placental cells. In various embodiments, at least
about 10%, at least about 20%, at least about 30%, at least about
40%, at least about 50%, or at least about 60% of cells in said
cell population are isolated CD200.sup.+, HLA-G placental stem
cells. In certain embodiments, at least about 70% of cells in said
cell population are isolated CD200.sup.+, HLA-G placental stem
cells. In certain other embodiments, at least about 90%, 95%, or
99% of said cells are isolated CD200.sup.+, HLA-G.sup.- placental
stem cells. In a specific embodiment of the cell populations, said
isolated CD200.sup.+, HLA-G placental stem cells are also
CD73.sup.+ and CD105.sup.+. In another specific embodiment, said
isolated CD200.sup.+, HLA-G.sup.- placental stem cells are also
CD34.sup.-, CD38.sup.- or CD45.sup.-. In another specific
embodiment, said isolated CD200.sup.+, HLA-G.sup.- placental stem
cells are also CD34.sup.-, CD38.sup.-, CD45.sup.-, CD73.sup.+ and
CD105.sup.+. In another specific embodiment, said cell population
is isolated away from placental cells that are not stem cells. In
another specific embodiment, said isolated CD200.sup.+, HLA-G.sup.-
placental stem cells are isolated away from placental cells that do
not display these markers.
[0184] In another embodiment, the isolated placental stem cells
useful in the methods and compositions described herein are
CD73.sup.+, CD105.sup.+, and CD200.sup.+. In another specific
embodiment, the isolated placental stem cells are HLA-G.sup.-. In
another specific embodiment, the isolated placental stem cells are
CD34.sup.-, CD38.sup.- or CD45.sup.-. In another specific
embodiment, the isolated placental stem cells are CD34.sup.-,
CD38.sup.- and CD45.sup.-. In another specific embodiment, the
isolated placental stem cells are CD34.sup.-, CD38.sup.-,
CD45.sup.-, and HLA-G.sup.-. In another specific embodiment, the
isolated placental stem cells are isolated away from placental
cells that are not the isolated placental stem cells. In another
specific embodiment, the isolated placental stem cells are isolated
away from placental cells that do not display these markers.
[0185] In another embodiment, a cell population useful in the
methods and compositions described herein is a population of cells
comprising, e.g., that is enriched for, isolated CD73.sup.+,
CD105.sup.+, CD200.sup.+ placental stem cells. In various
embodiments, at least about 10%, at least about 20%, at least about
30%, at least about 40%, at least about 50%, or at least about 60%
of cells in said cell population are isolated CD73.sup.+,
CD105.sup.+, CD200.sup.+ placental stem cells. In another
embodiment, at least about 70% of said cells in said population of
cells are isolated CD73.sup.+, CD105.sup.+, CD200.sup.+ placental
stem cells. In another embodiment, at least about 90%, 95% or 99%
of cells in said population of cells are isolated CD73.sup.+,
CD105.sup.+, CD200.sup.+ placental stem cells. In a specific
embodiment of said populations, the isolated placental stem cells
are HLA-G.sup.-. In another specific embodiment, the isolated
placental stem cells are additionally CD34.sup.-, CD38.sup.- or
CD45.sup.-. In another specific embodiment, the isolated placental
stem cells are additionally CD34.sup.-, CD38.sup.- and CD45.sup.-.
In another specific embodiment, the isolated placental stem cells
are additionally CD34.sup.-, CD38.sup.-, CD45.sup.-, and
HLA-G.sup.-. In another specific embodiment, said population of
placental cells is isolated away from placental cells that are not
stem cells. In another specific embodiment, said population of
placental stem cells is isolated away from placental cells that do
not display these characteristics.
[0186] In certain other embodiments, the isolated placental stem
cells are one or more of CD10.sup.+, CD29.sup.+, CD34.sup.-,
CD38.sup.-, CD44.sup.+, CD45.sup.-, CD54.sup.+, CD90.sup.+,
SH2.sup.+, SH3.sup.+, SH4.sup.+, SSEA3.sup.-, SSEA4.sup.-,
OCT-4.sup.+, HLA-G.sup.- or ABC-p.sup.+. In a specific embodiment,
the isolated placental stem cells are CD10.sup.+, CD29.sup.+,
CD34.sup.-, CD38.sup.-, CD44.sup.+, CD45.sup.-, CD54.sup.+,
CD90.sup.+, SH2.sup.+, SH3.sup.+, SH4.sup.+, SSEA3.sup.-,
SSEA4.sup.-, and OCT-4.sup.+. In another specific embodiment, the
isolated placental stem cells are CD10.sup.+, CD29.sup.+,
CD34.sup.-, CD38.sup.-, CD45.sup.-, CD54.sup.+, SH2.sup.+,
SH3.sup.+, and SH4.sup.+. In another specific embodiment, the
isolated placental stem cells are CD10.sup.+, CD29.sup.+,
CD34.sup.-, CD38.sup.-, CD45.sup.-, CD54.sup.+, SH2.sup.+,
SH3.sup.+, SH4.sup.+ and OCT-4.sup.+. In another specific
embodiment, the isolated placental stem cells are CD10.sup.+,
CD29.sup.+, CD34.sup.-, CD38.sup.-, CD44.sup.+, CD45.sup.-,
CD54.sup.+, CD90.sup.+, HLA-G.sup.-, SH2.sup.+, SH3.sup.+,
SH4.sup.+. In another specific embodiment, the isolated placental
stem cells are OCT-4.sup.- and ABC-p.sup.+. In another specific
embodiment, the isolated placental stem cells are SH2.sup.+,
SH3.sup.+, SH4.sup.+ and OCT-4.sup.+. In another embodiment, the
isolated placental stem cells are OCT-4.sup.+, CD34.sup.-,
SSEA3.sup.-, and SSEA4.sup.-. In a specific embodiment, said
isolated OCT-4.sup.+, CD34.sup.-, SSEA3.sup.-, and SSEA4.sup.-
placental stem cells are additionally CD10.sup.+, CD29.sup.+,
CD34.sup.-, CD44.sup.+, CD45.sup.-, CD54.sup.+, CD90.sup.+,
SH2.sup.+, SH3.sup.+, and SH4.sup.+. In another embodiment, the
isolated placental stem cells are OCT-4.sup.+ and CD34.sup.-, and
either SH3.sup.+ or SH4.sup.+. In another embodiment, the isolated
placental stem cells are CD34.sup.- and either CD10.sup.+,
CD29.sup.+, CD44.sup.+, CD54.sup.+, CD90.sup.+, or OCT-4.sup.+.
[0187] In another embodiment, the isolated placental stem cells
useful in the methods and compositions described herein are
CD200.sup.+ and OCT-4.sup.+. In a specific embodiment, the isolated
placental stem cells are CD73.sup.+ and CD105.sup.+. In another
specific embodiment, said isolated placental stem cells are
HLA-G.sup.-. In another specific embodiment, said isolated
CD200.sup.+, OCT-4.sup.+ placental stem cells are CD34.sup.-,
CD38.sup.- or CD45.sup.-. In another specific embodiment, said
isolated CD200.sup.+, OCT-4.sup.+ placental stem cells are
CD34.sup.-, CD38.sup.- and CD45.sup.-. In another specific
embodiment, said isolated CD200.sup.+, OCT-4.sup.+ placental stem
cells are CD34.sup.-, CD38.sup.-, CD45.sup.-, CD73.sup.+,
CD105.sup.+ and HLA-G.sup.-. In another specific embodiment, said
isolated CD200.sup.+, OCT-4.sup.+ placental stem cells are isolated
away from placental cells that are not stem cells. In another
specific embodiment, said isolated CD200.sup.+, OCT-4.sup.+
placental stem cells are isolated away from placental cells that do
not display these characteristics.
[0188] In another embodiment, a cell population useful in the
methods and compositions described herein is a population of cells
comprising, e.g., that is enriched for, CD200.sup.+, OCT-4.sup.+
placental stem cells. In various embodiments, at least about 10%,
at least about 20%, at least about 30%, at least about 40%, at
least about 50%, or at least about 60% of cells in said cell
population are isolated CD200.sup.+, OCT-4.sup.+ placental stem
cells. In another embodiment, at least about 70% of said cells are
said isolated CD200.sup.+, OCT-4.sup.+ placental stem cells. In
another embodiment, at least about 80%, 90%, 95%, or 99% of cells
in said cell population are said isolated CD200.sup.+, OCT-4.sup.+
placental stem cells. In a specific embodiment of the isolated
populations, said isolated CD200.sup.+, OCT-4.sup.+ placental stem
cells are additionally CD73.sup.+ and CD105.sup.+. In another
specific embodiment, said isolated CD200.sup.+, OCT-4.sup.+
placental stem cells are additionally HLA-G. In another specific
embodiment, said isolated CD200.sup.+, OCT-4.sup.+ placental stem
cells are additionally CD34.sup.-, CD38.sup.- and CD45.sup.-. In
another specific embodiment, said isolated CD200.sup.+, OCT-4.sup.+
placental stem cells are additionally CD34.sup.-, CD38.sup.-,
CD45.sup.-, CD73.sup.+, CD105.sup.+ and HLA-G.sup.-. In another
specific embodiment, said cell population is isolated away from
placental cells that are not isolated CD200.sup.+, OCT-4.sup.+
placental stem cells. In another specific embodiment, said cell
population is isolated away from placental cells that do not
display these markers.
[0189] In another embodiment, the isolated placental stem cells
useful in the methods and compositions described herein are
CD73.sup.+, CD105.sup.+ and HLA-G.sup.-. In another specific
embodiment, the isolated CD73.sup.+, CD105.sup.+ and HLA-G.sup.-
placental stem cells are additionally CD34.sup.-, CD38.sup.- or
CD45.sup.-. In another specific embodiment, the isolated
CD73.sup.+, CD105.sup.+, HLA-G placental stem cells are
additionally CD34.sup.-, CD38.sup.- and CD45.sup.-. In another
specific embodiment, the isolated CD73.sup.+, CD105.sup.+,
HLA-G.sup.- placental stem cells are additionally OCT-4.sup.+. In
another specific embodiment, the isolated CD73.sup.+, CD105.sup.+,
HLA-G.sup.- placental stem cells are additionally CD200.sup.+. In
another specific embodiment, the isolated CD73.sup.+, CD105.sup.+,
HLA-G placental stem cells are additionally CD34.sup.-, CD38.sup.-,
CD45.sup.-, OCT-4.sup.+ and CD200.sup.+. In another specific
embodiment, said the isolated CD73.sup.+, CD105.sup.+, HLA-G.sup.-
placental stem cells are isolated away from placental cells that
are not the isolated CD73.sup.+, CD105.sup.+, HLA-G.sup.- placental
stem cells. In another specific embodiment, said the isolated
CD73.sup.+, CD105.sup.+, HLA-G.sup.- placental stem cells are
isolated away from placental cells that do not display these
markers.
[0190] In another embodiment, a cell population useful in the
methods and compositions described herein is a population of cells
comprising, e.g., that is enriched for, isolated CD73.sup.+,
CD105.sup.+ and HLA-G placental stem cells. In various embodiments,
at least about 10%, at least about 20%, at least about 30%, at
least about 40%, at least about 50%, or at least about 60% of cells
in said population of cells are isolated CD73.sup.+, CD105.sup.+,
HLA-G.sup.- placental stem cells. In another embodiment, at least
about 70% of cells in said population of cells are isolated
CD73.sup.+, CD105.sup.+, HLA-G.sup.- placental stem cells. In
another embodiment, at least about 90%, 95% or 99% of cells in said
population of cells are isolated CD73.sup.+, CD105.sup.+,
HLA-G.sup.- placental stem cells. In a specific embodiment of the
above populations, said isolated CD73.sup.+, CD105.sup.+,
HLA-G.sup.- placental stem cells are additionally CD34.sup.-,
CD38.sup.- or CD45.sup.-. In another specific embodiment, said
isolated CD73.sup.+, CD105.sup.+, HLA-G.sup.- placental stem cells
are additionally CD34.sup.-, CD38.sup.- and CD45.sup.-. In another
specific embodiment, said isolated CD73.sup.+, CD105.sup.+,
HLA-G.sup.- placental stem cells are additionally OCT-4.sup.+. In
another specific embodiment, said isolated CD73.sup.+, CD105.sup.+,
HLA-G.sup.- placental stem cells are additionally CD200.sup.+. In
another specific embodiment, said isolated CD73.sup.+, CD105.sup.+,
HLA-G.sup.- placental stem cells are additionally CD34.sup.-,
CD38.sup.-, CD45.sup.-, OCT-4.sup.+ and CD200.sup.+. In another
specific embodiment, said cell population is isolated away from
placental cells that are not CD73.sup.+, CD105.sup.+, HLA-G.sup.-
placental stem cells. In another specific embodiment, said cell
population is isolated away from placental cells that do not
display these markers.
[0191] In another embodiment, the isolated placental stem cells
useful in the methods and compositions described herein are
isolated HLA-A,B,C.sup.+, CD45.sup.-, CD133.sup.- and CD34.sup.-
placental stem cells. In another embodiment, a cell population
useful in the methods and compositions described herein is a
population of cells comprising isolated placental cells, wherein at
least about 70%, at least about 80%, at least about 90%, at least
about 95% or at least about 99% of cells in said isolated
population of cells are isolated HLA-A,B,C.sup.+, CD45.sup.-,
CD133.sup.- and CD34.sup.- placental stem cells. In a specific
embodiment, said isolated placental stem cells or population of
isolated placental stem cells are isolated away from placental
cells that are not HLA-A,B,C.sup.+, CD45.sup.-, CD133.sup.- and
CD34.sup.- placental stem cells. In another specific embodiment of
any of the placental stem cells described herein, said isolated
placental stem cells are non-maternal in origin. In another
specific embodiment, said isolated population of placental stem
cells are substantially free of matcrnal components; e.g., at least
about 40%, 45%, 5-0%, 55%, 60%, 65%, 70%, 75%, 90%, 85%, 90%, 95%,
98% or 99% of said cells in said isolated population of placental
cells are non-maternal in origin.
[0192] In another embodiment, the isolated placental stem cells
useful in the methods and compositions described herein are
isolated CD10.sup.+, CD13.sup.+, CD33.sup.+, CD45.sup.-,
CD117.sup.- and CD133.sup.- placental stem cells. In another
embodiment, a cell population useful in the methods and
compositions described herein is a population of cells comprising
said isolated placental stem cells, wherein at least about 70%, at
least about 80%, at least about 90%, at least about 95% or at least
about 99% of cells in said population of cells are said isolated
CD10.sup.+, CD13.sup.+, CD33.sup.+, CD45.sup.-, CD117.sup.- and
CD133.sup.- placental stem cells. In a specific embodiment, said
isolated placental stem cells or population of isolated placental
stem cells are isolated away from placental cells that are not said
isolated placental cells. In another specific embodiment, said
isolated CD10.sup.+, CD13.sup.+, CD33.sup.+, CD45.sup.-,
CD117.sup.- and CD133.sup.- placental stem cells are non-maternal
in origin, i.e., have the fetal genotype. In another specific
embodiment, at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
90%, 85%, 90%, 95%, 98% or 99% of said cells in said isolated
population of placental stem cells, are non-maternal in origin. In
another specific embodiment, said isolated placental stem cells or
population of isolated placental stem cells are isolated away from
placental cells that do not display these characteristics.
[0193] In another embodiment, the isolated placental stem cells
useful in the methods and compositions described herein are
isolated CD10.sup.+, CD33.sup.-, CD44.sup.+, CD45.sup.-, and
CD117.sup.- placental stem cells. In another embodiment, a cell
population useful for the in the methods and compositions described
herein is a population of cells comprising, e.g., enriched for,
said isolated placental stem cells, wherein at least about 70%, at
least about 80%, at least about 90%, at least about 95% or at least
about 99% of cells in said population of cells are isolated
CD10.sup.-, CD33.sup.-, CD44.sup.+, CD45.sup.-, and CD117.sup.-
placental stem cells. In a specific embodiment, said isolated
placental stem cells or population of isolated placental stem cells
are isolated away from placental cells that are not said placental
stem cells. In another specific embodiment, said isolated placental
stem cells are non-maternal in origin. In another specific
embodiment, at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
90%, 85%, 90%, 95%, 98% or 99% of said placental stem cells in said
cell population are non-maternal in origin. In another specific
embodiment, said isolated placental stem cells or population of
isolated placental stem cells are isolated away from placental
cells that do not display these markers.
[0194] In another embodiment, the isolated placental stem cells
useful in the methods and compositions described herein are
isolated CD10.sup.-, CD13.sup.-, CD33.sup.-, CD45.sup.-, and
CD117.sup.- placental stem cells. In another embodiment, a cell
population useful for in the methods and compositions described
herein is a population of cells comprising, e.g., enriched for,
isolated CD10.sup.-, CD13.sup.-, CD33.sup.-, CD45.sup.-, and
CD117.sup.- placental stem cells, wherein at least about 70%, at
least about 80%, at least about 90%, at least about 95% or at least
about 99% of cells in said population are CD10.sup.-, CD13.sup.-,
CD33.sup.-, CD45.sup.-, and CD117.sup.- placental stem cells. In a
specific embodiment, said isolated placental stem cells or
population of isolated placental stem cells are isolated away from
placental cells that are not said placental stem cells. In another
specific embodiment, said isolated placental stem cells are
non-maternal in origin. In another specific embodiment, at least
about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 90%, 85%, 90%, 95%,
98% or 99% of said cells in said cell population are non-maternal
in origin. In another specific embodiment, said isolated placental
stem cells or population of isolated placental stem cells is
isolated away from placental cells that do not display these
characteristics.
[0195] In another embodiment, the isolated placental stem cells
useful in the methods and compositions described herein are HLA
A,B,C+, CD45.sup.-, CD34.sup.-, and CD133.sup.-, and are
additionally CD10.sup.+, CD13.sup.+, CD38.sup.+, CD44.sup.+,
CD90.sup.+, CD105.sup.+, CD200.sup.+ and/or HLA-G.sup.-, and/or
negative for CD117. In another embodiment, a cell population useful
in the methods described herein is a population of cells comprising
said isolated placental stem cells, wherein at least about 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 98% or about 99% of the cells in said population are
isolated placental stem cells that are HLA A,B,C.sup.-, CD45.sup.-,
CD34.sup.-, CD133.sup.-, and that are additionally positive for
CD10, CD13, CD38, CD44, CD90, CD105, CD200, and/or negative for
CD117 and/or HLA-G. In a specific embodiment, said isolated
placental stem cells or population of isolated placental stem cells
are isolated away from placental cells that are not said placental
stem cells. In another specific embodiment, said isolated placental
stem cells are non-maternal in origin. In another specific
embodiment, at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
90%, 85%, 90%, 95%, 98% or 99% of said cells in said cell
population are non-maternal in origin. In another specific
embodiment, said isolated placental stem cells or population of
isolated placental stem cells are isolated away from placental
cells that do not display these markers.
[0196] In another embodiment, the isolated placental stem cells
useful in the methods and compositions described herein are
CD200.sup.+ and CD10.sup.+, as determinable by antibody binding,
and CD117.sup.-, as determinable by both antibody binding and
RT-PCR. In another embodiment, the isolated placental stem cells
useful in the methods and compositions described herein are
CD10.sup.+, CD29.sup.-, CD54.sup.+, CD200.sup.+, HLA-G.sup.-, MHC
class I.sup.+ and .beta.-2-microglobulin.sup.+. In another
embodiment, isolated placental stem cells useful in the methods and
compositions described herein are placental cells wherein the
expression of at least one cellular marker is at least two-fold
higher than for a mesenchymal stem cell (e.g., a bone
marrow-derived mesenchymal stem cell). In another specific
embodiment, said isolated placental stem cells are non-maternal in
origin. In another specific embodiment, at least about 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 90%, 85%, 90%, 95%, 98% or 99% of
said cells in said cell population are non-maternal in origin.
[0197] In another embodiment, the isolated placental stem cells
useful in the methods and compositions described herein are one or
more of CD10.sup.+, CD29.sup.+, CD44.sup.+, CD45.sup.-,
CD54/ICAM.sup.+, CD62E.sup.-, CD62L.sup.-, CD62P.sup.-, CD80.sup.-,
CD86.sup.-, CD103.sup.-, CD104.sup.-, CD105.sup.+,
CD106/VCAM.sup.+, CD144/VE-cadherin.sup.low, CD184/CXCR4.sup.-,
.beta.2-microglobulin.sup.low, MHC-I.sup.low, MHC-II.sup.-,
HLA-G.sup.low, and/or PDL1.sup.low. In a specific embodiment, the
isolated placental stem cells are at least CD29.sup.+ and
CD54.sup.+. In another specific embodiment, the isolated placental
stem cells are at least CD44.sup.+ and CD106.sup.+. In another
specific embodiment, the isolated placental stem cells are at least
CD29.sup.+.
[0198] In another embodiment, a cell population useful in the
methods and compositions described herein comprises isolated
placental stem cells, wherein at least 50%, 60%, 70%, 80%, 90%,
95%, 98% or 99% of the cells in said cell population are isolated
placental stem cells that are one or more of CD10.sup.+,
CD29.sup.+, CD44.sup.+, CD45.sup.-, CD54/ICAM.sup.+, CD62E.sup.-,
CD62L.sup.-, CD62P.sup.-, CD80.sup.-, CD86.sup.-, CD103.sup.-,
CD104.sup.-, CD105.sup.+, CD106/VCAM.sup.+,
CD144/VE-cadherin.sup.dim, CD184/CXCR4.sup.-,
.beta.2-microglobulin.sup.dim, HLA-I.sup.dim, HLA-II.sup.-,
HLA-G.sup.dim, and/or PDL1.sup.dim. In another specific embodiment,
at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% of cells in said
cell population are CD10.sup.+, CD29.sup.+, CD44.sup.+, CD45.sup.-,
CD54/ICAM.sup.+, CD62-E.sup.-, CD62-L.sup.-, CD62-P.sup.-,
CD80.sup.-, CD86.sup.-, CD103.sup.-, CD104.sup.-, CD105.sup.+,
CD106/VCAM.sup.+, CD144/VE-cadherin.sup.dim, CD184/CXCR4.sup.-,
.beta.2-microglobulin.sup.dim, MHC-I.sup.dim, MHC-II.sup.-,
HLA-G.sup.dim, and PDL1.sup.dim placental stem cells.
[0199] In another embodiment, the isolated placental stem cells
useful in the methods and compositions described herein are one or
more, or all, of CD10.sup.+, CD29.sup.+, CD34.sup.-, CD38.sup.-,
CD44.sup.+, CD45.sup.-, CD54.sup.+, CD90.sup.+, SH2.sup.+,
SH3.sup.+, SH4.sup.+, SSEA3.sup.-, SSEA4.sup.-, OCT-4.sup.+, and
ABC-p.sup.+, where ABC-p is a placenta-specific ABC transporter
protein (also known as breast cancer resistance protein (BCRP) and
as mitoxantrone resistance protein (MXR)), wherein said isolated
placental stem cells are obtained by perfusion of a mammalian,
e.g., human, placenta that has been drained of cord blood and
perfused to remove residual blood.
[0200] In another specific embodiment of any of the embodiments of
placental stem cells described herein, the cells are negative for
telomerase gene expression, negative for telomerase activity, or
both. Telomerase gene expression can be detected using, e.g.,
detection of telomerase RNA using, e.g., dot blots or slot blots;
or a telomere repeat amplification protocol (TRAP) assay (e.g.,
TRAPEZE.RTM. ELISA, fluorometric or gel-based assay kits from
Millipore).
[0201] In another specific embodiment of any of the embodiments of
placental stem cells described herein, the placental stem cells are
positive for vimentin, e.g., at least 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, or 98% of said placental stem cells express
vimentin. Vimentin can be detected, e.g., by flow cytometry using
one or more antibodies to vimentin, e.g., that are available from
Abcam; by in situ fluorescent staining, or the like.
[0202] In another specific embodiment of any of the embodiments of
placental stem cells described herein, the placental stem cells do
not secrete detectable amounts of human chorionic gonadotropin
(hCG). Human chorionic gonadotropin can be detected, e.g., by ELISA
or immunofluorescence using, for example, hCG monoclonal antibody
HCG1 (Abcam), or polyclonal anti-hCG antibodies (Abcam, Novus
Biologicals).
[0203] In another embodiment of any of the isolated placental stem
cells described herein, a population of the isolated placental stem
cells comprises CD56.sup.+ tissue culture plastic-adherent
placental cells that are not natural killer cells. In a specific
embodiment, the population comprises about 1% to about 30% of said
CD56.sup.+ placental cells in said population of isolated placental
stem cells, as determinable by flow cytometry using CD56-FITC
(fluorescein isothiocyanate). In another specific embodiment, the
population comprises about 16% to about 62% of said CD56.sup.+
placental cells in said population of isolated placental stem
cells, as determinable by flow cytometry using CD56-APC
(allophycocyanin).
[0204] In another specific embodiment of any of the above
characteristics, expression of the cellular marker (e.g., cluster
of differentiation or immunogenic marker) is determinable by flow
cytometry; in another specific embodiment, expression of the marker
is determinable by RT-PCR.
[0205] Gene profiling confirms that isolated placental stem cells
are distinguishable from other cells, e.g., mesenchymal stem cells,
e.g., bone marrow-derived mesenchymal stem cells. The isolated
placental stem cells described herein can be distinguished from,
e.g., mesenchymal stem cells on the basis of the expression of one
or more genes, the expression of which is significantly higher in
the isolated placental stem cells, or in certain isolated umbilical
cord stem cells, in comparison to bone marrow-derived mesenchymal
stem cells. In particular, the isolated placental stem cells,
useful in the methods of treatment provided herein, can be
distinguished from mesenchymal stem cells, e.g., bone
marrow-derived mesenchymal stem cells, on the basis of the
expression of one or more genes, the expression of which is
significantly higher (that is, at least twofold higher) in the
isolated placental stem cells than in an equivalent number of bone
marrow-derived mesenchymal stem cells, wherein the one or more
genes are ACTG2, ADARB1, AMIGO2, ARTS-1, B4GALT6, BCHE, C11orf9,
CD200, COL4A1, COL4A2, CPA4, DMD, DSC3, DSG2, ELOVL2, F2RL1,
FLJ10781, GATA6, GPR126, GPRC5B, HLA-G, ICAM1, IER3, IGFBP7, IL1A,
IL6, IL18, KRT18, KRT8, LIPG, LRAP, MATN2, MEST, NFE2L3, NUAK1,
PCDH7, PDLIM3, PKP2, RTN1, SERPINB9, ST3GAL6, ST6GALNAC5, SLC12A8,
TCF21, TGFB2, VTN, ZC3H12A, or a combination of any of the
foregoing, when the cells are grown under equivalent conditions.
See, e.g., U.S. Patent Application Publication No. 2007/0275362,
the disclosure of which is incorporated herein by reference in its
entirety. In certain specific embodiments, said expression of said
one ore more genes is determined, e.g., by RT-PCR or microarray
analysis, e.g., using a U133-A microarray (Affymetrix). In another
specific embodiment, said isolated placental stem cells express
said one or more genes when cultured for a number of population
doublings, e.g., anywhere from about 3 to about 35 population
doublings, in a medium comprising DMEM-LG (e.g., from Gibco); 2%
fetal calf serum (e.g., from Hyclone Labs.); 1.times.
insulin-transferrin-selenium (ITS); 1.times. linoleic acid-bovine
serum albumin (LA-BSA); 10.sup.-9 M dexamethasone (e.g., from
Sigma); 10.sup.-4 M ascorbic acid 2-phosphate (e.g., from Sigma);
epidermal growth factor 10 ng/mL (e.g., from R&D Systems); and
platelet-derived growth factor (PDGF-BB) 10 ng/mL (e.g., from
R&D Systems). In another specific embodiment, the isolated
placental stem cell-specific gene is CD200. It should be understood
that generally, expression of a particular gene, e.g., any of the
genes listed herein, is assessed by analysis of the aggregate
expression of the gene in a population of placental stem cells.
[0206] Specific sequences for these genes can be found in GenBank,
e.g., at accession nos. NM.sub.--001615 (ACTG2), BC065545 (ADARB1),
(NM.sub.--181847 (AMIGO2), AY358590 (ARTS-1), BC074884 (B4GALT6),
BC008396 (BCHE), BC020196 (C11orf9), BC031103 (CD200),
NM.sub.--001845 (COL4A1), NM.sub.--001846 (COL4A2), BC052289
(CPA4), BC094758 (DMD), AF293359 (DSC3), NM.sub.--001943 (DSG2),
AF338241 (ELOVL2), AY336105 (F2RL1), NM.sub.--018215 (FLJ10781),
AY416799 (GATA6), BC075798 (GPR126), NM.sub.--016235 (GPRC5B),
AF340038 (ICAM1), BC000844 (IER3), BC066339 (IGFBP7), BC013142
(IL1A), BT019749 (IL6), BC007461 (IL18), (BC072017) KRT18, BC075839
(KRT8), BC060825 (LIPG), BC065240 (LRAP), BC010444 (MATN2),
BC011908 (MEST), BC068455 (NFE2L3), NM.sub.--014840 (NUAK1),
AB006755 (PCDH7), NM.sub.--014476 (PDLIM3), BC126199 (PKP-2),
BC090862 (RTN1), BC002538 (SERPINB9), BC023312 (ST3GAL6), BC001201
(ST6GALNAC5), BC126160 or BC065328 (SLC12A8), BC025697 (TCF21),
BC096235 (TGFB2), BC005046 (VTN), and BC005001 (ZC3H12A) as of
March 2008.
[0207] In certain specific embodiments, said isolated placental
stem cells express each of ACTG2, ADARB1, AMIGO2, ARTS-1, B4GALT6,
BCHE, C11orf9, CD200, COL4A1, COL4A2, CPA4, DMD, DSC3, DSG2,
ELOVL2, F2RL1, FLJ10781, GATA6, GPR126, GPRC5B, HLA-G, ICAM1, IER3,
IGFBP7, IL1A, IL6, IL18, KRT18, KRT8, LIPG, LRAP, MATN2, MEST,
NFE2L3, NUAK1, PCDH7, PDLIM3, PKP2, RTN1, SERPINB9, ST3GAL6,
ST6GALNAC5, SLC12A8, TCF21, TGFB2, VTN, and ZC3H12A at a higher
level, e.g., a detectably higher level, than an equivalent number
of bone marrow-derived mesenchymal stem cells, when the cells are
grown under equivalent conditions.
[0208] In specific embodiments, the placental stem cells express
CD200 and ARTS1 (aminopeptidase regulator of type 1 tumor necrosis
factor); CD200 and NUAK1, ARTS-1 and LRAP (leukocyte-derived
arginine aminopeptidase); IL6 (interleukin-6) and TGFB2
(transforming growth factor, beta 2); IL6 and KRT18 (keratin 18);
IER3 (immediate early response 3), MEST (mesoderm specific
transcript homolog) and TGFB2; CD200 and IER3; CD200 and IL6; CD200
and KRT18; CD200 and LRAP; CD200 and MEST; CD200 and NFE2L3
(nuclear factor (erythroid-derived 2)-like 3); or CD200 and TGFB2
at a higher level, e.g., a detectably higher level, than an
equivalent number of bone marrow-derived mesenchymal stem cells
(BM-MSCs) wherein said bone marrow-derived mesenchymal stem cells
have undergone a number of passages in culture equivalent to the
number of passages said isolated placental stem cells have
undergone. In other specific embodiments, the placental stem cells
express ARTS-1, CD200, IL6 and LRAP; ARTS-1, IL6, TGFB2, IER3,
KRT18 and MEST; CD200, IER3, IL6, KRT18, LRAP, MEST, NFE2L3, and
TGFB2; ARTS-1, CD200, IER3, IL6, KRT18, LRAP, MEST, NFE2L3, and
TGFB2; or IER3, MEST and TGFB2 at a higher level, e.g., a
detectably higher level, than an equivalent number of bone
marrow-derived mesenchymal stem cells BM-MSCs, wherein said bone
marrow-derived mesenchymal stem cells have undergone a number of
passages in culture equivalent to the number of passages said
isolated placental stem cells have undergone.
[0209] Expression, e.g., differential expression as compared to
bone marrow-derived mesenchymal stem cells, of the above-referenced
genes can be assessed by standard techniques. For example, probes
based on the sequence of the gene(s) can be individually selected
and constructed by conventional techniques. Expression of the genes
can be assessed, e.g., on a microarray comprising probes to one or
more of the genes, e.g., an Affymetrix GENECHIP.RTM. Human Genome
U133A 2.0 array, or an Affymetrix GENECHIP.RTM. Human Genome U133
Plus 2.0 (Santa Clara, Calif.). Expression of these genes can be
assessed even if the sequence for a particular GenBank accession
number is amended because probes specific for the amended sequence
can readily be generated using well-known standard techniques.
[0210] The level of expression of these genes can be used to
confirm the identity of a population of isolated placental stem
cells, to identify a population of cells as comprising at least a
plurality of isolated placental stem cells, or the like.
Populations of isolated placental stem cells, the identity of which
is confirmed, can be clonal, e.g., populations of isolated
placental stem cells expanded from a single isolated placental stem
cell, or a mixed population of stem cells, e.g., a population of
cells comprising isolated placental stem cells that are expanded
from multiple isolated placental stem cells, or a population of
cells comprising isolated placental stem cells, as described
herein, and at least one other type of cell.
[0211] The level of expression of these genes can be used to select
populations of isolated placental stem cells. For example, a
population of placental stem cells, e.g., clonally-expanded
placental stem cells, may be selected if the expression of one or
more of the genes listed above is significantly higher in a sample
from the population of placental stem cells than in an equivalent
population of mesenchymal stem cells, e.g., bone marrow-derived
mesenchymal stem cells.
[0212] Isolated placental stem cells can be selected on the basis
of the level of expression of one or more such genes as compared to
the level of expression in said one or more genes in, e.g., a
mesenchymal stem cell control, for example, the level of expression
in said one or more genes in an equivalent number of bone
marrow-derived mesenchymal stem cells. In one embodiment, the level
of expression of said one or more genes in a sample comprising an
equivalent number of mesenchymal stem cells is used as a control.
In another embodiment, the control, for isolated placental stem
cells tested under certain conditions, is a numeric value
representing the level of expression of said one or more genes in
mesenchymal stem cells under said conditions.
[0213] The isolated placental stem cells described herein, in
certain embodiments, display the above characteristics (e.g.,
combinations of cell surface markers and/or gene expression
profiles) in primary culture, or during proliferation in medium
comprising, e.g., DMEM-LG (Gibco), 2% fetal calf serum (FCS)
(Hyclone Laboratories), lx insulin-transferrin-selenium (ITS),
1.times. lenolenic-acid-bovine-serum-albumin (LA-BSA), 10.sup.-9 M
dexamethasone (Sigma), 10.sup.-4M ascorbic acid 2-phosphate
(Sigma), epidermal growth factor (EGF) 10 ng/ml (R&D Systems),
platelet derived-growth factor (PDGF-BB) 10 ng/ml (R&D
Systems), and 100 U penicillin/1000 U streptomycin.
[0214] In certain embodiments of any of the placental stem cells
disclosed herein, the cells are human. In certain embodiments of
any of the placental stem cells disclosed herein, the cellular
marker characteristics or gene expression characteristics are human
markers or human genes.
[0215] In another specific embodiment of said isolated placental
stem cells or populations of cells comprising the isolated
placental stem cells, said placental stem cells or population have
been expanded, for example, passaged at least, about, or no more
than, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, or 20 times, or proliferated for at least, about, or no
more than, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22,
24, 26, 28, 30, 32, 34, 36, 38 or 40 population doublings. In
another specific embodiment of said isolated placental stem cells
or populations of cells comprising the isolated placental stem
cells, said cells or population are primary isolates. In another
specific embodiment of the isolated placental stem cells, or
populations of cells comprising isolated placental stem cells, that
are disclosed herein, said isolated placental stem cells are fetal
in origin (that is, have the fetal genotype).
[0216] In certain embodiments, said isolated placental stem cells
do not differentiate during culturing in growth medium, i.e.,
medium formulated to promote proliferation, e.g., during
proliferation in growth medium. In another specific embodiment,
said isolated placental stem cells do not require a feeder layer in
order to proliferate. In another specific embodiment, said isolated
placental stem cells do not differentiate in culture in the absence
of a feeder layer, solely because of the lack of a feeder cell
layer.
[0217] In another embodiment, cells useful in the methods and
compositions described herein are isolated placental stem cells,
wherein a plurality of said isolated placental stem cells are
positive for aldehyde dehydrogenase (ALDH), as assessed by an
aldehyde dehydrogenase activity assay. Such assays are known in the
art (see, e.g., Bostian and Betts, Biochem. J., 173, 787, (1978)).
In a specific embodiment, said ALDH assay uses ALDEFLUOR.RTM.
(Aldagen, Inc., Ashland, Oreg.) as a marker of aldehyde
dehydrogenase activity. In a specific embodiment, said plurality is
between about 3% and about 25% of cells in said population of
cells. In another embodiment, provided herein is a population of
isolated umbilical cord cells, e.g., multipotent isolated umbilical
cord cells, wherein a plurality of said isolated umbilical cord
cells are positive for aldehyde dehydrogenase, as assessed by an
aldehyde dehydrogenase activity assay that uses ALDEFLUOR.RTM. as
an indicator of aldehyde dehydrogenase activity. In a specific
embodiment, said plurality is between about 3% and about 25% of
cells in said population of cells. In another embodiment, said
population of isolated placental stem cells or isolated umbilical
cord stem cells shows at least three-fold, or at least five-fold,
higher ALDH activity than a population of bone marrow-derived
mesenchymal stem cells having about the same number of cells and
cultured under the same conditions.
[0218] In a specific embodiment of any of the above embodiments of
the placental stem cells useful in the methods provided herein, the
placental stem cells expresses any one, or any combination of, the
flow cytometric markers and/or gene expression markers described
herein. In certain embodiments, at least 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95% or 98% of placental stem cells in an
isolated population of the isolated placental stem cells described
herein expresses any one, or any combination of, the flow
cytometric markers and/or gene expression markers described
herein.
[0219] In certain embodiments of any of the populations of cells
comprising the isolated placental stem cells described herein, the
placental stem cells in said populations of cells are substantially
free of cells having a maternal genotype; e.g., at least 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the
placental stem cells in said population have a fetal genotype. In
certain other embodiments of any of the populations of cells
comprising the isolated placental stem cells described herein, the
populations of cells comprising said placental stem cells are
substantially free of cells having a maternal genotype; e.g., at
least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
98% or 99% of the cells in said population have a fetal
genotype.
[0220] In a specific embodiment of any of the above isolated
placental stem cells or cell populations of isolated placental stem
cells, the karyotype of the cells, or at least about 95% or about
99% of the cells in said population, is normal. In another specific
embodiment of any of the above placental stem cells, the placental
stem cells, or cells in the population of cells, are non-maternal
in origin.
[0221] Different populations of isolated placental stem cells
bearing any of the above combinations of markers, can be combined
in any ratio. Any two or more populations of the above isolated
placental stem cells can be combined to form an isolated placental
stem cell population. For example, an population of isolated
placental stem cells can comprise a first population of isolated
placental stem cells defined by one of the marker combinations
described above, and a second population of isolated placental stem
cells defined by another of the marker combinations described
above, wherein said first and second populations are combined in a
ratio of about 1:99, 2:98, 3:97, 4:96, 5:95, 10:90, 20:80, 30:70,
40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4, 97:3, 98:2,
or about 99:1. In like fashion, any three, four, five or more
populations of the above-described isolated placental stem cells
can be combined.
[0222] Isolated placental stem cells useful in the methods and
compositions described herein can be obtained, e.g., by disruption
of placental tissue, with or without enzymatic digestion (see
Section 5.3.3) or perfusion (see Section 5.3.4). For example,
populations of isolated placental cells, from which placental stem
cells can be isolated, can be produced according to a method
comprising perfusing a mammalian placenta that has been drained of
cord blood and perfused to remove residual blood; perfusing said
placenta with a perfusion solution; and collecting said perfusion
solution, wherein said perfusion solution after perfusion comprises
a population of placental cells that comprises isolated placental
stem cells; and isolating a plurality of said isolated placental
cells from said population of cells. In a specific embodiment, the
perfusion solution is passed through both the umbilical vein and
umbilical arteries and collected after it exudes from the placenta.
In another specific embodiment, the perfusion solution is passed
through the umbilical vein and collected from the umbilical
arteries, or passed through the umbilical arteries and collected
from the umbilical vein.
[0223] In various embodiments, the isolated placental stem cells,
contained within a population of cells obtained from perfusion of a
placenta, are at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or at
least 99.5% of said population of placental cells. In another
specific embodiment, the isolated placental stem cells collected by
perfusion comprise fetal and maternal cells. In another specific
embodiment, the isolated placental stem cells collected by
perfusion are at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or at
least 99.5% fetal cells.
[0224] In another specific embodiment, provided herein is a
composition comprising a population of the isolated placental stem
cells, as described herein, collected by perfusion, wherein said
composition comprises at least a portion of the perfusion solution
used to collect the isolated placental stem cells.
[0225] The placental stem cells described herein can also be
isolated by digestion of placental tissue with one or more
tissue-disrupting enzymes to obtain a population of placental cells
comprising the placental stem cells, and isolating, or
substantially isolating, the placental stem cells from the
remainder of said placental cells. The whole, or part of, the
placenta can be digested to obtain the isolated placental stem
cells described herein. In other specific embodiment, the
tissue-disrupting enzyme is trypsin or collagenase. In various
embodiments, the isolated placental stem cells, contained within a
population of cells obtained from digesting a placenta, are at
least 50%, 60%, 70%, 80%, 90%, 95%, 99% or at least 99.5% of said
population of placental cells.
[0226] Populations of the isolated placental stem cells described
above can comprise about, at least, or no more than,
1.times.10.sup.5, 5.times.10.sup.5, 1.times.10.sup.6,
5.times.10.sup.6, 1.times.10.sup.7, 5.times.10.sup.7,
1.times.10.sup.8, 5.times.10.sup.8, 1.times.10.sup.9,
5.times.10.sup.9, 1.times.10.sup.10, 5.times.10.sup.10,
1.times.10.sup.11 or more of the isolated placental stem cells.
Populations of isolated placental stem cells useful in the methods
of treatment described herein comprise at least 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% viable isolated placental
stem cells, e.g., as determinable by, e.g., trypan blue
exclusion.
[0227] The placental stem cells described herein, useful in the
methods provided herein, display the above characteristics (e.g.,
combinations of cell surface markers and/or gene expression
profiles) in primary culture, or during proliferation in medium
comprising 60% DMEM-LG (Gibco), 40% MCDB-201 (Sigma), 2% fetal calf
serum (FCS) (Hyclone Laboratories), 1.times.
insulin-transferrin-selenium (ITS), 1.times.
lenolenic-acid-bovine-serum-albumin (LA-BSA), 10.sup.-9 M
dexamethasone (Sigma), 10.sup.-4 M ascorbic acid 2-phosphate
(Sigma), epidermal growth factor (EGF) 10 ng/ml (R&D Systems),
platelet derived-growth factor (PDGF-BB) 10 ng/ml (R&D
Systems), and 100 U penicillin/1000 U streptomycin.
[0228] 5.2.3 Growth in Culture
[0229] The growth of the isolated placental stem cells described
herein in Section 5.2.2 in certain embodiments depends in part upon
the particular medium selected for growth. Under optimum
conditions, the isolated placental stem cells typically double in
number in about 1-3 days. During culture, the isolated placental
stem cells described herein adhere to a substrate in culture, e.g.
the surface of a tissue culture container (e.g., tissue culture
dish plastic, fibronectin-coated plastic, and the like) and form a
monolayer. In specific embodiments, said placental stem cells
double in culture when cultured at 37.degree. C. in 95% air/5%
CO.sub.2 in medium comprising 60% DMEM-LG (Gibco) and 40% MCDB-201
(Sigma) supplemented with 2% fetal calf serum (FCS) (Hyclone
Laboratories), 1.times. insulin-transferrin-selenium (ITS),
1.times. lenolenic-acid-bovine-serum-albumin (LA-BSA), 10.sup.-9 M
dexamethasone (Sigma), 10.sup.-4 M ascorbic acid 2-phosphate
(Sigma), epidermal growth factor (EGF) 10 ng/ml (R&D Systems),
platelet derived-growth factor (PDGF-BB) 10 ng/ml (R&D
Systems), and 100 U penicillin/1000 U streptomycin; or in medium
comprising DMEM-LG (Gibco) supplemented with 2%-10% fetal calf
serum (FCS) (Hyclone Laboratories), 1.times.ITS, 1.times. (LA-BSA),
10.sup.-9 M dexamethasone (Sigma), 10.sup.-4 M ascorbic acid
2-phosphate (Sigma), epidermal growth factor (EGF) 10 ng/ml
(R&D Systems), platelet derived-growth factor (PDGF-BB) 10
ng/ml (R&D Systems), and 100 U penicillin/1000 U
streptomycin.
5.3 Methods of Obtaining Isolated Placental Stem Cells
[0230] 5.3.1 Cell Collection Composition
[0231] Placental stem cells are obtained from a mammalian placenta
using a physiologically-acceptable solution, e.g., a cell
collection composition. An exemplary cell collection composition is
described in detail in related U.S. Patent Application Publication
No. 2007/0190042, the disclosure of which is incorporated herein by
reference in its entirety
[0232] The cell collection composition can comprise any
physiologically-acceptable solution suitable for the collection
and/or culture of cells, e.g., the isolated placental stem cells
described herein, for example, a saline solution (e.g.,
phosphate-buffered saline, Kreb's solution, modified Kreb's
solution, Eagle's solution, 0.9% NaCl, etc.), a culture medium
(e.g., DMEM, H.DMEM, etc.), and the like.
[0233] The cell collection composition can comprise one or more
components that tend to preserve isolated placental stem cells,
that is, prevent the isolated placental stem cells from dying, or
delay the death of the isolated placental stem cells, reduce the
number of isolated placental stem cells in a population of cells
that die, or the like, from the time of collection to the time of
culturing. Such components can be, e.g., an apoptosis inhibitor
(e.g., a caspase inhibitor or JNK inhibitor); a vasodilator (e.g.,
magnesium sulfate, an antihypertensive drug, atrial natriuretic
peptide (ANP), adrenocorticotropin, corticotropin-releasing
hormone, sodium nitroprusside, hydralazine, adenosine triphosphate,
adenosine, indomethacin or magnesium sulfate, a phosphodiesterase
inhibitor, etc.); a necrosis inhibitor (e.g.,
2-(1H-Indol-3-yl)-3-pentylamino-maleimide, pyrrolidine
dithiocarbamate, or clonazepam); a TNF-.alpha. inhibitor; and/or an
oxygen-carrying perfluorocarbon (e.g., perfluorooctyl bromide,
perfluorodecyl bromide, etc.).
[0234] The cell collection composition can comprise one or more
tissue-degrading enzymes, e.g., a metalloprotease, a serine
protease, a neutral protease, an RNase, or a DNase, or the like.
Such enzymes include, but are not limited to, collagenases (e.g.,
collagenase I, II, III or IV, a collagenase from Clostridium
histolyticum, etc.); dispase, thermolysin, elastase, trypsin,
LIBERASE, hyaluronidase, and the like.
[0235] The cell collection composition can comprise a
bacteriocidally or bacteriostatically effective amount of an
antibiotic. In certain non-limiting embodiments, the antibiotic is
a macrolide (e.g., tobramycin), a cephalosporin (e.g., cephalexin,
cephradine, cefuroxime, cefprozil, cefaclor, cefixime or
cefadroxil), a clarithromycin, an erythromycin, a penicillin (e.g.,
penicillin V) or a quinolone (e.g., ofloxacin, ciprofloxacin or
norfloxacin), a tetracycline, a streptomycin, etc. In a particular
embodiment, the antibiotic is active against Gram(+) and/or Gram(-)
bacteria, e.g., Pseudomonas aeruginosa, Staphylococcus aureus, and
the like. In one embodiment, the antibiotic is gentamycin, e.g.,
about 0.005% to about 0.01% (w/v) in culture medium
[0236] The cell collection composition can also comprise one or
more of the following compounds: adenosine (about 1 mM to about 50
mM); D-glucose (about 20 mM to about 100 mM); magnesium ions (about
1 mM to about 50 mM); a macromolecule of molecular weight greater
than 20,000 daltons, in one embodiment, present in an amount
sufficient to maintain endothelial integrity and cellular viability
(e.g., a synthetic or naturally occurring colloid, a polysaccharide
such as dextran or a polyethylene glycol present at about 25 g/l to
about 100 g/l, or about 40 g/l to about 60 g/l); an antioxidant
(e.g., butylated hydroxyanisole, butylated hydroxytoluene,
glutathione, vitamin C or vitamin E present at about 25 .mu.M to
about 100 .mu.M); a reducing agent (e.g., N-acetylcysteine present
at about 0.1 mM to about 5 mM); an agent that prevents calcium
entry into cells (e.g., verapamil present at about 2 .mu.M to about
25 .mu.M); nitroglycerin (e.g., about 0.05 g/L to about 0.2 g/L);
an anticoagulant, in one embodiment, present in an amount
sufficient to help prevent clotting of residual blood (e.g.,
heparin or hirudin present at a concentration of about 1000 units/l
to about 100,000 units/l); or an amiloride containing compound
(e.g., amiloride, ethyl isopropyl amiloride, hexamethylene
amiloride, dimethyl amiloride or isobutyl amiloride present at
about 1.0 .mu.M to about 5 .mu.M).
[0237] 5.3.2 Collection and Handling of Placenta
[0238] Generally, a human placenta is recovered shortly after its
expulsion after birth. In a preferred embodiment, the placenta is
recovered from a patient after informed consent and after a
complete medical history of the patient is taken and is associated
with the placenta. Preferably, the medical history continues after
delivery. Such a medical history can be used to coordinate
subsequent use of the placenta or the isolated placental stem cells
harvested therefrom. For example, isolated human placental stem
cells can be used, in light of the medical history, for
personalized medicine for the infant associated with the placenta,
or for parents, siblings or other relatives of the infant.
[0239] Prior to recovery of isolated placental stem cells, the
umbilical cord blood and placental blood are preferably removed. In
certain embodiments, after delivery, the cord blood in the placenta
is recovered. The placenta can be subjected to a conventional cord
blood recovery process. Typically a needle or cannula is used, with
the aid of gravity, to exsanguinate the placenta (see, e.g.,
Anderson, U.S. Pat. No. 5,372,581; Hessel et al., U.S. Pat. No.
5,415,665). The needle or cannula is usually placed in the
umbilical vein and the placenta can be gently massaged to aid in
draining cord blood from the placenta. Such cord blood recovery may
be performed commercially, e.g., LifeBank USA, Cedar Knolls, N.J.
Preferably, the placenta is gravity drained without further
manipulation so as to minimize tissue disruption during cord blood
recovery.
[0240] Typically, a placenta is transported from the delivery or
birthing room to another location, e.g., a laboratory, for recovery
of cord blood and collection of placental stem cells by, e.g.,
perfusion or tissue dissociation. The placenta is preferably
transported in a sterile, thermally insulated transport device
(maintaining the temperature of the placenta between 20-28.degree.
C.), for example, by placing the placenta, with clamped proximal
umbilical cord, in a sterile zip-lock plastic bag, which is then
placed in an insulated container. In another embodiment, the
placenta is transported in a cord blood collection kit
substantially as described in pending U.S. Pat. No. 7,147,626, the
disclosure of which is incorporated by reference herein.
Preferably, the placenta is delivered to the laboratory four to
twenty-four hours following delivery. In certain embodiments, the
proximal umbilical cord is clamped, preferably within 4-5 cm
(centimeter) of the insertion into the placental disc prior to cord
blood recovery. In other embodiments, the proximal umbilical cord
is clamped after cord blood recovery but prior to further
processing of the placenta.
[0241] The placenta, prior to cell collection, can be stored under
sterile conditions and at either room temperature or at a
temperature of 5.degree. C. to 25.degree. C. The placenta may be
stored for a period of for a period of four to twenty-four hours,
up to forty-eight hours, or longer than forty eight hours, prior to
perfusing the placenta to remove any residual cord blood. In one
embodiment, the placenta is harvested from between about zero hours
to about two hours post-expulsion. The placenta is preferably
stored in an anticoagulant solution at a temperature of 5.degree.
C. to 25.degree. C. Suitable anticoagulant solutions are well known
in the art. For example, a solution of heparin or warfarin sodium
can be used. In a preferred embodiment, the anticoagulant solution
comprises a solution of heparin (e.g., 1% w/w in 1:1000 solution).
The exsanguinated placenta is preferably stored for no more than 36
hours before placental stem cells are collected.
[0242] The mammalian placenta or a part thereof, once collected and
prepared generally as above, can be treated in any art-known
manner, e.g., can be perfused or disrupted, e.g., digested with one
or more tissue-disrupting enzymes, to obtain isolated placental
stem cells.
[0243] 5.3.3 Physical Disruption and Enzymatic Digestion of
Placental Tissue
[0244] In one embodiment, stem cells are collected from a mammalian
placenta by physical disruption of part of all of the organ. For
example, the placenta, or a portion thereof, may be, e.g., crushed,
sheared, minced, diced, chopped, macerated or the like. The tissue
can then be cultured to obtain a population of isolated placental
stem cells. Typically, the placental tissue is disrupted using,
e.g., culture medium, a saline solution, or a stem cell collection
composition (see Section 5.5.1 and below).
[0245] Typically, isolated placental stem cells can be obtained by
disruption of a small block of placental tissue, e.g., a block of
placental tissue that is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,
30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800,
900 or about 1000 cubic millimeters in volume. Any method of
physical disruption can be used, provided that the method of
disruption leaves a plurality, more preferably a majority, and more
preferably at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the
cells in said organ viable, as determinable by, e.g., trypan blue
exclusion.
[0246] The isolated placental stem cells can generally be collected
from a placenta, or portion thereof, at any time within about the
first three days post-expulsion, but preferably between about 8
hours and about 18 hours post-expulsion.
[0247] In a specific embodiment, the disrupted tissue is cultured
in tissue culture medium suitable for the proliferation of isolated
placental stem cells (see, e.g., Section 5.6, below, describing the
culture of placental stem cells, e.g., PDACs).
[0248] In another specific embodiment, placental stem cells are
isolated e.g., in part, by physical disruption of placental tissue,
wherein the physical disruption includes enzymatic digestion, which
can be accomplished by use of one or more tissue-digesting enzymes.
The placenta, or a portion thereof, may also be physically
disrupted and digested with one or more enzymes, and the resulting
material then immersed in, or mixed into, a cell collection
composition.
[0249] A preferred cell collection composition comprises one or
more tissue-disruptive enzyme(s). Enzymes that can be used to
disrupt placenta tissue include papain, deoxyribonucleases, serine
proteases, such as trypsin, chymotrypsin, collagenase, dispase or
elastase. Serine proteases may be inhibited by alpha 2
microglobulin in serum and therefore the medium used for digestion
is usually serum-free. EDTA and DNase are commonly used in enzyme
digestion procedures to increase the efficiency of cell recovery.
The digestate is preferably diluted so as to avoid trapping cells
within the viscous digest.
[0250] Any combination of tissue digestion enzymes can be used.
Typical concentrations for digestion using trypsin include, 0.1% to
about 2% trypsin, e.g., about 0.25% trypsin. Proteases can be used
in combination, that is, two or more proteases in the same
digestion reaction, or can be used sequentially in order to
liberate placental stem cells. For example, in one embodiment, a
placenta, or part thereof, is digested first with an appropriate
amount of collagenase I at about 1 to about 2 mg/ml for, e.g., 30
minutes, followed by digestion with trypsin, at a concentration of
about 0.25%, for, e.g., 10 minutes, at 37.degree. C. Serine
proteases are preferably used consecutively following use of other
enzymes.
[0251] In another embodiment, the tissue can further be disrupted
by the addition of a chelator, e.g., ethylene glycol
bis(2-aminoethyl ether)-N,N,N'N'-tetraacetic acid (EGTA) or
ethylenediaminetetraacetic acid (EDTA) to the stem cell collection
composition comprising the stem cells, or to a solution in which
the tissue is disrupted and/or digested prior to isolation of the
placental stem cells with the stem cell collection composition.
[0252] Following digestion, the digestate is washed, for example,
three times with culture medium, and the washed cells are seeded
into culture flasks. The cells are then isolated by differential
adherence, and characterized for, e.g., viability, cell surface
markers, differentiation, and the like.
[0253] It will be appreciated that where an entire placenta, or
portion of a placenta comprising both fetal and maternal cells (for
example, where the portion of the placenta comprises the chorion or
cotyledons), the placental stem cells isolated can comprise a mix
of placental stem cells derived from both fetal and maternal
sources. Where a portion of the placenta that comprises no, or a
negligible number of, maternal cells (for example, amnion), the
placental stem cells isolated therefrom will comprise almost
exclusively fetal placental stem cells (that is, placental stem
cells having the genotype of the fetus).
[0254] Placental stem cells, e.g., the placental stem cells
described in Section 5.2.2, above, can be isolated from disrupted
placental tissue by differential trypsinization (see Section 5.3.5,
below) followed by culture in one or more new culture containers in
fresh proliferation medium, optionally followed by a second
differential trypsinization step.
[0255] 5.3.4 Placental Perfusion
[0256] Placental stem cells, e.g., the placental stem cells
described in Section 5.2.2, above, can also be isolated, e.g., in
part, by perfusion of the mammalian placenta. Methods of perfusing
mammalian placenta to obtain placental stem cells are disclosed,
e.g., in U.S. Pat. Nos. 7,045,148 and 7,255,729, in U.S. Patent
Application Publication Nos. 2007/0275362 and 2007/0190042, the
disclosures of each of which are incorporated herein by reference
in their entireties.
[0257] Placental stem cells can be collected, e.g., isolated, by
perfusion, e.g., through the placental vasculature, using, e.g., a
cell collection composition as a perfusion solution. In one
embodiment, a mammalian placenta is perfused by passage of
perfusion solution through either or both of the umbilical artery
and umbilical vein. The flow of perfusion solution through the
placenta may be accomplished using, e.g., gravity flow into the
placenta. Preferably, the perfusion solution is forced through the
placenta using a pump, e.g., a peristaltic pump. The umbilical vein
can be, e.g., cannulated with a cannula, e.g., a TEFLON.RTM. or
plastic cannula, that is connected to a sterile connection
apparatus, such as sterile tubing. The sterile connection apparatus
is connected to a perfusion manifold.
[0258] In preparation for perfusion, the placenta is preferably
oriented (e.g., suspended) in such a manner that the umbilical
artery and umbilical vein are located at the highest point of the
placenta. The placenta can be perfused by passage of a perfusion
fluid through the placental vasculature and surrounding tissue. The
placenta can also be perfused by passage of a perfusion fluid into
the umbilical vein and collection from the umbilical arteries, or
passage of a perfusion fluid into the umbilical arteries and
collection from the umbilical vein.
[0259] In one embodiment, for example, the umbilical artery and the
umbilical vein are connected simultaneously, e.g., to a pipette
that is connected via a flexible connector to a reservoir of the
perfusion solution. The perfusion solution is passed into the
umbilical vein and artery. The perfusion solution exudes from
and/or passes through the walls of the blood vessels into the
surrounding tissues of the placenta, and is collected in a suitable
open vessel from the surface of the placenta that was attached to
the uterus of the mother during gestation. The perfusion solution
may also be introduced through the umbilical cord opening and
allowed to flow or percolate out of openings in the wall of the
placenta which interfaced with the maternal uterine wall. Placental
stem cells that are collected by this method, which can be referred
to as a "pan" method, are typically a mixture of fetal and maternal
cells.
[0260] In another embodiment, the perfusion solution is passed
through the umbilical veins and collected from the umbilical
artery, or is passed through the umbilical artery and collected
from the umbilical veins. Placental stem cells collected by this
method, which can be referred to as a "closed circuit" method, are
typically almost exclusively fetal.
[0261] It will be appreciated that perfusion using the pan method,
that is, whereby perfusate is collected after it has exuded from
the maternal side of the placenta, results in a mix of fetal and
maternal cells. As a result, the cells collected by this method can
comprise a mixed population of placental stem cells, of both fetal
and maternal origin. In contrast, perfusion solely through the
placental vasculature in the closed circuit method, whereby
perfusion fluid is passed through one or two placental vessels and
is collected solely through the remaining vessel(s), results in the
collection of a population of placental stem cells almost
exclusively of fetal origin.
[0262] The closed circuit perfusion method can, in one embodiment,
be performed as follows. A post-partum placenta is obtained within
about 48 hours after birth. The umbilical cord is clamped and cut
above the clamp. The umbilical cord can be discarded, or can
processed to recover, e.g., umbilical cord stem cells, and/or to
process the umbilical cord membrane for the production of a
biomaterial. The amniotic membrane can be retained during
perfusion, or can be separated from the chorion, e.g., using blunt
dissection with the fingers. If the amniotic membrane is separated
from the chorion prior to perfusion, it can be, e.g., discarded, or
processed, e.g., to obtain stem cells by enzymatic digestion, or to
produce, e.g., an amniotic membrane biomaterial, e.g., the
biomaterial described in U.S. Application Publication No.
2004/0048796, the disclosure of which is incorporated by reference
herein in its entirety. After cleaning the placenta of all visible
blood clots and residual blood, e.g., using sterile gauze, the
umbilical cord vessels are exposed, e.g., by partially cutting the
umbilical cord membrane to expose a cross-section of the cord. The
vessels are identified, and opened, e.g., by advancing a closed
alligator clamp through the cut end of each vessel. The apparatus,
e.g., plastic tubing connected to a perfusion device or peristaltic
pump, is then inserted into each of the placental arteries. The
pump can be any pump suitable for the purpose, e.g., a peristaltic
pump. Plastic tubing, connected to a sterile collection reservoir,
e.g., a blood bag such as a 250 mL collection bag, is then inserted
into the placental vein. Alternatively, the tubing connected to the
pump is inserted into the placental vein, and tubes to a collection
reservoir(s) are inserted into one or both of the placental
arteries. The placenta is then perfused with a volume of perfusion
solution, e.g., about 750 ml of perfusion solution. Cells in the
perfusate are then collected, e.g., by centrifugation. In certain
embodiments, the placenta is perfused with perfusion solution,
e.g., 100-300 mL perfusion solution, to remove residual blood prior
to perfusion to collect placental stem cells. In another
embodiment, the placenta is not perfused with perfusion solution to
remove residual blood prior to perfusion to collect placental stem
cells.
[0263] In one embodiment, the proximal umbilical cord is clamped
during perfusion, and more preferably, is clamped within 4-5 cm
(centimeter) of the cord's insertion into the placental disc.
[0264] The first collection of perfusion fluid from a mammalian
placenta during the exsanguination process is generally colored
with residual red blood cells of the cord blood and/or placental
blood. The perfusion fluid becomes more colorless as perfusion
proceeds and the residual cord blood cells are washed out of the
placenta. Generally from 30 to 100 ml (milliliter) of perfusion
fluid is adequate to initially exsanguinate the placenta, but more
or less perfusion fluid may be used depending on the observed
results.
[0265] The volume of perfusion liquid used to isolate placental
stem cells may vary depending upon the number of cells to be
collected, the size of the placenta, the number of collections to
be made from a single placenta, etc. In various embodiments, the
volume of perfusion liquid may be from 50 mL to 5000 mL, 50 mL to
4000 mL, 50 mL to 3000 mL, 100 mL to 2000 mL, 250 mL to 2000 mL,
500 mL to 2000 mL, or 750 mL to 2000 mL. Typically, the placenta is
perfused with 700-800 mL of perfusion liquid following
exsanguination.
[0266] The placenta can be perfused a plurality of times over the
course of several hours or several days. Where the placenta is to
be perfused a plurality of times, it may be maintained or cultured
under aseptic conditions in a container or other suitable vessel,
and perfused with the cell collection composition, or a standard
perfusion solution (e.g., a normal saline solution such as
phosphate buffered saline ("PBS")) with or without an anticoagulant
(e.g., heparin, warfarin sodium, coumarin, bishydroxycoumarin),
and/or with or without an antimicrobial agent (e.g.,
3-mercaptoethanol (0.1 mM); antibiotics such as streptomycin (e.g.,
at 40-100 g/ml), penicillin (e.g., at 40 U/ml), amphotericin B
(e.g., at 0.5 .mu.g/ml). In one embodiment, an isolated placenta is
maintained or cultured for a period of time without collecting the
perfusate, such that the placenta is maintained or cultured for 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, or 24 hours, or 2 or 3 or more days before perfusion
and collection of perfusate. The perfused placenta can be
maintained for one or more additional time(s), e.g., 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24 or more hours, and perfused a second time with, e.g., 700-800 mL
perfusion fluid. The placenta can be perfused 1, 2, 3, 4, 5 or more
times, for example, once every 1, 2, 3, 4, 5 or 6 hours. In a
preferred embodiment, perfusion of the placenta and collection of
perfusion solution, e.g., cell collection composition, is repeated
until the number of recovered nucleated cells falls below 100
cells/ml. The perfusates at different time points can be further
processed individually to recover time-dependent populations of
cells, e.g., stem cells. Perfusates from different time points can
also be pooled. In a preferred embodiment, placental stem cells are
collected at a time or times between about 8 hours and about 18
hours post-expulsion.
[0267] Placental stem cells can be isolated from placenta by
perfusion with a solution comprising one or more proteases or other
tissue-disruptive enzymes. In a specific embodiment, a placenta or
portion thereof (e.g., amniotic membrane, amnion and chorion,
placental lobule or cotyledon, umbilical cord, or combination of
any of the foregoing) is brought to 25-37.degree. C., and is
incubated with one or more tissue-disruptive enzymes in 200 mL of a
culture medium for 30 minutes. Cells from the perfusate are
collected, brought to 4.degree. C., and washed with a cold
inhibitor mix comprising 5 mM EDTA, 2 mM dithiothreitol and 2 mM
beta-mercaptoethanol. The placental stem cells are washed after
several minutes with a cold (e.g., 4.degree. C.) stem cell
collection composition.
[0268] In certain embodiments, perfusion (whether by pan method or
closed-circuit method) is carried out open under a sterile hood in,
e.g., a pan. In certain other embodiments, perfusion is carried out
within a closed environment, e.g., a sterile bag containing the
placenta. In certain other embodiments, the placenta is folded,
e.g., folded in half, or substantially in half, once or a plurality
of times during perfusion. In specific embodiments, the folding is
accomplished by hand, or mechanically.
[0269] Perfusion, carried out as described above, results in the
collection of placental perfusate, a solution comprising a
heterogeneous population of different placental cells, which
population comprises the tissue culture plastic adhesive placental
stem cells described above in Section 5.2.2, as well as
hematopoietic placental stem cells, e.g., CD34 placental stem
cells, which are not tissue culture plastic adherent.
[0270] 5.3.5 Isolation, Sorting, and Characterization of Placental
Stem Cells
[0271] The isolated placental stem cells, e.g., the tissue culture
plastic-adherent cells described in Section 5.2.2, above, whether
obtained by perfusion or physical disruption, e.g., by enzymatic
digestion, can initially be purified from (i.e., be isolated from)
other cells by Ficoll gradient centrifugation. Such centrifugation
can follow any standard protocol for centrifugation speed, etc. In
one embodiment, for example, cells collected from the placenta are
recovered from perfusate by centrifugation at 5000.times.g for 15
minutes at room temperature, which separates cells from, e.g.,
contaminating debris and platelets. In certain embodiments, the
Ficoll is used at a density of from about 1.070 g/ml to about 1.090
g/mL, e.g., about 1.073 g/mL, 1.077 g/mL, or about 1.084 g/mL; the
placental stem cells will collect on top of the gradient at these
densities. In another embodiment, placental perfusate is
concentrated to about 200 ml, gently layered over Ficoll, and
centrifuged at about 1100.times.g for 20 minutes at 22.degree. C.,
and the low-density interface layer of cells is collected for
further processing.
[0272] Cell pellets can be resuspended in fresh stem cell
collection composition, or a medium suitable for cell maintenance,
e.g., stem cell maintenance, for example, IMDM serum-free medium
containing 2 U/ml heparin and 2 mM EDTA (GibcoBRL, NY). The total
mononuclear cell fraction can be isolated, e.g., using Lymphoprep
(Nycomed Pharma, Oslo, Norway) according to the manufacturer's
recommended procedure.
[0273] Placental stem cells obtained by perfusion or digestion can,
for example, be further, or initially, isolated by differential
trypsinization using, e.g., a solution of 0.05% trypsin with 0.2%
EDTA (Sigma, St. Louis Mo.). Differential trypsinization is
possible because the isolated placental stem cells, which are
tissue culture plastic-adherent, typically detach from the plastic
surfaces within about five minutes whereas other adherent
populations typically require more than 20-30 minutes incubation.
The detached placental stem cells can be harvested following
trypsinization and trypsin neutralization, using, e.g., Trypsin
Neutralizing Solution (TNS, Cambrex). In one embodiment of
isolation of adherent cells, aliquots of, for example, about
5-10.times.10.sup.6 cells are placed in each of several T-75
flasks, preferably fibronectin-coated T75 flasks. In such an
embodiment, the cells can be cultured with commercially available
Mesenchymal Stem Cell Growth Medium (MSCGM) (Cambrex), and placed
in a tissue culture incubator (37.degree. C., 5% CO.sub.2). After
10 to 15 days, non-adherent cells are removed from the flasks by
washing with PBS. The PBS is then replaced by MSCGM.
[0274] The number and type of cells collected from a mammalian
placenta can be monitored, for example, by measuring changes in
morphology and cell surface markers using standard cell detection
techniques such as flow cytometry, cell sorting,
immunocytochemistry (e.g., staining with tissue specific or
cell-marker specific antibodies) fluorescence activated cell
sorting (FACS), magnetic activated cell sorting (MACS), by
examination of the morphology of cells using light or confocal
microscopy, and/or by measuring changes in gene expression using
techniques well known in the art, such as PCR and gene expression
profiling. These techniques can be used, too, to identify cells
that are positive for one or more particular markers. For example,
using antibodies to CD73, one can determine, using the techniques
above, whether a cell comprises a detectable amount of CD73; if so,
the cell is CD73.sup.+. Likewise, if a cell produces enough OCT-4
RNA to be detectable by RT-PCR, or significantly more OCT-4 RNA
than an adult (terminally-differentiated) cell (e.g., a dermal
fibroblast), the cell is OCT-4.sup.+. In a specific embodiment, the
cell is positive for a particular mRNA if the mRNA is amplified
above background by RT-PCR, using an appropriate primer pair, in 35
cycles or less. Antibodies to cell surface markers (e.g., CD
markers such as CD34) and the sequence of stem cell-specific genes,
such as OCT-4, are well-known in the art.
[0275] Placental stem cells, particularly cells that have been
isolated by Ficoll separation, differential adherence, or a
combination of both, may be sorted, e.g., 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). 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.
[0276] In one sorting scheme, placental stem cells, e.g., PDACs,
are sorted on the basis of expression of one or more of the markers
CD34, CD44, CD45, CD73, CD90, CD105, CD133, CD166, CD200 and/or
KDR; that is, the placental stem cells are sorted on the basis of
expression of one or more of CD44, CD73, CD90, CD105, CD166 or
CD200, and/or lack of expression of CD34, CD45, CD133, or KDR. This
can be accomplished in connection with procedures to select such
cells on the basis of their adherence properties in culture. For
example, tissue culture plastic adherence selection can be
accomplished before or after sorting on the basis of marker
expression. In one embodiment, for example, cells are sorted first
on the basis of their expression of CD34; CD34.sup.- cells are
retained, and CD34.sup.- cells that are additionally one or more of
CD73.sup.+, CD90.sup.+ or CD200.sup.+ are separated from all other
CD34.sup.- cells. In another embodiment, cells from placenta are
sorted based on their expression of CD200; for example, cells
displaying CD200 are isolated for further use. Cells that express,
e.g., CD200 can, in a specific embodiment, be further sorted based
on their expression of CD73 and/or CD105, or epitopes recognized by
antibodies SH2, SH3 or SH4, or lack of expression of CD34, CD38 or
CD45. For example, in another embodiment, placental stem cells are
sorted by expression, or lack thereof, of CD200, CD73, CD105, CD34,
CD38 and CD45, and placental stem cells that are CD200.sup.+,
CD73.sup.+, CD105.sup.+, CD34.sup.-, CD38.sup.- and CD45.sup.- are
isolated from other placental cells for further use.
[0277] Sorting of cells can be used to confirm the identity of a
population of placental stem cells. For example, the tissue culture
plastic-adherent placental stem cells, e.g., PDACs, described
herein can be cultured for one or more passages, then collected and
a sample characterized using antibodies to CD34, CD44, CD45, CD73,
CD90, CD105, and/or CD200, to determine if, e.g., 70%, 75%, 80%,
85%, 90%, 95% or 98% of the cultured cells are CD34.sup.-,
CD44.sup.+, CD45.sup.-, CD73.sup.+, CD90.sup.+, CD105.sup.+, and/or
CD200.sup.+.
[0278] In specific embodiments of any of the above embodiments of
sorted placental stem cells, at least 50%, 60%, 70%, 80%, 90% or
95% of the cells in a cell population remaining after sorting are
said isolated placental stem cells. Placental stem cells can be
sorted by one or more of any of the markers described in Section
5.2.2, above. In a specific embodiment, for example, placental
cells that are (1) adherent to tissue culture plastic, and (2)
CD10.sup.+, CD34.sup.- and CD105.sup.+ are sorted from (i.e.,
isolated from) other placental cells. In another specific
embodiment, placental cells that are (1) adherent to tissue culture
plastic, and (2) CD10.sup.+, CD34.sup.-, CD105.sup.+ and
CD200.sup.+ are sorted from (i.e., isolated from) other placental
cells. In another specific embodiment, placental cells that are (1)
adherent to tissue culture plastic, and (2) CD10.sup.+, CD34.sup.-,
CD45.sup.-, CD90.sup.+, CD105.sup.+ and CD200.sup.+ are sorted from
(i.e., isolated from) other placental cells. Placental stem cells
need not be sorted according to a particular cellular marker, or
set of cellular markers, to be "isolated," however.
[0279] With respect to nucleotide sequence-based detection and/or
analysis of placental stem cells, sequences for the markers listed
herein are readily available in publicly-available databases such
as GenBank or EMBL.
[0280] With respect to antibody-mediated detection and sorting of
placental stem cells, e.g., placental stem cells or placental
multipotent cells, any antibody, specific for a particular marker,
can be used, in combination with any fluorophore or other label
suitable for the detection and sorting of cells (e.g.,
fluorescence-activated cell sorting). Antibody/fluorophore
combinations to specific markers include, but are not limited to,
fluorescein isothiocyanate (FITC) conjugated monoclonal antibodies
against HLA-G (available from Serotec, Raleigh, N.C.), CD10
(available from BD Immunocytometry Systems, San Jose, Calif.), CD44
(available from BD Biosciences Pharmingen, San Jose, Calif.), and
CD105 (available from R&D Systems Inc., Minneapolis, Minn.);
phycoerythrin (PE) conjugated monoclonal antibodies against CD44,
CD200, CD117, and CD13 (BD Biosciences Pharmingen);
phycoerythrin-Cy7 (PE Cy7) conjugated monoclonal antibodies against
CD33 and CD10 (BD Biosciences Pharmingen); allophycocyanin (APC)
conjugated streptavidin and monoclonal antibodies against CD38 (BD
Biosciences Pharmingen); and Biotinylated CD90 (BD Biosciences
Pharmingen). Other antibodies that can be used include, but are not
limited to, CD133-APC (Miltenyi), KDR-Biotin (CD309, Abcam),
Cytokeratin-Fitc (Sigma or Dako), HLA ABC-Fitc (BD), HLA
DR,DQ,DP-PE (BD), .beta.-2-microglobulin-PE (BD), CD80-PE (BD) and
CD86-APC (BD). Other antibody/label combinations that can be used
include, but are not limited to, CD45-PerCP (peridin chlorophyll
protein); CD44-PE; CD10-F (fluorescein); HLA-G-F and
7-amino-actinomycin-D (7-AAD); HLA-ABC-F; and the like. This list
is not exhaustive, and other antibodies from other suppliers are
also commercially available.
[0281] Isolated placental stem cells can be assayed for CD117 or
CD133 using, for example, phycoerythrin-Cy5 (PE Cy5) conjugated
streptavidin and biotin conjugated monoclonal antibodies against
CD117 or CD133; however, using this system, the cells can appear to
be positive for CD117 or CD133, respectively, because of a
relatively high background.
[0282] The isolated placental stem cells can be labeled with an
antibody to a single marker and detected and/sorted. Placental stem
cells can also be simultaneously labeled with multiple antibodies
to different markers.
[0283] In another embodiment, magnetic beads can be used to
separate cells, e.g., separate placental stem cells from other
placental cells. The cells may be sorted using a magnetic activated
cell sorting (MACS) technique, a method for separating particles
based on their ability to bind magnetic beads (0.5-100 .mu.m
diameter). A variety of useful modifications can be performed on
the magnetic microspheres, including covalent addition of antibody
that specifically recognizes a particular cell surface molecule or
hapten. The beads are then mixed with the cells to allow binding.
Cells are then passed through a magnetic field to separate out
cells having the specific cell surface marker. In one embodiment,
these cells can then isolated and re-mixed with magnetic beads
coupled to an antibody against additional cell surface markers. The
cells are again passed through a magnetic field, isolating cells
that bound both the antibodies. Such cells can then be diluted into
separate dishes, such as microtiter dishes for clonal
isolation.
[0284] Isolated placental stem cells can also be characterized
and/or sorted based on cell morphology and growth characteristics.
For example, isolated placental stem cells can be characterized as
having, and/or selected on the basis of, e.g., a fibroblastoid
appearance in culture. The isolated placental stem cells can also
be characterized as having, and/or be selected, on the basis of
their ability to form embryoid-like bodies. In one embodiment, for
example, placental stem cells that are fibroblastoid in shape,
express CD73 and CD105, and produce one or more embryoid-like
bodies in culture are isolated from other placental cells. In
another embodiment, OCT-4.sup.+ placental stem cells that produce
one or more embryoid-like bodies in culture are isolated from other
placental cells.
[0285] The isolated placental stem cells can be assessed for
viability, proliferation potential, and longevity using standard
techniques known in the art, such as trypan blue exclusion assay,
fluorescein diacetate uptake assay, propidium iodide uptake assay
(to assess viability); and thymidine uptake assay, MTT
(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) 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.
[0286] Isolated placental stem cells, e.g., the isolated placental
stem cells described in Section 5.2.2, above, can also be separated
from other placental cells using other techniques known in the art,
e.g., selective growth of desired cells (positive selection),
selective destruction of unwanted cells (negative selection);
separation based upon differential cell agglutinability in the
mixed population as, for example, with soybean agglutinin;
freeze-thaw procedures; filtration; conventional and zonal
centrifugation; centrifugal elutriation (counter-streaming
centrifugation); unit gravity separation; countercurrent
distribution; electrophoresis; and the like.
5.4 Culture of Isolated Placental Stem Cells
[0287] 5.4.1 Culture Media
[0288] Isolated placental cells, or placental tissue from which
placental stem cells grow out, can be used to initiate, or seed,
cultures of placental stem cells. Cells are generally transferred
to sterile tissue culture vessels either uncoated or coated with
extracellular matrix or ligands such as laminin, collagen (e.g.,
native or denatured), gelatin, fibronectin, ornithine, vitronectin,
and extracellular membrane protein (e.g., MATRIGEL.RTM. (BD
Discovery Labware, Bedford, Mass.)). Similar procedures may be used
for BM-MSC culture.
[0289] Isolated placental cells, e.g., isolated placental stem
cells, can be cultured in any medium, and under any conditions,
recognized in the art as acceptable for the culture of cells, e.g.,
stem cells. Preferably, the culture medium comprises serum, e.g.,
bovine calf serum, human serum, or the like. The isolated placental
stem cells can be cultured in, for example, DMEM-LG (Dulbecco's
Modified Essential Medium, low glucose)/MCDB 201 (chick fibroblast
basal medium) containing ITS (insulin-transferrin-selenium), LA+BSA
(linoleic acid-bovine serum albumin), dexamethasone L-ascorbic
acid, PDGF, EGF, IGF-1, and penicillin/streptomycin; DMEM-HG (high
glucose) comprising 10% fetal bovine serum (FBS); DMEM-HG
comprising 15% FBS; IMDM (Iscove's modified Dulbecco's medium)
comprising 10% FBS, 10% horse serum, and hydrocortisone; M199
comprising 1% to 20% FBS, EGF, and heparin; .alpha.-MEM (minimal
essential medium) comprising 10% FBS, GLUTAMAX.TM. and gentamicin;
DMEM comprising 10% FBS, GLUTAMAX.TM. and gentamicin, etc.
[0290] Other media in that can be used to culture placental stem
cells include DMEM (high or low glucose), Eagle's basal medium,
Ham's F10 medium (F10), Ham's F-12 medium (F12), Iscove's modified
Dulbecco's medium, Mesenchymal Stem Cell Growth Medium (MSCGM),
Liebovitz's L-15 medium, MCDB, DMEM/F12, RPMI 1640, advanced DMEM
(Gibco), DMEM/MCDB201 (Sigma), and CELL-GRO FREE.
[0291] The culture medium can be supplemented with one or more
components including, for example, serum (e.g., fetal bovine serum
(FBS), preferably about 2-15% (v/v); equine (horse) serum (ES);
human serum (HS)); beta-mercaptoethanol (BME), preferably about
0.001% (v/v); one or more growth factors, for example,
platelet-derived growth factor (PDGF), epidermal growth factor
(EGF), basic fibroblast growth factor (bFGF), insulin-like growth
factor-1 (IGF-1), leukemia inhibitory factor (LIF), vascular
endothelial growth factor (VEGF), and erythropoietin (EPO); amino
acids, including L-valine; and one or more antibiotic and/or
antimycotic agents to control microbial contamination, such as, for
example, penicillin G, streptomycin sulfate, amphotericin B,
gentamicin, and nystatin, either alone or in combination.
[0292] In certain embodiments, the placental stem cells suitable
for use in the methods described herein can be cultured in medium
comprising DMEM medium supplemented with fetal bovine serum (FBS,
e.g., 1.9% FBS v/v), linoleic acid-albumin (e.g., 0.01% v/v)
(Sigma), insulin-transferrin-selenium (0.97% v/v) (Invitrogen,
Carlsbad, Calif.), gentamicin (e.g., 48 .mu.g/mL) (Invitrogen),
L-ascorbic acid 2-phosphate sesquimagnesium salt (e.g., 97 .mu.M)
(Sigma), dexamethasone 48 nM (Sigma), recombinant human PDGF-BB
(e.g., 9.7 ng/ml) (Invitrogen), and recombinant human EGF (e.g.,
9.7 ng/ml) (Invitrogen).
[0293] The isolated placental stem cells can be cultured in
standard tissue culture conditions, e.g., in tissue culture dishes
or multiwell plates. The isolated placental stem cells can also be
cultured using a hanging drop method. In this method, isolated
placental stem cells are suspended at about 1.times.10.sup.4 cells
per mL in about 5 mL of medium, and one or more drops of the medium
are placed on the inside of the lid of a tissue culture container,
e.g., a 100 mL Petri dish. The drops can be, e.g., single drops, or
multiple drops from, e.g., a multichannel pipetter. The lid is
carefully inverted and placed on top of the bottom of the dish,
which contains a volume of liquid, e.g., sterile PBS sufficient to
maintain the moisture content in the dish atmosphere, and the
placental stem cells are cultured.
[0294] In one embodiment, isolated placental stem cells are
cultured in the presence of a compound that acts to maintain an
undifferentiated phenotype in the isolated placental stem cells. In
this context, "undifferentiated" does not require complete
non-differentiation, e.g., encompasses a relatively
undifferentiated phenotype as compared to terminally differentiated
cells, or placental stem cells caused to differentiation such as to
express one or more characteristics of a terminally differentiated
cell. In a specific embodiment, the compound is a substituted
3,4-dihydropyridimol[4,5-d]pyrimidine. In another specific
embodiment, the compound is a compound having the following
chemical structure:
##STR00001##
The compound can be contacted with isolated placental stem cells at
a concentration of, for example, between about 1 .mu.M to about 10
.mu.M.
[0295] 5.4.2 Expansion and Proliferation of Placental Stem
Cells
[0296] Once placental stem cells have been isolated (e.g.,
separated from at least 50% of the placental cells with which the
placental stem cells are normally associated in vivo), the cell or
population of cells can be proliferated and expanded in vitro. For
example, isolated placental stem cells can be cultured in tissue
culture containers, e.g., dishes, flasks, multiwell plates, or the
like, for a sufficient time for the cells to proliferate to 70-90%
confluence, that is, until the cells and their progeny occupy
70-90% of the culturing surface area of the tissue culture
container.
[0297] The isolated placental stem cells can be seeded in culture
vessels at a density that allows cell growth. For example, the
cells may be seeded at low density (e.g., about 1,000 to about
5,000 cells/cm.sup.2) to high density (e.g., about 50,000 or more
cells/cm.sup.2). In a preferred embodiment, the cells are cultured
in the presence of about 0 to about 5 percent by volume CO.sub.2 in
air. In some preferred embodiments, the cells are cultured at about
2 to about 25 percent O.sub.2 in air, preferably about 5 to about
20 percent O.sub.2 in air. The cells preferably are cultured at
about 25.degree. C. to about 40.degree. C., preferably 37.degree.
C. The cells are preferably cultured in an incubator. The culture
medium can be static or agitated, for example, using a bioreactor.
Placental stem cells, in certain embodiments, are grown under low
oxidative stress (e.g., with addition of glutathione, ascorbic
acid, catalase, tocopherol, N-acetylcysteine, or the like).
[0298] Once confluence of less than 100%, for example, 70% to 90%
is obtained, the cells may be passaged. For example, the cells can
be enzymatically treated, e.g., trypsinized, using techniques
well-known in the art, to separate them from the tissue culture
surface. After removing the cells by pipetting and counting the
cells, about 10,000-100,000 cells/cm.sup.2 are passaged to a new
culture container containing fresh culture medium. Typically, the
new medium is the same type of medium from which the isolated
placental stem cells were removed. The isolated placental stem
cells can be passaged about, at least, or no more than 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 times, or more.
[0299] 5.4.3 Populations of Isolated Placental Stem Cells
[0300] Also provided herein are populations of isolated placental
stem cells, e.g., the isolated placental stem cells described in
Section 5.2.2, above, useful in the methods and compositions
described herein. Populations of isolated placental stem cells can
be isolated directly from one or more placentas; that is, the cell
population can be a population of placental cells comprising the
isolated placental cells, wherein the isolated placental stem cells
are obtained from, or contained within, perfusate, or obtained
from, or contained within, disrupted placental tissue, e.g.,
placental tissue digestate (that is, the collection of cells
obtained by enzymatic digestion of a placenta or part thereof). The
isolated placental stem cells described herein can also be cultured
and expanded to produce populations of the isolated placental stem
cells. Populations of placental cells comprising the isolated
placental stem cells can also be cultured and expanded to produce
placental stem cell populations.
[0301] Placental stem cell populations useful in the methods of
treatment provided herein comprise the isolated placental stem
cells, for example, the isolated placental stem cells as described
in Section 5.2.2 herein. In various embodiments, at least 10%, 20%,
30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the cells in a
placental cell population are the isolated placental stem cells.
That is, a population of the isolated placental cells can comprise,
e.g., as much as 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90% cells that are not the isolated placental stem cells.
[0302] Isolated placental cell populations, comprising the isolated
placental stem cells, useful in the methods and compositions
described herein can be produced by, e.g., selecting isolated
placental cells, whether derived from enzymatic digestion or
perfusion, that express particular markers and/or particular
culture or morphological characteristics. In various embodiments,
for example, provided herein is a method of producing a cell
population by selecting placental cells that comprise placental
stem cells that (a) adhere to a substrate, and (b) express any one,
or any combination of, the flow cytometric markers and/or gene
expression characteristics described herein, e.g., in Section 5.2,
above.
[0303] In another aspect, populations of placental stem cells,
e.g., the placental stem cells described in Section 5.2, above, can
be produced by selecting for both marker expression characteristics
and the ability of the population of placental stem cells, e.g., a
sample of the population of placental stem cells, to suppress,
e.g., detectably suppress, the proliferation of cells of a
bone-related cancer. In various embodiments, the cells of a
bone-related cancer are multiple myeloma cells, chondrosarcoma
cells, bone cancer cells, neuroblastoma cells, osteosarcoma cells,
Ewing's sarcoma cells, chordoma cells, malignant fibrous
histiocytoma of bone cells, prostate cancer cells, or fibrosarcoma
of bone cells. Such a selection can be applied, for example, to
different populations of placental stem cells, e.g., batches or
lots of placental stem cells in order to identify populations that
satisfy, for example, certain predetermined criteria for
effectiveness.
[0304] Selection of cell populations comprising placental stem
cells having any of the marker combinations described in Section
5.2.2, above, can be isolated or obtained in similar fashion.
[0305] In any of the above embodiments, selection of the isolated
cell populations can additionally comprise selecting placental stem
cells that express ABC-p (a placenta-specific ABC transporter
protein; see, e.g., Allikmets et al., Cancer Res. 58(23):5337-9
(1998)). The method can also comprise selecting cells exhibiting at
least one characteristic specific to, e.g., a mesenchymal stem
cell, for example, expression of CD44, expression of CD90, or
expression of a combination of the foregoing.
[0306] In the above embodiments, the substrate can be any surface
on which culture and/or selection of cells, e.g., isolated
placental stem cells, can be accomplished. Typically, the substrate
is plastic, e.g., tissue culture dish or multiwell plate plastic.
Tissue culture plastic can be coated with a biomolecule, e.g.,
laminin or fibronectin.
[0307] Isolated placental stem cells can be selected by any means
known in the art of cell selection. For example, cells can be
selected using an antibody or antibodies to one or more cell
surface markers, for example, in flow cytometry or FACS. Selection
can be accomplished using antibodies in conjunction with magnetic
beads. Antibodies that are specific for certain stem cell-related
markers are known in the art. For example, antibodies to OCT-4
(Abcam, Cambridge, Mass.), CD200 (Abcam), HLA-G (Abcam), CD73 (BD
Biosciences Pharmingen, San Diego, Calif.), CD105 (Abcam; BioDesign
International, Saco, Me.), etc. Antibodies to other markers are
also available commercially, e.g., CD34, CD38 and CD45 are
available from, e.g., StemCell Technologies or BioDesign
International.
[0308] Isolated placental stem cell populations can comprise
placental cells that are not stem cells, or cells that are not
placental cells.
[0309] The isolated placental stem cell populations provided herein
can be combined with one or more populations of non-stem cells or
non-placental cells. For example, a population of isolated
placental stem cells can be combined with blood (e.g., placental
blood or umbilical cord blood), blood-derived stem cells (e.g.,
stem cells derived from placental blood or umbilical cord blood),
umbilical cord stem cells, populations of blood-derived nucleated
cells, bone marrow-derived mesenchymal cells, bone-derived stem
cell populations, crude bone marrow, adult (somatic) stem cells,
populations of stem cells contained within tissue, cultured stem
cells, populations of fully-differentiated cells (e.g.,
chondrocytes, fibroblasts, amniotic cells, osteoblasts, muscle
cells, cardiac cells, etc.) and the like. In a specific embodiment,
a population of cells useful in the methods and compositions
described herein comprises isolated placental stem cells and
isolated umbilical cord stem cells. Cells in an isolated placental
stem cell population can be combined with a plurality of cells of
another type in ratios of about 100,000,000:1, 50,000,000:1,
20,000,000:1, 10,000,000:1, 5,000,000:1, 2,000,000:1, 1,000,000:1,
500,000:1, 200,000:1, 100,000:1, 50,000:1, 20,000:1, 10,000:1,
5,000:1, 2,000:1, 1,000:1, 500:1, 200:1, 100:1, 50:1, 20:1, 10:1,
5:1, 2:1, 1:1; 1:2; 1:5; 1:10; 1:100; 1:200; 1:500; 1:1,000;
1:2,000; 1:5,000; 1:10,000; 1:20,000; 1:50,000; 1:100,000;
1:500,000; 1:1,000,000; 1:2,000,000; 1:5,000,000; 1:10,000,000;
1:20,000,000; 1:50,000,000; or about 1:100,000,000, comparing
numbers of total nucleated cells in each population. Cells in an
isolated placental stem cell population can be combined with a
plurality of cells of a plurality of cell types, as well.
[0310] In one embodiment, an isolated population of placental stem
cells is combined with a plurality of hematopoietic stem cells.
Such hematopoietic stem cells can be, for example, contained within
unprocessed placental, umbilical cord blood or peripheral blood; in
total nucleated cells from placental blood, umbilical cord blood or
peripheral blood; in an isolated population of CD34.sup.+ cells
from placental blood, umbilical cord blood or peripheral blood; in
unprocessed bone marrow; in total nucleated cells from bone marrow;
in an isolated population of CD34.sup.+ cells from bone marrow, or
the like.
[0311] In other embodiments, a population of the placental stem
cells described herein, e.g., the PDACs described in Section 5.2.2,
above, are combined with osteogenic placental adherent cells
(OPACs), e.g., the OPACs described in U.S. Patent Application No.
2010/0047214, the disclosure of which is hereby incorporated by
reference in its entirety. In other embodiments, a population of
the placental stem cells described herein, e.g., the PDACs
described in Section 5.2.2, above, are combined with placental
perfusate and/or natural killer cells, e.g., natural killer cells
from placental perfusate, e.g., placental intermediate natural
killer cells, e.g., as described in U.S. Patent application
Publication No. 2009/0252710, the disclosure of which is hereby
incorporated by reference in its entirety.
5.5 Preservation of Placental Stem Cells
[0312] Isolated placental stem cells, e.g., the isolated placental
stem cells described above, can be preserved, that is, placed under
conditions that allow for long-term storage, or conditions that
inhibit cell death by, e.g., apoptosis or necrosis.
[0313] Placental stem cells can be preserved using, e.g., a
composition comprising an apoptosis inhibitor, necrosis inhibitor
and/or an oxygen-carrying perfluorocarbon, as described in related
U.S. Application Publication No. 2007/0190042, the disclosure of
which is incorporated herein by reference in its entirety. In one
embodiment, a method of preserving a population of cells, e.g.,
placental stem cells, comprises contacting said population of cells
with a cell collection composition comprising an inhibitor of
apoptosis and an oxygen-carrying perfluorocarbon, wherein said
inhibitor of apoptosis is present in an amount and for a time
sufficient to reduce or prevent apoptosis in the population of
cells, as compared to a population of cells not contacted with the
inhibitor of apoptosis. In a specific embodiment, said inhibitor of
apoptosis is a caspase inhibitor. In another specific embodiment,
said inhibitor of apoptosis is a JNK inhibitor. In another specific
embodiment, said JNK inhibitor does not modulate differentiation or
proliferation of said cells. In another embodiment, said cell
collection composition comprises said inhibitor of apoptosis and
said oxygen-carrying perfluorocarbon in separate phases. In another
embodiment, said cell collection composition comprises said
inhibitor of apoptosis and said oxygen-carrying perfluorocarbon in
an emulsion. In another embodiment, the cell collection composition
additionally comprises an emulsifier, e.g., lecithin. In another
embodiment, said apoptosis inhibitor and said perfluorocarbon are
between about 0.degree. C. and about 25.degree. C. at the time of
contacting the cells. In another specific embodiment, said
apoptosis inhibitor and said perfluorocarbon are between about
2.degree. C. and 10.degree. C., or between about 2.degree. C. and
about 5.degree. C., at the time of contacting the cells. In another
specific embodiment, said contacting is performed during transport
of said population of cells. In another specific embodiment, said
contacting is performed during freezing and thawing of said
population of cells, e.g., placental stem cells.
[0314] Populations of placental useful in the methods and
compositions described herein, cells can be preserved, e.g., by a
method comprising contacting said population of cells with an
inhibitor of apoptosis and an organ-preserving compound, wherein
said inhibitor of apoptosis is present in an amount and for a time
sufficient to reduce or prevent apoptosis in the population of
cells, as compared to a population of cells not contacted with the
inhibitor of apoptosis. In a specific embodiment, the
organ-preserving compound is UW solution (e.g., as described in
U.S. Pat. No. 4,798,824; also known as ViaSpan; see also Southard
et al., Transplantation 49(2):251-257 (1990)) or a solution
described in Stern et al., U.S. Pat. No. 5,552,267, the disclosure
of which is hereby incorporated by reference in their entireties.
In another embodiment, said organ-preserving compound is
hydroxyethyl starch, lactobionic acid, raffinose, or a combination
thereof. In another embodiment, the cell collection composition
additionally comprises an oxygen-carrying perfluorocarbon, either
in two phases or as an emulsion.
[0315] In another embodiment of the method, placental stem cells
are contacted with a cell collection composition comprising an
apoptosis inhibitor and oxygen-carrying perfluorocarbon,
organ-preserving compound, or combination thereof, during
perfusion. In another embodiment, said cells are contacted during a
process of tissue disruption, e.g., enzymatic digestion. In another
embodiment, placental stem cells are contacted with said cell
collection compound after collection by perfusion, or after
collection by tissue disruption, e.g., enzymatic digestion.
[0316] Typically, during placental stem cell collection, enrichment
and isolation, it is preferable to minimize or eliminate cell
stress due to hypoxia and mechanical stress. In another embodiment
of the method, therefore, a cell, or population of cells, e.g.,
placental stem cells, is exposed to a hypoxic condition during
collection, enrichment or isolation for less than six hours during
said preservation, wherein a hypoxic condition is a concentration
of oxygen that is less than normal blood oxygen concentration. In
another specific embodiment, said population of cells is exposed to
said hypoxic condition for less than two hours during said
preservation. In another specific embodiment, said population of
cells is exposed to said hypoxic condition for less than one hour,
or less than thirty minutes, or is not exposed to a hypoxic
condition, during collection, enrichment or isolation. In another
specific embodiment, said population of cells is not exposed to
shear stress during collection, enrichment or isolation.
[0317] Placental stem cells can be cryopreserved, e.g., in
cryopreservation medium in small containers, e.g., ampoules.
Suitable cryopreservation medium includes, but is not limited to,
culture medium including, e.g., growth medium, or cell freezing
medium, for example commercially available cell freezing medium,
e.g., C2695, C2639 or C6039 (Sigma). Cryopreservation medium
preferably comprises DMSO (dimethylsulfoxide), at a concentration
of about 2% to about 15% (v/v), e.g., about 10% (v/v).
Cryopreservation medium may comprise additional agents, for
example, methylcellulose and/or glycerol. Placental stem cells are
preferably cooled at about 1.degree. C./min during
cryopreservation. Cryopreservation can be accomplished by bringing
the cells to a temperature of about -80.degree. C. to about
-180.degree. C., preferably about -125.degree. C. to about
-140.degree. C. Cryopreserved cells can be transferred to liquid
nitrogen prior to thawing for use. In some embodiments, for
example, once the ampoules have reached about -90.degree. C., they
are transferred to a liquid nitrogen storage area. Cryopreservation
can also be done using a controlled-rate freezer. Cryopreserved
cells preferably are thawed at a temperature of about 25.degree. C.
to about 40.degree. C., preferably to a temperature of about
37.degree. C.
[0318] Bone marrow-derived mesenchymal stem cells can be preserved
by any of the above methods, as well.
5.6 Compositions Comprising Isolated Placental Stem Cells
[0319] The placental stem cells described herein, e.g., in Section
5.2.2, can be combined with any physiologically-acceptable or
medically-acceptable compound, composition or device for use in the
methods and compositions described herein. In certain embodiments,
the composition is a pharmaceutically-acceptable composition, e.g.,
a composition comprising placental stem cells in a
pharmaceutically-acceptable carrier. Any of the compositions
described herein can additionally comprise isolated bone
marrow-derived mesenchymal stem cells, or bone marrow comprising
BM-MSCs, e.g., the BM-MSCs described in U.S. Pat. No.
5,486,359.
[0320] In certain embodiments, a composition comprising the
isolated placental stem cells additionally comprises a matrix,
e.g., a decellularized matrix or a synthetic matrix. In another
specific embodiment, said matrix is a three-dimensional scaffold.
In another specific embodiment, said matrix comprises collagen,
gelatin, laminin, fibronectin, pectin, ornithine, or vitronectin.
In another ore specific embodiment, the matrix is an amniotic
membrane or an amniotic membrane-derived biomaterial. In another
specific embodiment, said matrix comprises an extracellular
membrane protein. In another specific embodiment, said matrix
comprises a synthetic compound. In another specific embodiment,
said matrix comprises a bioactive compound. In another specific
embodiment, said bioactive compound is a growth factor, cytokine,
antibody, or organic molecule of less than 5,000 daltons.
[0321] In another embodiment, a composition useful in the methods
of treatment provided herein comprises medium conditioned by any of
the foregoing placental stem cells, or any of the foregoing
placental stem cell populations.
[0322] 5.6.1 Cryopreserved Cells
[0323] The isolated placental stem cells useful in the methods and
compositions described herein can be preserved, for example,
cryopreserved for later use. Methods for cryopreservation of cells,
such as stem cells, are well known in the art. Isolated placental
stem cell populations can be prepared in a form that is easily
administrable to an individual, e.g., an isolated placental stem
cell population that is contained within a container that is
suitable for medical use. Such a container can be, for example, a
syringe, sterile plastic bag, flask, jar, or other container from
which the isolated placental cell population can be easily
dispensed. For example, the container can be a blood bag or other
plastic, medically-acceptable bag suitable for the intravenous
administration of a liquid to a recipient. The container is
preferably one that allows for cryopreservation of the isolated
placental stem cells.
[0324] The cryopreserved isolated placental stem cells can comprise
isolated placental cells derived from a single donor, or from
multiple donors. The isolated placental stem cell population can be
completely HLA-matched to an intended recipient, or partially or
completely HLA-mismatched.
[0325] Thus, in one embodiment, isolated placental stem cells can
be used in the methods and described herein in the form of a
composition comprising a tissue culture plastic-adherent placental
stem cell population in a container. In a specific embodiment, the
isolated placental stem cells are cryopreserved. In another
specific embodiment, the container is a bag, flask, or jar. In
another specific embodiment, said bag is a sterile plastic bag. In
another specific embodiment, said bag is suitable for, allows or
facilitates intravenous administration of said isolated placental
stem cell population, e.g., by intravenous infusion. The bag can
comprise multiple lumens or compartments that are interconnected to
allow mixing of the isolated placental stem cells and one or more
other solutions, e.g., a drug, prior to, or during, administration.
In another specific embodiment, the composition comprises one or
more compounds that facilitate cryopreservation of the placental
stem cells. In another specific embodiment, said isolated placental
stem cells are contained within a physiologically-acceptable
aqueous solution. In another specific embodiment, said
physiologically-acceptable aqueous solution is a 0.9% NaCl
solution. In another specific embodiment, said isolated placental
stem cells comprise placental stem cells that are HLA-matched to a
recipient of said placental stem cells. In another specific
embodiment, said combined cell population comprises placental stem
cells that are at least partially HLA-mismatched to a recipient of
said placental stem cells. In another specific embodiment, said
isolated stem placental stem cells are derived from a plurality of
donors.
[0326] In certain embodiments, the isolated placental stem cells in
the container are any of the isolated placental stem cells
described in Section 5.2.2 herein, wherein said cells have been
cryopreserved, and are contained within a container.
[0327] In a specific embodiment of any of the foregoing
cryopreserved isolated placental stem cells, said container is a
bag. In various specific embodiments, said container comprises
about, at least, or at most 1.times.10.sup.6 said isolated
placental stem cells, 5.times.10.sup.6 said isolated placental stem
cells, 1.times.10.sup.7 said isolated placental stem cells,
5.times.10.sup.7 said isolated placental stem cells,
1.times.10.sup.8 said isolated placental stem cells,
5.times.10.sup.8 said isolated placental stem cells,
1.times.10.sup.9 said isolated placental stem cells,
5.times.10.sup.9 said isolated placental stem cells,
1.times.10.sup.10 said isolated placental stem cells, or
1.times.10.sup.10 said isolated placental stem cells. In other
specific embodiments of any of the foregoing cryopreserved
populations, said isolated placental stem cells have been passaged
about, at least, or no more than 5 times, no more than 10 times, no
more than 15 times, or no more than 20 times. In another specific
embodiment of any of the foregoing cryopreserved isolated placental
stem cells, said isolated placental stem cells have been expanded
within said container.
[0328] 5.6.2 Genetically Engineered Placental Stem Cells
[0329] Further provided herein are placental stem cells, wherein
the placental stem cells have been genetically engineered to
produce recombinant or exogenous cytokincs associated with tumor
suppression. For example, in various embodiments, the placental
stem cells are engineered to express detectable amounts of
exogenous protein, wherein said exogenous protein is one or more of
a bone morphogenetic protein (BMP), activin A, osteonectin,
osteoprotegerin, or a connexin. Sequences encoding activin A can be
found, e.g., at GenBank Accession No. NM.sub.--002191. Sequences
encoding osteonectin can be found, e.g., at GenBank Accession No.
NM.sub.--003118. Sequences encoding osteoprotegerin can be found,
e.g., at GenBank Accession No. NM.sub.--002546.
[0330] In specific embodiments, the connexin is connexin 26 (Cx26)
or connexin 43 (Cx43). Sequences encoding Cx26 or Cx43 can be
found, e.g., at GenBank Accession Nos. NM.sub.--004004 and
NM.sub.--000165, respectively.
[0331] In specific embodiments, said bone morphogenetic protein is
one or more of BMP1 (bone morphogenetic protein 1), BMP2, BMP3,
BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9 (GDF2; Growth
Differentiation Factor-2), BMP10, BMP11 (GDF11), BMP12 (GDF7),
BMP13 (GDF6), BMP14 (GDF5), or BMP15, or any combination thereof.
Sequences encoding BMPs can be found, e.g., in GenBank, e.g.,
GenBank Accession No. NM.sub.--001199 (BMP1), NM.sub.--001200
(BMP2), NM.sub.--001201 (BMP3), NM.sub.--001202 (BMP4),
NM.sub.--021073 (BMP5), NM.sub.--021073 (BMP6), NM.sub.--001719
(BMP7), NM.sub.--181809 (BMP8a), NM.sub.--001720 (BMP8b),
NM.sub.--016204 (BMP9/GDF2), NM.sub.--014482 (BMP10),
NM.sub.--005811 (BMP11/GDF11), NM.sub.--182828 (BMP12/GDF7),
NM.sub.--001001557 (BMP13/GDF6), NM.sub.--000557 (BMP14/GDF5), or
NM.sub.--005448 (BMP15).
[0332] In other embodiments, provided herein are isolated placental
stem cells, wherein the placental stem cells are engineered to
express exogenous IFN-.beta. or IL-2. In a specific embodiment,
said placental stem cells express exogenous IFN-.beta. or IL-2 in
an amount that results in greater, e.g., detectably greater,
suppression of tumor cell proliferation, when said tumor cells are
contacted with said placental stem cells, compared to placental
stem cells not expressing exogenous IFN-.beta. or IL-2.
[0333] Methods for genetically engineering cells, for example with
retroviral vectors, adenoviral vectors, adeno-associated viral
vectors, polyethylene glycol, or other methods known to those
skilled in the art, can be used. These include using expression
vectors which transport and express nucleic acid molecules in the
cells. (See Geoddel; Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif. (1990)). Vector
DNA can be introduced into prokaryotic or eukaryotic cells via
conventional transformation or transfection techniques. Suitable
methods for transforming or transfecting host cells can be found in
Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd
Edition, Cold Spring Harbor Laboratory press (1989), and other
laboratory textbooks.
[0334] Placental stem cells can be genetically modified by
introducing DNA or RNA into the cell, e.g., DNA or RNA encoding a
protein of interest, by methods including viral transfer, including
the use of DNA or RNA viral vectors, such as retroviruses
(including lentiviruses), Simian virus 40 (SV40), adenovirus,
Sindbis virus, and bovine papillomavirus for example; chemical
transfer, including calcium phosphate transfection and DEAE dextran
transfection methods; membrane fusion transfer, using DNA-loaded
membrane vesicles such as liposomes, red blood cell ghosts, and
protoplasts, for example; or physical transfer techniques, such as
microinjection, electroporation, or naked DNA transfer. The
placental stem cells can be genetically altered by insertion of
exogenous DNA, or by substitution of a segment of the cellular
genome with exogenous DNA. Insertion of exogenous DNA sequence(s)
can be accomplished, e.g., by homologous recombination or by viral
integration into the host cell genome, or by incorporating the DNA
into the cell, particularly into its nucleus, using a plasmid
expression vector and a nuclear localization sequence. The DNA can
comprise one or more promoters that allow positive or negative
induction of expression of the protein of interest using certain
chemicals/drugs, e.g., tetracycline; the promoters can, in other
embodiments, be constitutive.
[0335] Calcium phosphate transfection can be used to introduce,
e.g., plasmid DNA containing a polynucleotide sequence encoding the
protein of interest, into a cell, e.g., a placental stem cell. In
certain embodiments, DNA is combined with a solution of calcium
chloride, then added to a phosphate-buffered solution. Once a
precipitate has formed, the solution is added directly to cultured
cells. Treatment with DMSO or glycerol can be used to improve
transfection efficiency, and levels of stable transfectants can be
improved using bis-hydroxyethylamino ethanesulfonate (BES). Calcium
phosphate transfection systems are commercially available (e.g.,
PROFECTION.RTM., Promega Corp., Madison, Wis.). DEAE-dextran
transfection may also be used.
[0336] Isolated placental stem cells may also be genetically
modified by microinjection. In certain embodiments, a glass
micropipette is guided into the nucleus of cells under a light
microscope to inject DNA or RNA.
[0337] Placental stem cells can also be genetically modified using
clectroporation. In certain embodiments, DNA or RNA is added to a
suspension of cultured cells, and the DNA/RNA-cell suspension is
placed between two electrodes and subjected to an electrical pulse,
causing a transient permeability in the cell's outer membrane that
is manifested by the appearance of pores across the membrane.
[0338] Liposomal delivery of DNA or RNA to genetically modify the
cells can be performed using cationic liposomes, optionally
including dioleoyl phosphatidylethanolamine (DOPE) or dioleoyl
phosphatidylcholine (DOPC), e.g., LIPOFECTIN.RTM. (Life
Technologies, Inc.). Other commercially-available delivery systems
include EFFECTENE.TM. (Qiagen), DOTAP (Roche Molecular
Biochemicals), FUGENE 6.TM.. (Roche Molecular Biochemicals), and
TRANSFECTAM.RTM. (Promega).
[0339] Viral vectors can be used to genetically alter placental
stem cells by delivery of, e.g., target genes, polynucleotides,
antisense molecules, or ribozyme sequences into the cells.
Retroviral vectors are effective for transducing rapidly-dividing
cells, although a number of retroviral vectors have been developed
to effectively transfer DNA into non-dividing cells as well.
Packaging cell lines for retroviral vectors are known to those of
skill in the art. In certain embodiments, a retroviral DNA vector
contains two retroviral LTRs such that a first LTR is located 5' to
the SV40 promoter, which is operationally linked to the target gene
sequence cloned into a multicloning site, followed by a 3' second
LTR. Once formed, the retroviral DNA vector is transferred into a
packaging cell line using calcium phosphate-mediated transfection,
as previously described. Following approximately 48 hours of virus
production, the viral vector, now containing the target gene
sequence, is harvested. Methods of transfecting cells using
lentiviral vectors, recombinant herpes viruses, adenoviral vectors,
or alphavirus vectors are known in the art.
[0340] Successful transfection or transduction of target cells can
be demonstrated using genetic markers, in a technique that is known
to those of skill in the art. The green fluorescent protein of
Aequorea victoria, for example, has been shown to be an effective
marker for identifying and tracking genetically modified
hematopoietic cells. Alternative selectable markers include the
.beta.-Gal gene, truncated nerve growth factor receptor, or drug
selectable markers (including but not limited to NEO, MTX, or
hygromycin).
[0341] Bone marrow-derived mesenchymal stem cells can be
genetically modified by any of the methods, and/or by any of the
genes, disclosed above.
[0342] 5.6.3 Pharmaceutical Compositions
[0343] Populations of isolated placental stem cells, or populations
of cells comprising the isolated placental stem cells, can be
formulated into pharmaceutical compositions for use in vivo, e.g.,
in the methods of treatment provided herein. Such pharmaceutical
compositions comprise a population of isolated placental stem
cells, or a population of cells comprising isolated placental stem
cells, in a pharmaceutically-acceptable carrier, e.g., a saline
solution or other accepted physiologically-acceptable solution for
in vivo administration. Pharmaceutical compositions comprising the
isolated placental stem cells described herein can comprise any, or
any combination, of the isolated placental stem cells described
elsewhere herein. The pharmaceutical compositions can comprise
fetal, maternal, or both fetal and maternal isolated placental stem
cells. The pharmaceutical compositions provided herein can further
comprise isolated placental stem cells obtained from a single
individual or placenta, or from a plurality of individuals or
placentae.
[0344] The pharmaceutical compositions provided herein can comprise
any number of isolated placental stem cells. For example, a single
unit dose of isolated placental stem cells can comprise, in various
embodiments, about, at least, or no more than 1.times.10.sup.5,
5.times.10.sup.5, 1.times.10.sup.6, 5.times.10.sup.6,
1.times.10.sup.7, 5.times.10.sup.7, 1.times.10.sup.8,
5.times.10.sup.8, 1.times.10.sup.9, 5.times.10.sup.9,
1.times.10.sup.10, 5.times.10.sup.10, 1.times.10.sup.11 or more
isolated placental stem cells, or from 1.times.10.sup.5 to
5.times.10.sup.5, 5.times.10.sup.5 to 1.times.10.sup.6,
1.times.10.sup.6 to 5.times.10.sup.6, 5.times.10.sup.6 to
1.times.10.sup.7, 1.times.10.sup.7 to 5.times.10.sup.7,
5.times.10.sup.7 to 1.times.10.sup.8, 1.times.10.sup.8 to
5.times.10.sup.8, 5.times.10.sup.8 to 1.times.10.sup.9,
1.times.10.sup.9 to 5.times.10.sup.9, 5.times.10.sup.9 to
1.times.10.sup.10, 1.times.10.sup.10 to 5.times.10.sup.10, or
5.times.10.sup.10 to 1.times.10.sup.11 isolated placental stem
cells.
[0345] The pharmaceutical compositions provided herein comprise
populations of placental stem cells that comprise 50% viable cells
or more (that is, at least 50% of the cells in the population are
functional or living). Preferably, at least 60% of the cells in the
population are viable. More preferably, at least 70%, 80%, 90%,
95%, or 99% of the cells in the population in the pharmaceutical
composition are viable.
[0346] The pharmaceutical compositions provided herein can comprise
one or more compounds that, e.g., facilitate engraftment (e.g.,
anti-T-cell receptor antibodies, an immunosuppressant, or the
like); stabilizers such as albumin, dextran 40, gelatin,
hydroxyethyl starch, plasmalyte, and the like.
[0347] When formulated as an injectable solution, in one
embodiment, the pharmaceutical composition comprises about 1% to
1.5% HSA and about 2.5% dextran. In a preferred embodiment, the
pharmaceutical composition comprises from about 5.times.10.sup.6
cells per milliliter to about 2.times.10.sup.7 cells per milliliter
in a solution comprising 5% HSA and 10% dextran, optionally
comprising an immunosuppressant, e.g., cyclosporine A at, e.g., 10
mg/kg.
[0348] In other embodiments, the pharmaceutical composition, e.g.,
a solution, comprises isolated placental stem cells, wherein said
pharmaceutical composition comprises between about
1.0.+-.0.3.times.10.sup.6 cells per milliliter to about
5.0.+-.1.5.times.10.sup.6 cells per milliliter. In other
embodiments, the pharmaceutical composition comprises between about
1.5.times.10.sup.6 cells per milliliter to about
3.75.times.10.sup.6 cells per milliliter. In other embodiments, the
pharmaceutical composition comprises between about 1.times.10.sup.6
cells/mL to about 50.times.10.sup.6 cells/mL, about
1.times.10.sup.6 cells/mL to about 40.times.10.sup.6 cells/mL,
about 1.times.10.sup.6 cells/mL to about 30.times.10.sup.6
cells/mL, about 1.times.10.sup.6 cells/mL to about
20.times.10.sup.6 cells/mL, about 1.times.10.sup.6 cells/mL to
about 15.times.10.sup.6 cells/mL, or about 1.times.10.sup.6
cclls/mL to about 10.times.10.sup.6 cells/mL. In certain
embodiments, the pharmaceutical composition comprises no visible
cell clumps (i.e., no macro cell clumps), or substantially no such
visible clumps. As used herein, "macro cell clumps" means an
aggregation of cells visible without magnification, e.g., visible
to the naked eye, and generally refers to a cell aggregation larger
than about 150 microns In some embodiments, the pharmaceutical
composition comprises about 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%,
5.5%, 6.0%, 6.5%, 7.0%, 7.5% 8.0%, 8.5%, 9.0%, 9.5% or 10% dextran,
e.g., dextran-40. In a specific embodiment, said composition
comprises about 7.5% to about 9% dextran-40. In a specific
embodiment, said composition comprises about 5.5% dextran-40. In
certain embodiments, the pharmaceutical composition comprises from
about 1% to about 15% human serum albumin (HSA). In specific
embodiments, the pharmaceutical composition comprises about 1%, 2%,
3%, 4%, 5%, 65, 75, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% HSA. In
a specific embodiment, said cells have been cryopreserved and
thawed. In another specific embodiment, said cells have been
filtered through a 70 .mu.M to 100 .mu.M filter. In another
specific embodiment, said composition comprises no visible cell
clumps. In another specific embodiment, said composition comprises
fewer than about 200 cell clumps per 10.sup.6 cells, wherein said
cell clumps are visible only under a microscope, e.g., a light
microscope. In another specific embodiment, said composition
comprises fewer than about 150 cell clumps per 10.sup.6 cells,
wherein said cell clumps are visible only under a microscope, e.g.,
a light microscope. In another specific embodiment, said
composition comprises fewer than about 100 cell clumps per 10.sup.6
cells, wherein said cell clumps are visible only under a
microscope, e.g., a light microscope.
[0349] In a specific embodiment, the pharmaceutical composition
comprises about 1.0.+-.0.3.times.10.sup.6 cells per milliliter,
about 5.5% dextran-40 (w/v), about 10% HSA (w/v), and about 5% DMSO
(v/v).
[0350] In other embodiments, the pharmaceutical composition
comprises a plurality of isolated placental stem cells in a
solution comprising 10% dextran-40, wherein the pharmaceutical
composition comprises between about 1.0.+-.0.3.times.10.sup.6 cells
per milliliter to about 5.0.+-.1.5.times.10.sup.6 cells per
milliliter, and wherein said composition comprises no cell clumps
visible with the unaided eye (i.e., comprises no macro cell
clumps). In some embodiments, the pharmaceutical composition
comprises between about 1.5.times.10.sup.6 cells per milliliter to
about 3.75.times.10.sup.6 cells per milliliter. In a specific
embodiment, said cells have been cryopreserved and thawed. In
another specific embodiment, said cells have been filtered through
a 70 .mu.M to 100 .mu.M filter. In another specific embodiment,
said composition comprises fewer than about 200 micro cell clumps
(that is, cell clumps visible only with magnification) per 10.sup.6
cells. In another specific embodiment, the pharmaceutical
composition comprises fewer than about 150 micro cell clumps per
10.sup.6 cells. In another specific embodiment, the pharmaceutical
composition comprises fewer than about 100 micro cell clumps per
10.sup.6 cells. In another specific embodiment, the pharmaceutical
composition comprises less than 15%, 14%, 13%, 12%, 11%, 10%, 9%,
8%, 7%, 6%, 5%, 4%, 3%, or 2% DMSO, or less than 1%, 0.9%, 0.8%,
0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% DMSO.
[0351] Further provided herein are compositions comprising
placental stem cells, wherein said compositions are produced by one
of the methods disclosed herein. For example, in one embodiment,
the pharmaceutical composition comprises cells, e.g., placental
stem cells, wherein the pharmaceutical composition is produced by a
method comprising filtering a solution comprising cells, e.g.,
placental stem cells, to form a filtered cell-containing solution;
diluting the filtered cell-containing solution with a first
solution to about 1 to 50.times.10.sup.6, 1 to 40.times.10.sup.6, 1
to 30.times.10.sup.6, 1 to 20.times.10.sup.6, 1 to
15.times.10.sup.6, or 1 to 10.times.10.sup.6 cells per milliliter,
e.g., prior to cryopreservation; and diluting the resulting
filtered cell-containing solution with a second solution comprising
dextran, but not comprising human serum albumin (HSA) to produce
said composition. In certain embodiments, said diluting is to no
more than about 15.times.10.sup.6 cells per milliliter. In certain
embodiments, said diluting is to no more than about
10.+-.3.times.10.sup.6 cells per milliliter. In certain
embodiments, said diluting is to no more than about
7.5.times.10.sup.6 cells per milliliter. In other certain
embodiments, if the filtered cell-containing solution, prior to the
dilution, comprises less than about 15.times.10.sup.6 cells per
milliliter, filtration is optional. In other certain embodiments,
if the filtered cell-containing solution, prior to the dilution,
comprises less than about 10.+-.3.times.10.sup.6 cells per
milliliter, filtration is optional. In other certain embodiments,
if the filtered cell-containing solution, prior to the dilution,
comprises less than about 7.5.times.10.sup.6 cells per milliliter,
filtration is optional.
[0352] In a specific embodiment, the cells, e.g., placental stem
cells, are cryopreserved between said diluting with a first
dilution solution and said diluting with said second dilution
solution. In another specific embodiment, the first dilution
solution comprises dextran and HSA. The dextran in the first
dilution solution or second dilution solution can be dextran of any
molecular weight, e.g., dextran having a molecular weight of from
about 10 kDa to about 150 kDa. In some embodiments, said dextran in
said first dilution solution or said second solution is about 2.5%,
3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5% 8.0%,
8.5%, 9.0%, 9.5% or 10% dextran. In another specific embodiment,
the dextran in said first dilution solution or said second dilution
solution is dcxtran-40. In another specific embodiment, the dextran
in said first dilution solution and said second dilution solution
is dextran-40. In another specific embodiment, said dextran-40 in
said first dilution solution is 5.0% dextran-40. In another
specific embodiment, said dextran-40 in said first dilution
solution is 5.5% dextran-40. In another specific embodiment, said
dextran-40 in said second dilution solution is 10% dextran-40. In
another specific embodiment, said HSA in said solution comprising
HSA is 1 to 15% HSA. In another specific embodiment, said HSA in
said solution comprising HSA is about 1%, 2%, 3%, 4%, 5%, 6%, 7%,
8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% HSA. In another specific
embodiment, said HSA in said solution comprising HSA is 10% HSA. In
another specific embodiment, said first dilution solution comprises
HSA. In another specific embodiment, said HSA in said first
dilution solution is 10% HSA. In another specific embodiment, said
first dilution solution comprises a cryoprotectant. In another
specific embodiment, said cryoprotectant is DMSO. In another
specific embodiment, said dextran-40 in said second dilution
solution is about 10% dextran-40. In another specific embodiment,
said composition comprising cells comprises about 7.5% to about 9%
dextran. In another specific embodiment, the pharmaceutical
composition comprises from about 1.0.+-.0.3.times.10.sup.6 cells
per milliliter to about 5.0.+-.1.5.times.10.sup.6 cells per
milliliter. In another specific embodiment, the pharmaceutical
composition comprises from about 1.5.times.10.sup.6 cells per
milliliter to about 3.75.times.10.sup.6 cells per milliliter.
[0353] In another embodiment, the pharmaceutical composition is
made by a method comprising (a) filtering a cell-containing
solution comprising placental stem cells prior to cryopreservation
to produce a filtered cell-containing solution; (b) cryopreserving
the cells in the filtered cell-containing solution at about 1 to
50.times.10.sup.6, 1 to 40.times.10.sup.6, 1 to 30.times.10.sup.6,
1 to 20.times.10.sup.6, 1 to 15.times.10.sup.6, or 1 to
10.times.10.sup.6 cells per milliliter; (c) thawing the cells; and
(d) diluting the filtered cell-containing solution about 1:1 to
about 1:11 (v/v) with a dextran-40 solution. In certain
embodiments, if the number of cells is less than about
10.+-.3.times.10.sup.6 cells per milliliter prior to step (a),
filtration is optional. In another specific embodiment, the cells
in step (b) are cryopreserved at about 10.+-.3.times.10.sup.6 cells
per milliliter. In another specific embodiment, the cells in step
(b) are cryopreserved in a solution comprising about 5% to about
10% dextran-40 and HSA. In certain embodiments, said diluting in
step (b) is to no more than about 15.times.10.sup.6 cells per
milliliter.
[0354] In another embodiment, the pharmaceutical composition is
made by a method comprising: (a) suspending placental stem cells in
a 5.5% dextran-40 solution that comprises 10% HSA to form a
cell-containing solution; (b) filtering the cell-containing
solution through a 70 .mu.M filter; (c) diluting the
cell-containing solution with a solution comprising 5.5%
dextran-40, 10% HSA, and 5% DMSO to about 1 to 50.times.10.sup.6, 1
to 40.times.10.sup.6, 1 to 30.times.10.sup.6, 1 to
20.times.10.sup.6, 1 to 15.times.10.sup.6, or 1 to
10.times.10.sup.6 cells per milliliter; (d) cryopreserving the
cells; (e) thawing the cells; and (f) diluting the cell-containing
solution 1:1 to 1:11 (v/v) with 10% dextran-40. In certain
embodiments, said diluting in step (c) is to no more than about
15.times.10.sup.6 cells per milliliter. In certain embodiments,
said diluting in step (c) is to no more than about 10
3.times.10.sup.6 cells/mL. In certain embodiments, said diluting in
step (c) is to no more than about 7.5.times.10.sup.6 cells/mL.
[0355] In another embodiment, the composition comprising cells is
made by a method comprising: (a) centrifuging a plurality of cells,
e.g., placental stem cells, to collect the cells; (b) resuspending
the cells in 5.5% dextran-40; (c) centrifuging the cells to collect
the cells; (d) resuspending the cells in a 5.5% dextran-40 solution
that comprises 10% HSA; (e) filtering the cells through a 70 .mu.M
filter; (f) diluting the cells in 5.5% dextran-40, 10% HSA, and 5%
DMSO to about 1 to 50.times.10.sup.6, 1 to 40.times.10.sup.6, 1 to
30.times.10.sup.6, 1 to 20.times.10.sup.6, 1 to 15.times.10.sup.6,
or 1 to 10.times.10.sup.6 cells per milliliter; (g) cryopreserving
the cells; (h) thawing the cells; and (i) diluting the cells 1:1 to
1:11 (v/v) with 10% dextran-40. In certain embodiments, said
diluting in step (f) is to no more than about 15.times.10.sup.6
cells per milliliter. In certain embodiments, said diluting in step
(f) is to no more than about 10.+-.3.times.10.sup.6 cells/mL. In
certain embodiments, said diluting in step (f) is to no more than
about 7.5.times.10.sup.6 cells/mL. In other certain embodiments, if
the number of cells is less than about 10.+-.3.times.10.sup.6 cells
per milliliter, filtration is optional.
[0356] Other injectable formulations, suitable for the
administration of cellular products, may be used.
[0357] The pharmaceutical compositions useful in the methods of the
invention can comprise any of the placental stem cells described
herein, e.g., as described in Section 5.2.2, above. In one
embodiment, the pharmaceutical composition comprises isolated
placental stem cells that are substantially, or completely,
non-maternal in origin, that is, have the fetal genotype; e.g., at
least about 90%, 95%, 98%, 99% or about 100% are non-maternal in
origin. In certain embodiments, a pharmaceutical composition
comprises a population of isolated placental stem cells that are,
in non-limiting examples, CD10.sup.+, CD34.sup.-, CD105.sup.+ and
CD200.sup.+; CD200.sup.+ and HLA-G.sup.-; CD73.sup.+, CD105.sup.+,
and CD200.sup.+; CD200.sup.+ and OCT-4.sup.+; or CD73.sup.+,
CD105.sup.+ and HLA-G.sup.-; or a combination of the foregoing,
wherein at least 70%, 80%, 90%, 95% or 99% of said isolated
placental stem cells are non-maternal in origin. In another
embodiment, a pharmaceutical composition comprises a population of
isolated placental stem cells that are CD10.sup.+, CD105.sup.+ and
CD34.sup.-; CD10.sup.+, CD105.sup.+, CD200.sup.+ and CD34.sup.-;
CD10.sup.+, CD105.sup.+, CD200.sup.+, CD34.sup.- and at least one
of CD90.sup.+ or CD45.sup.-; CD10.sup.+, CD90.sup.+, CD105.sup.+,
CD200.sup.+, CD34.sup.- and CD45.sup.-; CD10.sup.+, CD90.sup.+,
CD105.sup.+, CD200.sup.+, CD34.sup.- and CD45.sup.-; CD200.sup.+
and HLA-G.sup.-; CD73.sup.+, CD105.sup.+, and CD200.sup.+;
CD200.sup.+ and OCT-4.sup.+; CD73.sup.+, CD105.sup.+ and
HLA-G.sup.-; one or more of CD117.sup.-, CD133.sup.-, KDR.sup.-,
CD80.sup.-, CD86.sup.-, HLA-A,B,C.sup.+, HLA-DP,DQ,DR.sup.- and/or
PDL1.sup.+; or a combination of the foregoing, wherein at least
70%, 80%, 90%, 95% or 99% of said isolated placental stem cells are
non-maternal in origin. In a specific embodiment, the
pharmaceutical composition additionally comprises a stem cell that
is not obtained from a placenta.
[0358] Isolated placental stem cells in the compositions, e.g.,
pharmaceutical compositions, provided herein, can comprise
placental stem cells derived from a single donor, or from multiple
donors. The isolated placental stem cells can be completely
HLA-matched to an intended recipient, or partially or completely
HLA-mismatched.
[0359] The pharmaceutical compositions provided herein can further
comprise BM-MSCs. In certain embodiments, the placental stem cells
and BM-MSCs are present in the pharmaceutical composition at a
ratio of, e.g., 99:1, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30,
65:35, 60:40, 55:45, 50:50, 45:55, 40:60, 35:65, 30:70, 25:75,
20:80, 15:85, 10:90, 5:95 or 1:99 by numbers of cells, or between
99:1 and 95:5, between 95:5 and 90:10, between 90:10 and 85:15,
between 85:15 and 80:20, between 80:20 and 75:25, between 75:25 and
70:30, between 70:30 and 65:35, between 65:35 and 60:40, between
60:40 and 55:45, between 55:45 and 50:50, between 50:50 and 45:55,
between 45:55 and 40:60, between 40:60 and 35:65, between 35:65 and
30:70, between 30:70 and 25:75, between 25:75 and 20:80, between
20:80 and 15:85, between 10:90 and 5:95, or between 5:95 and 1:99,
by numbers of cells.
[0360] 5.6.4 Matrices Comprising Isolated Placental Stem Cells
[0361] Further provided herein are compositions comprising
matrices, hydrogels, scaffolds, and the like that comprise
placental stem cells. Such compositions can be used in the place
of, or in addition to, cells in liquid suspension. In certain
embodiments, the isolated placental stem cells are combined with
platelet rich plasma. In other embodiments, the isolated placental
stem cells are combined with alginate.
[0362] The isolated placental stem cells described herein can be
seeded onto a natural matrix, e.g., a placental biomaterial such as
an amniotic membrane material. Such an amniotic membrane material
can be, e.g., amniotic membrane dissected directly from a mammalian
placenta; fixed or heat-treated amniotic membrane, substantially
dry (i.e., <20% H.sub.2O) amniotic membrane, chorionic membrane,
substantially dry chorionic membrane, substantially dry amniotic
and chorionic membrane, and the like. Preferred placental
biomaterials on which isolated placental stem cells can be seeded
are described in Hariri, U.S. Application Publication No.
2004/0048796, the disclosure of which is incorporated herein by
reference in its entirety.
[0363] The isolated placental stem cells described herein can be
suspended in a hydrogel solution suitable for, e.g., injection.
Suitable hydrogels for such compositions include self-assembling
peptides, such as RAD16. In one embodiment, a hydrogel solution
comprising the cells can be allowed to harden, for instance in a
mold, to form a matrix having cells dispersed therein for
implantation. Isolated placental stem cells in such a matrix can
also be cultured so that the cells are mitotically expanded prior
to implantation. The hydrogel is, e.g., an organic polymer (natural
or synthetic) that is cross-linked via covalent, ionic, or hydrogen
bonds to create a three-dimensional open-lattice structure that
entraps water molecules to form a gel. Hydrogel-forming materials
include polysaccharides such as alginate and salts thereof,
peptides, polyphosphazines, and polyacrylates, which are
crosslinked ionically, or block polymers such as polyethylene
oxide-polypropylene glycol block copolymers which are crosslinked
by temperature or pH, respectively. In some embodiments, the
hydrogel or matrix is biodegradable.
[0364] In some embodiments, the formulation comprises an in situ
polymerizable gel (see, e.g., U.S. Patent Application Publication
2002/0022676, the disclosure of which is incorporated herein by
reference in its entirety; Anseth et al., J. Control Release,
78(1-3):199-209 (2002); Wang et al., Biomaterials, 24(22):3969-80
(2003).
[0365] In some embodiments, the polymers are at least partially
soluble in aqueous solutions, such as water, buffered salt
solutions, or aqucous alcohol solutions, that have charged side
groups, or a monovalent ionic salt thereof. Examples of polymers
having acidic side groups that can be reacted with cations are
poly(phosphazenes), poly(acrylic acids), poly(methacrylic acids),
copolymers of acrylic acid and methacrylic acid, poly(vinyl
acetate), and sulfonated polymers, such as sulfonated polystyrene.
Copolymers having acidic side groups formed by reaction of acrylic
or methacrylic acid and vinyl ether monomers or polymers can also
be used. Examples of acidic groups are carboxylic acid groups,
sulfonic acid groups, halogenated (preferably fluorinated) alcohol
groups, phenolic OH groups, and acidic OH groups.
[0366] The isolated placental stem cells described herein or
co-cultures thereof can be seeded onto a three-dimensional
framework or scaffold and implanted in vivo. Such a framework can
be implanted in combination with any one or more growth factors,
cells, drugs or other components that, e.g., stimulate tissue
formation.
[0367] Examples of scaffolds that can be used include nonwoven
mats, porous foams, or self assembling peptides. Nonwoven mats can
be formed using fibers comprised of a synthetic absorbable
copolymer of glycolic and lactic acids (e.g., PGA/PLA) (VICRYL,
Ethicon, Inc., Somerville, N.J.). Foams, composed of, e.g.,
poly(.epsilon.-caprolactone)/poly(glycolic acid) (PCL/PGA)
copolymer, formed by processes such as freeze-drying, or
lyophilization (see, e.g., U.S. Pat. No. 6,355,699), can also be
used as scaffolds.
[0368] In another embodiment, isolated placental stem cells can be
seeded onto, or contacted with, a felt, which can be, e.g.,
composed of a multifilament yarn made from a bioabsorbable material
such as PGA, PLA, PCL copolymers or blends, or hyaluronic acid.
[0369] The isolated placental stem cells provided herein can, in
another embodiment, be seeded onto foam scaffolds that may be
composite structures. Such foam scaffolds can be molded into a
useful shape, such as that of a portion of a specific structure,
e.g., a bone containing a lesion. In some embodiments, the
framework is treated, e.g., with 0.1M acetic acid followed by
incubation in polylysine, PBS, and/or collagen, prior to
inoculation of the cells in order to enhance cell attachment.
External surfaces of a matrix may be modified to improve the
attachment or growth of cells and differentiation of tissue, such
as by plasma-coating the matrix, or addition of one or more
proteins (e.g., collagens, elastic fibers, reticular fibers),
glycoproteins, glycosaminoglycans (e.g., heparin sulfate,
chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate,
keratin sulfate, etc.), a cellular matrix, and/or other materials
such as, but not limited to, gelatin, alginates, agar, agarose, and
plant gums, and the like.
[0370] In some embodiments, the scaffold comprises, or is treated
with, materials that render it non-thrombogenic. These treatments
and materials may also promote and sustain endothelial growth,
migration, and extracellular matrix deposition. Examples of these
materials and treatments include but are not limited to natural
materials such as basement membrane proteins such as laminin and
Type IV collagen, synthetic materials such as EPTFE, and segmented
polyurethaneurea silicones, such as PURSPAN.TM. (The Polymer
Technology Group, Inc., Berkeley, Calif.). The scaffold can also
comprise anti-thrombotic agents such as heparin; the scaffolds can
also be treated to alter the surface charge (e.g., coating with
plasma) prior to seeding with isolated placental stem cells.
[0371] The placental stem cells provided herein can also be seeded
onto, or contacted with, a physiologically-acceptable ceramic
material including, but not limited to, mono-, di-, tri-,
alpha-tri-, beta-tri-, and tetra-calcium phosphate, hydroxyapatite,
fluoroapatites, calcium sulfates, calcium fluorides, calcium
oxides, calcium carbonates, magnesium calcium phosphates,
biologically active glasses such as BIOGLASS.RTM., and mixtures
thereof. Porous biocompatible ceramic materials currently
commercially available include SURGIBONE.RTM. (CanMedica Corp.,
Canada), ENDOBON.RTM. (Merck Biomaterial France, France),
CEROS.RTM. (Mathys, AG, Bettlach, Switzerland), and mineralized
collagen bone grafting products such as HEALOS.TM. (DePuy, Inc.,
Raynham, Mass.) and VITOSS.RTM., RHAKOSS.TM., and CORTOSS.RTM.
(Orthovita, Malvern, Pa.). The framework can be a mixture, blend or
composite of natural and/or synthetic materials.
[0372] In one embodiment, the isolated placental stem cells are
seeded onto, or contacted with, a suitable scaffold at about
0.5.times.10.sup.6 to about 8.times.10.sup.6 cells/mL.
5.7 Immortalized Placental Stem Cell Lines
[0373] The placental stem cells useful in the treatment of a
bone-related cancer, suppression of bone related cancer cell
proliferation, or suppression of osteoclast progenitor maturation
can be conditionally immortalized by transfection with any suitable
vector containing a growth-promoting gene, that is, a gene encoding
a protein that, under appropriate conditions, promotes growth of
the transfected cell, such that the production and/or activity of
the growth-promoting protein is regulatable by an external factor.
In a preferred embodiment the growth-promoting gene is an oncogene
such as, but not limited to, v-myc, N-myc, c-myc, p53, SV40 large T
antigen, polyoma large T antigen, E1a adenovirus or E7 protein of
human papillomavirus.
[0374] External regulation of the growth-promoting protein can be
achieved by placing the growth-promoting gene under the control of
an externally-regulatable promoter, e.g., a promoter the activity
of which can be controlled by, for example, modifying the
temperature of the transfected cells or the composition of the
medium in contact with the cells. in one embodiment, a tetracycline
(tet)-controlled gene expression system can be employed (see Gossen
et al., Proc. Natl. Acad. Sci. USA 89:5547-5551, 1992; Hoshimaru et
al., Proc. Natl. Acad. Sci. USA 93:1518-1523, 1996). In the absence
of tet, a tet-controlled transactivator (tTA) within this vector
strongly activates transcription from ph.sub.CMV*-1, a minimal
promoter from human cytomegalovirus fused to tet operator
sequences. tTA is a fusion protein of the repressor (tetR) of the
transposon-10-derived tet resistance operon of Escherichia coli and
the acidic domain of VP16 of herpes simplex virus. Low, non-toxic
concentrations of tet (e.g., 0.01-1.0 .mu.g/mL) almost completely
abolish transactivation by tTA.
[0375] In one embodiment, the vector further contains a gene
encoding a selectable marker, e.g., a protein that confers drug
resistance. The bacterial neomycin resistance gene (neo.sup.R) is
one such marker that may be employed within the present methods.
Cells carrying neo.sup.R may be selected by means known to those of
ordinary skill in the art, such as the addition of, e.g., 100-200
.mu.g/mL G418 to the growth medium.
[0376] Transfection can be achieved by any of a variety of means
known to those of ordinary skill in the art including, but not
limited to, retroviral infection. In general, a cell culture may be
transfected by incubation with a mixture of conditioned medium
collected from the producer cell line for the vector and DMEM/F12
containing N2 supplements. For example, a placental stem cell
culture prepared as described above may be infected after, e.g.,
five days in vitro by incubation for about 20 hours in one volume
of conditioned medium and two volumes of DMEM/F12 containing N2
supplements. Transfected cells carrying a selectable marker may
then be selected as described above.
[0377] Following transfection, the cells are passaged onto a
surface that permits proliferation, e.g., allows at least 30% of
the cells to double in a 24 hour period. Preferably, the substrate
is a polyomithine/laminin substrate, consisting of tissue culture
plastic coated with polyornithine (10 .mu.g/mL) and/or laminin (10
.mu.g/mL), a polylysine/laminin substrate or a surface treated with
fibronectin. Cultures are then fed every 3-4 days with growth
medium, which may or may not be supplemented with one or more
proliferation-enhancing factors. Proliferation-enhancing factors
may be added to the growth medium when cultures are less than 50%
confluent.
[0378] The conditionally-immortalized placental stem cell lines can
be passaged using standard techniques, such as by trypsinization,
when 80-95% confluent. Up to approximately the twentieth passage,
it is, in some embodiments, beneficial to maintain selection (by,
for example, the addition of G418 for cells containing a neomycin
resistance gene). Cells may also be frozen in liquid nitrogen for
long-term storage.
[0379] Clonal cell lines can be isolated from a
conditionally-immortalized human placental stem cell line prepared
as described above. In general, such clonal cell lines may be
isolated using standard techniques, such as by limit dilution or
using cloning rings, and expanded. Clonal cell lines may generally
be fed and passaged as described above.
[0380] Conditionally-immortalized human placental stem cell lines,
which may, but need not, be clonal, may generally be induced to
differentiate by suppressing the production and/or activity of the
growth-promoting protein under culture conditions that facilitate
differentiation. For example, if the gene encoding the
growth-promoting protein is under the control of an
externally-regulatable promoter, the conditions, e.g., temperature
or composition of medium, may be modified to suppress transcription
of the growth-promoting gene. For the tetracycline-controlled gene
expression system discussed above, differentiation can be achieved
by the addition of tetracycline to suppress transcription of the
growth-promoting gene. In general, 1 .mu.g/mL tetracycline for 4-5
days is sufficient to initiate differentiation. To promote further
differentiation, additional agents may be included in the growth
medium.
[0381] BM-MSCs may also be immortalized using any of the above
methods.
[0382] 5.8 Kits
[0383] In another aspect, provided herein are kits, suitable for
the treatment of an individual who has a bone-related cancer, e.g.,
multiple myeloma or chondrosarcoma, or one of the other
bone-related cancers listed elsewhere herein, comprising, in a
container separate from remaining kit contents, tissue culture
plastic placental stem cells, e.g., the isolated placental stem
cells described in Section 5.2.2, above, and/or isolated bone
marrow-derived mesenchymal stem cells, and instructions for use.
Preferably, the placental stem cells and/or BM-MSCs are provided in
a pharmaceutically-acceptable solution, e.g., a solution suitable
for intralesional administration or a solution suitable for
intravenous administration.
[0384] In certain embodiments, the kits comprise one or more
components that facilitate delivery of the placental stem cells
and/or BM-MSCs to the individual. For example, in certain
embodiments, the kit comprises components that facilitate
intralesional delivery of the cells to the individual. In such
embodiments, the kit can comprise, e.g., syringes and needles
suitable for delivery of cells to the individual, and the like. In
such embodiments, the placental stem cells may be contained in the
kit in a bag, or in one or more vials. In certain other
embodiments, the kit comprises components that facilitate
intravenous or intra-arterial delivery of the placental cells to
the individual. In such embodiments, the placental stem cells may
be contained, e.g., within a bottle or bag (for example, a blood
bag or similar bag able to contain up to about 1.5 L solution
comprising the cells), and the kit additionally comprises tubing
and needles suitable for the delivery of cells to the
individual.
[0385] Additionally, the kit may comprise one or more compounds
that reduce pain or inflammation in the individual (e.g., an
analgesic, steroidal or non-steroidal anti-inflammatory compound,
or the like. The kit may also comprise an antibacterial or
antiviral compound (e.g., one or more antibiotics), a compound to
reduce anxiety in the individual (e.g., alaprazolam), a compound
that reduces an immune response in the individual (e.g.,
cyclosporine A), an antihistamine (diphenhydramine, loratadine,
desloratadine, quetiapine, fexofenadine, cetirizine, promethazine,
chlorepheniramine, levocetirizine, cimetidine, famotidine,
ranitidine, nizatidine, roxatidine, lafutidine, or the like).
[0386] Additionally, the kit can comprise disposables, e.g.,
sterile wipes, disposable paper goods, gloves, or the like, which
facilitate preparation of the individual for delivery, or which
reduce the likelihood of infection in the individual as a result of
the administration of the placental stem cells.
6. EXAMPLES
6.1 Example 1
Placental Stem Cells Promote Bone Formation In Vivo
[0387] This Example demonstrates the ability of isolated tissue
culture plastic-adherent placental stem cells to promote bone
formation.
[0388] Placental stem cells were obtained as follows. Briefly,
placental tissue measuring approximately 1.times.2.times.1 cm was
obtained and minced into approximately 1 mm.sup.3 pieces. These
pieces were digested with collagenase IA (2 mg/ml, Sigma) for 30
minutes, followed by digestion with trypsin-EDTA (0.25%, GIBCO BRL)
for 10 minutes, at 37.degree. C. in a water bath. The resulting
solution was centrifuged at 400 g for 10 minutes at room
temperature, followed by removal of the digestion solution. The
pellet was resuspended to approximately 10 volumes with PBS, and
centrifuged at 400 g for 10 minutes at room temperature. The
tissue/cell pellet was resuspended in 130 mL culture medium, and
the cells were seeded at 13 ml per fibronectin-coated T-75 flask.
Cells were incubated at 37.degree. C. with a humidified atmosphere
with 5% CO.sub.2. Cells used in the studies described herein, and
in following Examples, were cultured to passage 6 before use. Such
isolated placental stem cells are generally CD34.sup.-, CD10.sup.+,
CD105.sup.+, and CD200.sup.+. Examination with antibodies to CD44
and CD90 further showed the cells to be CD34.sup.-, CD10.sup.+,
CD44.sup.+, CD90.sup.+, CD105.sup.+, and CD200.sup.+.
[0389] Rats used in this study were approximately 6 weeks old at
the time of the study, and sixteen rats were assigned to each
group. Bilateral cranial defects (left and right; approximately 3
mm.times.5 mm) were created in 96 male Hsd:RH-Foxn.sup.rnu athymic
rats (Charles River, Wilmington, Mass.). Briefly, in the central
cranial area between the ears a transverse skin incision was made,
and a tissue expander was placed into the central region of the
rostral margin of the incision (skin flap). The expander opened the
incision and exposed the cranium. The periosteum was removed from
the parietal bones after the incision was made. The defect sites
were marked, and a Dremel drill at a medium speed was used to
gently carve out the margin of both defects, approximately 3 mm by
5 mm in area, in each parietal bone. The edges of the defect were
checked and gently smoothed using forceps if needed. Once cleaned
and cleared of excess fluid, the defect was treated
intralesionally, as described below. The dermis was then pulled
back over the cranium and the dermal incision closed using
sutures.
[0390] The treatment groups were as follows. One defect per rat was
repaired with HEALOS.RTM. (sponge-like biomimetic matrix comprising
cross-linked collagen and hydroxyapatite; DuPuy Spine, Inc.,
Raynham, Mass.) seeded with placental stem cells (5.times.10.sup.6
cells in 500 .mu.L), bone marrow-derived mesenchymal stem cells
(BM-MSCs; obtained from fresh bone marrow aspirate (AllCells,
Emeryville, Calif.)) (5.times.10.sup.6 cells in 500 .mu.L),
HEALOS.RTM. alone as a negative control, or HEALOS.RTM.
supplemented with bone morphogenetic protein 2 (BMP-2) (5 .mu.g per
explant) as a positive control. In other negative control rats, the
defect was not repaired. The remaining defect in each rat was
repaired using HEALOS.RTM. alone.
[0391] Three weeks after implantation, rats receiving
HEALOS.RTM.+BMP-2, HEALOS.RTM.+placental stem cells, and
HEALOS.RTM.+BM-MSCs all showed approximately the same level of
healing, and significantly greater healing of the cranial defect
than rats receiving HEALOS.RTM. alone, or receiving no repair. See
FIG. 1.
[0392] Thus, PDACs have the capacity to promote the healing of bone
lesions, one symptom of multiple myeloma progression.
6.2 Example 2
Placental Stem Cells Suppress Osteoclast Maturation
[0393] This Example demonstrates that tissue culture
plastic-adherent placental stem cells (PDACs) inhibit maturation of
osteoclast precursors. Suppression of osteoclast precursors would
provide a benefit to multiple myeloma patients suffering from bone
lesions (and attendant symptoms) caused by myeloma-induced
osteoclast overproduction.
[0394] Human osteoclast precursors, obtained by enriching
CD14.sup.+ cells from peripheral blood mononuclear cells (PBMCs)
using an EASYSEP.RTM. Human CD14 Positive Selection Kit (Cat
#18058), were prepared in 24 well plates and cultured in the medium
.alpha.MEM supplemented with macrophage colony stimulating factor
(M-CSF) and Receptor Activator for Nuclear Factor .kappa. B Ligand
(RANKL; see Yaccoby et al., Cancer Research 64(6):2016-2023
(2004)). Placental stem cells, isolated as described in Example 1,
or fetal mesenchymal stem cells (MSC), were cultured with
osteoclast precursors in noncontact conditions by seeding the cells
on 1 .mu.m TRANSWELLs.RTM. (COSTAR.RTM.; CORNING.RTM., New York)
(10,000 cells/TRANSWELL.RTM.) and coculturing the placental stem
cells or MSCs with the osteoclast precursors for 5-6 days. At the
end of the culturing, the TRANSWELLs.RTM. were removed and
osteoclast precursors and/or osteoclasts were examined for evidence
of apoptosis by staining for annexin V and propidium iodide (PI)
using an annexin V/PI kit (Caltag Labs., Burlingame, Calif.).
Annexin V binds phosphatidylserine, which is transported from the
inner leaflet of the plasma membrane to the outer leaflet during
apoptosis; cells with intact plasma membranes exclude propidium
iodide. Thus, cells that are positive for annexin staining but not
PI staining are early apoptotic cells; cells positive for both
annexin staining and PI staining are late apoptotic cells.
[0395] The placental stem cells were found to significantly induce
apoptosis and reduce viability of osteoclast precursors compared to
controls in which multiple myeloma cells were grown without
placental stem cells, as shown by increased Annexin V and propidium
iodide staining.
[0396] Cells in the TRANSWELLs.RTM. were fixed with formalin and
stained for tartrate resistant acid phosphatase (TRAP; an
osteoclast marker). The numbers of multinucleated TRAP-expressing
osteoclasts were counted in each well. Placental stem cells,
isolated as described in Example 1, inhibited differentiation of
osteoclasts, as indicated by a lessening of TRAP staining in
histological sections, and by a significant (p<0.05) decrease in
the number of TRAP-positive osteoclasts (FIG. 2).
[0397] Thus, placental stem cells can not only repair bone lesions,
but can reduce the number and activity of osteoclasts that would
create or contribute to such lesions.
6.3 Example 3
Placental Stem Cells Inhibit the Growth of Multiple Myeloma
Cells
[0398] This Example demonstrates that tissue culture
plastic-adherent placental stem cells (PDACs) are able to suppress
the proliferation of multiple myeloma cells both in vitro and in
vivo.
[0399] 6.3.1 PDAC Suppression of Multiple Myeloma Cell
Proliferation In Vitro
[0400] Human multiple myeloma cell lines BN, JB, DNC and HLE (see
Li et al., Br. J. Haematol. 138(6):802-11 (2007)), and ARP1 were
established at the Myeloma Institute for Research and Therapy at
the University of Arkansas for Medical Sciences. The multiple
myeloma cell line U266 (Nilsson et al., Clin. Exp. Immunol. 7:477
(1970)) was obtained from the American type Culture Collection.
These cell lines were transfected with a luciferase/GFP lentiviral
construct by established methods (see Li et al., ibid.) to
facilitate tracking and analysis of tumor growth in the presence of
placental stem cells in cell-to-cell contact conditions. BN, JB and
DNC are stroma-dependent cell lines. Placental stem cells, fetal
MSC (FB-MSC), and MSC generated from bone marrow of patients with
multiple myeloma (Pt-MSC) were cultured in 96 well plates at about
10,000 cells/well). Multiple myeloma cells (10,000 cells/well) were
co-cultured with placental stem cells or MSCs for a week in RPMI
media supplemented with 10% FBS and antibiotics. At the end of
culture, growth of the multiple myeloma cells was determinable by
measurement of luciferase activity.
[0401] The results of this study are summarized in FIG. 3, showing
fold growth of multiple myeloma cells in the presence of placental
stem cells compared to growth in the presence of FB-MSC and
Pt-MSCs. Multiple myeloma cell growth in the presence of placental
stem cells varied depending on the particular cell line, but growth
for each cell line was significantly lower for cell lines
co-cultured with placental stem cells than for cell lines
co-cultured with fetal MSCs or patient MSCs.
[0402] MSCs, and placental stem cells isolated as described in
Example 1, were also induced to differentiate into osteoblasts
through incubation with DMEM/10% fetal bovine serum (FBS)
conditioned by osteoblast osteogenesis factors (e.g., ascorbic
acid, beta glycerophosphate and dexamethasone) for approximately
3-3.5 weeks (see Yaccoby et al., Haematologica 91(2): 192-199
(2006)). For testing effects on growth of multiple myeloma cell
lines, the plates were washed with PBS to remove osteoblastic
factors. Multiple myeloma cell growth in co-culture with
osteoblasts generated from FB-MSC or Pt-MSC was reduced as compared
to growth of multiple myeloma cells in co-culture with FB-MSC or
Pt-MSC. Differentiation of placental stem cells into osteoblasts
had no effect on growth of, or slightly reduced the growth of,
multiple myeloma cell lines as compared to co-culture with
placental stem cells. The experiment was repeated 3 times for most
of the cell lines.
[0403] To study the possible effect of cell-cell contact, cells
were cultured in a system in which cell-cell contact is prevented.
In particular, MSCs (FB-MSCs or BM-MSCs), or placental stem cells
isolated as described in Example 1, were cultured in a
TRANSWELL.RTM. system on the back side of 24-well TRANSWELL.RTM.
membranes, while multiple myeloma plasma cells were cultured on the
upper chamber of the TRANSWELL.RTM.. See FIG. 4. Primary multiple
myeloma cells from 6 patients were isolated using
CD138-immunomagnetic bead separation and co-cultured at 500,000
multiple myeloma cells/well with MSCs or placental stem cells
(100,000 cells/TRANSWELL.RTM.) for 6-10 days. CD138 is a marker of
plasma cells.
[0404] The effects of co-cultures on multiple myeloma cell
viability were determined by trypan blue exclusion and by an MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
assay. The MTT assay is a colorimetric assay for measuring the
activity of enzymes that reduce MTT to formazan, giving a purple
color. This reduction takes place only when mitochondrial reductase
enzymes are active, and therefore conversion is often used as a
measure of viable cells. In one experiment, multiple myeloma cells
were also subjected to annexin V/PI flow analysis, as described in
Example 2, above. Survival of primary multiple myeloma cells was
reduced in the TRANSWELL.RTM. co-culture with placental stem cells
as compared to survival in TRANSWELL.RTM. co-culture with fetal
MSC, for myeloma cells from most patients tested. See FIG. 5.
[0405] 6.3.2 Placental Stem Cell Suppression of Multiple Myeloma
Cell Proliferation and Increase in Bone Mass In Vivo
[0406] The pLEGFP retroviral vector containing an Enhanced Green
Fluorescent Protein (EGFP) coding sequence (Clontech, Palo Alto,
Calif., USA) was used to transiently transfect the packaging cell
line Phoenix Eco (ecotropic) using SuperFect (QIAGEN Inc.,
Valencia, Calif., USA). EGFP is a red-shifted variant of wild-type
Aequorea victoria green fluorescent protein that has been optimized
for brighter fluorescence and higher expression in mammalian cells.
Supernatants containing retroviral particles were collected 24-48
hours after transfection. To facilitate tracking, placental stem
cells were infected with the retroviral particles in the presence
of 8 .mu.g/ml polybrene for 12 hours at which time the media were
replaced with fresh culture medium. In some experiments, cells were
exposed to the supernatants containing the viral particles once
more before being selected by culturing them in the presence of
200-400 .mu.g/ml of G418 for 2-3 weeks.
[0407] As an alternative to using human bone tissue in a SCID-hu
model of primary human myeloma, a system in which rabbit bones were
implanted into SCID mice (SCID-rab mice), followed by introduction
of myeloma cells directly into the implanted bone, was used.
Myelomatous SCID-rab mice were constructed as previously described.
See Yata, K. and Yaccoby, S., et al, Leukemia 2004; 18:1891-1897.
CB.17/Icr-SCID mice (6-8-week old) were obtained from Harlan
Sprague Dawley (Indianapolis, Ind., USA) and pregnant New Zealand
rabbits from Myrtle Rabbitry (Thompson Station, Tenn., USA). The
3-4-week-old rabbits were deeply anesthetized with a high dose of
pentobarbital sodium and killed by cervical dislocation. The rabbit
femora and tibiae were cut into two pieces, with the proximal and
distal ends kept closed, while the vertebrae were cut into small
fragments (1.times.2 cm.sup.2).
[0408] For bone implantation, the right or left side of the SCID
mouse was rinsed with alcohol and blotted with sterile gauze. The
rabbit bone was inserted subcutaneously through a small (5 mm)
incision. The incision was then closed with sterile surgical
staples, and engraftment of the bones was allowed to take place for
6-8 weeks. In some experimental mice, two bones were simultaneously
implanted contralaterally in the same mouse. For each experiment,
10-50.times.10.sup.6 unseparated human patient-derived myeloma bone
marrow cells containing 17+/-8% plasma cells (PCs) or
3.3+/-1.6.times.10.sup.6 PCs in 50 .mu.l of phosphate-buffered
saline (PBS) were injected directly into the implanted rabbit bone.
At least two mice were used for each experiment. Mice were
periodically bled from the tail vein to measure changes in levels
of circulating human immunoglobulin (Ig) of the M-protein
isotype.
[0409] Establishment of myeloma growth was demonstrated by
increased levels of human monoclonal immunoglobulins (hIg) in mouse
sera, as seen by ELISA, and by radiographic evaluation of lytic
bone lesions. 5.times.10.sup.5 EGFP-expressing placental stem
cells, isolated as described in Example 1 prior to transformation,
were collected with the use of trypsin-EDTA and resuspended in 50
.mu.l PBS. The placental stem cells were injected directly into the
implanted bones in the SCID-rab mice. Experiments were continued
for 8-16 weeks post-injection. Changes in the bone mineral density
(BMD) of the implanted bones were determined using a PIXImus DEXA
densitometer (GE Medical Systems LUNAR, Madison, Wis.). The effect
of the placental stem cells on multiple myeloma cell proliferation
was determined by tracking the levels of human monoclonal
immunoglobulins (hIg) in mouse sera, as seen by ELISA.
[0410] Multiple myeloma cells from one patient (designated Patient
1) were found to grow in SCID-rab/SCID-hu mice, and could be
passaged to newly constructed SCID-rab/SCID-hu mice; however, they
were not able to grow independently or on stromal layer in vitro.
Six SCID-rab mice successfully engrafted with the multiple myeloma
cells were administered transfected placental stem cells
intralesionally, and six were administered a control (phosphate
buffered saline).
[0411] Growth of multiple myeloma cells was found to be
significantly inhibited at two and four weeks after injection of
placental stem cells, but not PBS, by detection of human monoclonal
immunoglobulins (hTg) in sera from the mice, as seen by ELISA
(p<0.00.sup.7; FIG. 6). Bioluminescence analysis in live animals
detected luciferase-expressing placental stem cells in these mice;
bioluminescence intensity at 14 days was reduced in all mice
administered placental stem cells (Table 1B). Further, X-rays taken
before administration of placental stem cells and 4 weeks after
treatment revealed increased bone mass following placental stem
cell injection into myelomatous bones, but reduced bone mass in
control PBS-treated bones (FIG. 7).
TABLE-US-00002 TABLE 1B Results of live bioluminescence
assays--numbers of counts per animal. Mouse 1 2 3 4 5 3 days 5.20
.times. 10.sup.6 6.50 .times. 10.sup.5 8.3 .times. 10.sup.6 2.30
.times. 10.sup.7 3.30 .times. 10.sup.6 14 days 2.68 .times.
10.sup.4 ND 2.80 .times. 10.sup.5 1.10 .times. 10.sup.6 2.00
.times. 10.sup.4
[0412] To test the effect of placental stem cells on the bone mass
density of nonmyelomatous bone, placental stem cells
(1.times.10.sup.6 cells/mouse) or vehicle were injected directly
into the implanted nonmyelomatous bones in SCID-rab mice. Injection
of the placental stem cells, but not vehicle, resulted in marked
increased of BMD of the implanted bone from pretreatment levels.
These data indicate that direct injection of placental stem cells
into myelomatous or nonmyelomatous bone resulted in increased local
bone mass, and that increased bone formation by placental stem
cells was associated with reduced myeloma burden.
[0413] Next we utilized myeloma cells from a second patient,
designated Patient 2, which are molecularly classified as a high
risk, MMSET subtype (associated with aggressive multiple myeloma
and a poor prognosis) and express moderate level of DKK1. Patient 2
myeloma cells did not grow in culture but were successfully
passaged in the SCID-rab model described above. Treatment was
initiated when myeloma growth was well established and osteolytic
lesions were evident. Placental stem cells were injected
intralesionally into the implanted bone (0.1-1.times.10.sup.6
placental stem cells/bone, 7 hosts/group) or subcutaneously using a
HyStem-C hydrogel carrier (5.times.10.sup.6 placental stem
cells/mouse, 6 mice). Analyzed 4 weeks after treatment,
intralesional injection of 0.5 and 1.times.10.sup.6 placental stem
cells resulted in increased BMD of the implanted bones from
pretreatment levels (p<0.01) or prevention of bone loss compared
to control group (p<0.02) (FIG. 8). Increased bone mass by
injection of 1.times.10.sup.6 placental stem cells was additionally
associated with reduced myeloma growth at near significant level
(p<0.08, FIG. 9).
[0414] The effect of placental stem cells and human fetal MSCs on
myeloma bone disease and tumor growth was also compared. Cells were
injected (1.times.10.sup.6 cells/mouse) directly into the implanted
bones of SCID-rab mice engrafted with Patient 2 myeloma cells (7
hosts/group). Placental stem cells and MSC treatment resulted in
increased BMD of the implanted bone as compared to pretreatment
level, however the effect of placental stem cells was more profound
(FIG. 10). Both placental stem cells and MSC treatment
significantly inhibited growth of patient #2 myeloma cells in the
SCID-rab model (FIG. 11). These results suggest that, while both
MSCs and placental stem cells are effective in increasing BMD of
myeloma-affected bones, placental stem cells have higher bone
anabolic potential than fetal MSCs.
[0415] Thus, this Example demonstrates that placental stem cells
can significantly reduce the viability of multiple myeloma cells,
particularly when administered intralesionally into myelomatous
individuals. Placental stem cells also reduce the viability of
multiple myeloma cells in vitro in conditions allowing cell-cell
contact, and in conditions preventing cell-cell contact. Coupled
with the ability of placental stem cells to repair bone, e.g., bone
lesions that are symptomatic of multiple myeloma, and to inhibit
osteoclast maturation, a major cause of the development of multiple
myeloma-related bone lesions, these results indicate that placental
stem cells can be a useful anti-multiple myeloma therapeutic.
6.4 Example 4
Placental Stem Cells Promote Multiple Myeloma Cell Cycle Arrest
[0416] This Example demonstrates that tissue culture
plastic-adherent placental stem cells (PDACs) suppress the growth
of multiple myeloma cells.
[0417] 6.4.1 Placental Stem Cells Suppress Multiple Myeloma Cell
Proliferation
[0418] To study the effect of placental stem cells on the growth of
multiple myeloma cells, placental stem cells, isolated as described
in Example 1, were co-cultured with 6 multiple myeloma cell lines
(MMCLs), designated U-266 (American Type Culture Collection (ATCC)
Catalog No. TIB-196), RPMI-8226 (ATCC Catalog No. CCL-155), L-363
(Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ)
Catalog No. ACC49), H929 (Gazdar, Blood 67:1542-1549 (1986)), LP-1
(DSMZ Catalog No. ACC41) and OPM-2 (DSMZ Catalog No. ACC-50). The
four multiple myeloma cell lines selected for these experiments
represent the heterogeneity of multiple myeloma cells, as seen by
differences in the production of immunoglobulins (see Table 2) and
differences in cellular marker expression (see Tables 3A-3C).
TABLE-US-00003 TABLE 2 Production of immunoglobulin types by
multiple myeloma cell lines Cell line IgA IgG Kappa chain Lambda
chain H-929 0.0 0.1 98.9 0.9 OPM-2 0.6 0.1 3.5 97.3 RPMI-8226 0.0
0.0 0.9 85.8 U266 0.0 0.1 1.2 99.5
Table 3A-3C: Cellular markers expressed by multiple myeloma cell
lines (expressed as percentage of cells expressing a marker).
TABLE-US-00004 TABLE 3A Sample CD38.sup.+ CD56.sup.+ CD19.sup.+
CD45.sup.+ CD11b.sup.+ CD40.sup.+ CD138.sup.+ H929 97.7 98.6 0.74
2.3 7.56 0.18 72.9 OPM-2 13.2 21.2 0.062 0.21 56.9 0.14 7.33 RPMI-
96.7 64.4 0.093 0.3 29.4 18.3 16.7 8226 U266 4.39 3.39 0.64 91.5
2.51 0.84 16.6
TABLE-US-00005 TABLE 3B CXCR4 CD49e Cell line CD58.sup.+
(CD184).sup.+ CD44.sup.+ (VLA5).sup.+ CD117.sup.+ CD20.sup.+ H929
99.8 0.65 99.7 51.1 27.1 0.54 OPM-2 29 8.14 21.2 0.35 0.53 0.31
RPMI- 95.8 4.2 6.48 67.6 7.73 0.057 8226 U266 100 83.5 48.3 0.55
0.28 1.21
TABLE-US-00006 TABLE 3C CD49d Cell line CD33.sup.+ CD54.sup.+
CD28.sup.+ (VLA4).sup.1 CD106.sup.+ CD11a.sup.+ H929 0.76 99.2 95.3
99.6 0.2 12.4 OPM-2 0.2 1.01 2.44 0.059 0.2 0.1 RPMI- 26.2 99.9
99.7 2.75 6.39 46.5 8226 U266 6.83 100 99.9 99.5 0.58 5.77
[0419] Passage 6 placental stem cells, isolated as described in
Example 1, were thawed with DMEM+10% fetal calf serum (FCS).
5.times.10.sup.4 placental stem cells were plated in 24-well plate
per well. After the placental stem cells grew to confluency, with a
one time medium change, 5.times.10.sup.4 MMCL cells per well were
plated on the top of the placental stem cells, and incubated at
37.degree. C. under 5% CO.sub.2 for 4-5 days. MMCL cells were
harvested on days 1, 2 and 5 of culture for further analysis. Cells
were counted using the EASYCOUNT.TM. System (Immunicon).
[0420] Results indicated that the placental stem cells achieved
significant growth inhibition of multiple myeloma cell lines U266
(p<0.001 at day 5 of co-culture), RPMI-8226 (p<0.03 at day 5
of co-culture), H929 (p<0.003 at day 4 of co-culture), and OPM-2
(p<0.01 at day 5 of co-culture), compared to these multiple
myeloma cells cultured alone. See FIG. 12. In separate experiments,
co-culture of L-363 cells with placental stem cells resulted in
substantial inhibition of growth (p<0.0.sup.6 at day 5 of
co-culture), and co-culture of LP-1 cells with placental stem cells
also resulted in inhibition of growth.
[0421] 6.4.2 Placental Stem Cells Downregulate Multiple Myeloma
Cell Expression of Genes Encoding Proteins that Play Key Roles in
NF-.kappa.B Signaling and B Cell Activation
[0422] To further characterize the growth inhibition of the
placental stem cells on the multiple myeloma cell lines, the
placental stem cells, isolated as described in Example 1, were
co-cultured with U-266, RPMI-8226, OPM-2 and H929 cells for 4 days,
then multiple myeloma cells co-cultured with placental stem cells,
or multiple myeloma cells cultured alone were collected by gentle
pipetting without disturbing placental stem cells followed by RNA
preparation and quantitative real-time PCR (qRT-PCR) analysis.
qRT-PCR was performed using 384-well microfluidic cards
(TAQMAN.RTM. Custom Array, Applied Biosystems), which enable
simultaneous real-time PCR reactions. The cards contained 300 genes
involved in cell cycle regulation, cellular growth and
proliferation, and hormonal immune response, including genes
involved in B cell signaling and NF-.kappa.B signaling. qRT-PCR was
performed using 7900HT Fast Real-Time PCR System (Applied
Biosystems), and data was analyzed using REALTIME STATMINER.RTM.
software.
[0423] Co-culture with the placental stem cells significantly
downregulated genes encoding key components of B cell activation,
including TRAF1 (TNF Receptor Associated Factor 1), TRAF6, and
genes encoding key components of the NF-.kappa.B signaling pathway,
including TIRAP (Toll-Interleukin 1 Receptor TIR domain containing
Adaptor Protein); p65/RelA, and RelB. See Table 4, below.
[0424] DKK1, a protein produced by multiple myeloma cells, inhibits
the activity of osteoblasts and tips the balance between
osteoblasts and osteoclasts in favor of bone resorption. After
co-culture with placental stem cells as above, DKK1 expression in
OPM-2 cells was downregulated as well. See Table 4.
TABLE-US-00007 TABLE 4 Fold change of gene expression in OPM-2
co-cultured with placental stem cells in comparison with OPM-2
alone. Standard deviation was calculated for means of fold change
for 2 replicates. Gene Fold Change STDEV DKK1 0.34 0.09 RELA 0.72
0.03 RELB 0.27 0.08 TIRAP 0.49 0.05 TRAF1 0.44 0.07 TRAF6 0.50 0.12
STDEV: Standard Deviation.
[0425] 6.4.3 Placental Stem Cells Downregulate Multiple Myeloma
Cell Expression of Genes Encoding Cyclins and CDKs, and Upregulate
Genes Encoding CDK Inhibitors
[0426] The effect of the placental stem cells (PDACs), isolated as
described in Example 1, on the expression of cyclins (CCNs) and
cyclin-dependent kinases (CDKs) was analyzed by qRT-PCR using
384-well microfluidic cards containing genes involved in cell cycle
regulation, as described above, and analyzed using Ingenuity
Pathways Analysis (INGENUITY.RTM. Systems, www.ingenuity.com). The
placental stem cells were found to decrease expression in the
multiple myeloma cell lines of genes encoding certain CCNs and
CDKs, and to increase expression of genes for certain CDK
inhibitors in a cell type-specific manner. For example, in multiple
myeloma cell line OPM-2, the CDKs CDK3, CDK5, and CDK7 were
downregulated; in multiple myeloma cell lines RPMI-8226 and U-266,
CDK4 was downregulated. In contrast, in multiple myeloma cell line
OPM-2, CDK inhibitors p16, and p19, and CDK inhibitor 3 were
upregulated; in multiple myeloma cell line RPMT-8226, CDK inhibitor
p19 was upregulated; in multiple myeloma cell line U266 p21 was
upregulated; and in multiple myeloma cell line H929, p19, p21 and
p27 were all upregulated.
[0427] A summary of changes in expression of cell cycle-related
genes is presented below in Tables 5A-5D.
Tables 5A-5D. Fold change of gene expression in multiple myeloma
cells co-cultured with placental stem cells in comparison with
multiple myeloma cells alone for multiple myeloma cell lines OPM-2,
U-266, RPMI-8226, and H929. Standard deviation was calculated for
means of fold change for 2 replicates.
TABLE-US-00008 TABLE 5A OPM-2 Fold Change STDEV CCNB3 0.39 0.07
CCNC 0.63 0.02 CCND1 0.01 0.00 CDK3 0.82 0.05 CDK5 0.82 0.00 CDK7
0.73 0.05 CDKN2A (p16) 1.55 0.26 CDKN2D (p19) 1.61 0.25 CDKN3 4.41
0.27
TABLE-US-00009 TABLE 5B U-266 Fold Change STDEV CCNB1 0.16 0.01
CCNB2 0.16 0.03 CCND1 0.23 0.01 CCND2 0.08 0.00 CDK4 0.38 0.01
CDKN1A (p21) 1.45 0.14 E2F3 0.80 0.02 E2F4 0.44 0.00 E2F5 0.30 0.00
E2F6 0.22 0.00
TABLE-US-00010 TABLE 5C RPMI-8226 Fold Change STDEV CCNB1 0.61 0.03
CCNB2 0.82 0.14 CCND1 0.64 0.06 CCND2 0.68 0.05 CDK2AP1 0.56 0.02
CDK4 0.56 0.03 CDKN2D (p19) 1.41 0.13 E2F3 0.67 0.02 E2F4 0.75 0.10
E2F5 0.52 0.02 E2F6 0.63 0.07
TABLE-US-00011 TABLE 5D H929 Fold Change STDEV CCNB1 0.52 0.03
CCNB2 0.71 0.07 CCNB3 0.54 0.37 CCNC 0.64 0.02 CDK10 0.40 0.00 CDK3
0.84 0.06 CDK5 0.83 0.09 CDK9 0.82 0.03 CDKN1A (p21) 3.71 0.99
CDKN1B (p27) 1.12 0.18 CDKN2D (p19) 1.18 0.08
[0428] Placental stem cells, isolated as described in Example 1,
were also found to decrease expression in the multiple myeloma cell
lines of genes encoding E2F family members 3, 4, 5 and 6 (proteins
that play a major role in the transition from G.sub.1 to S phase)
and phosphorylated Rb (Retinoblastoma protein). This finding is
significant because in the hypophosphorylated state, Rb acts as
tumor suppressor by inhibiting the factors of E2F family;
phosphorylated Rb, however, has little inhibitory function on cell
cycle progression.
[0429] To further investigate the effect of the placental stem
cells on multiple myeloma cell proliferation, the phosphorylation
state of Retinoblastoma protein (Rb) was analyzed by flow cytometry
using the J146-35 monoclonal antibody (BD Pharmingen, Cat #558549)
and the 3112-906 monoclonal antibody (Cat #558549, BD). Antibody
J146-35 recognizes Rb phosphorylated at serine 780 (pS780), which
affects Rb binding to E2F, and antibody J112-906 recognizes Rb
phosphorylated at serines 807 and 811 (pS807/pS811), which regulate
c-Abl binding and cell cycle progression. H929, LP1 and OPM2
co-cultured with the placental stem cells showed decreased RB
phosphorylation at pS780, and at pS807/pS811, relative to cells
cultured alone. See FIGS. 13A-13C.
[0430] The effect of the placental stem cells on the proliferation
of multiple myeloma cell lines was further assayed using
fluorescent the dyes BrdU and 7-AAD using an APC BrdU flow kit (Cat
#552598, BD biosciences). Co-culture with the placental stem cells
resulted in an increased percentage of multiple myeloma cells in
G0/G1 phase, and a decreased percentage of such cells in S phase,
for cell lines RPMI-8226, OPM-2 and U266, as compared to the
multiple myeloma cells cultured alone. See Table 6.
TABLE-US-00012 TABLE 6 Cell analysis from MMCL: placental stem cell
co-culture G0/G1 S phase H929 63.5 27.0 H929 + Placental Stem Cells
53.9 28.9 RPMI-8226 45.3 11.6 RPMI-8226 + Placental Stem Cells 64.9
9.9 OPM2 49.2 42.8 OPM2 + Placental Stem Cells 78.4 11.5 U266 43.0
19.2 U266 + Placental Stem Cells 65.9 9.3
[0431] Multiple myeloma cells secrete aberrantly high levels of
immunoglobulins. To study the effect of the placental stem cells on
immunoglobulin production by multiple myeloma cell lines, surface
or intracellular immunoglobulin production by MMCLs co-cultured
with the placental stem cells, or MMCLs cultured alone, was
analyzed by flow cytomctry. Decreased immunoglobulin production was
observed from multiple myeloma cell lines H929, OPM2 and LP1 when
co-cultured with the placental stem cells as compared to the
multiple myeloma cells cultured alone. For example, co-cultured
H929 cells showed decreased Kappa (.kappa.) immunoglobulin
production; co-cultured OPM2 cells showed decreased Lambda
(.lamda.) production; and co-cultured LP1 showed decreased surface
and intracellular Lambda and IgG and intracellular Kappa
production, compared to the cells when cultured alone. See Table
7.
TABLE-US-00013 TABLE 7 Change of geometric mean of Ig production in
MMCL: placental stem cell co-culture system Cell Line Ig Location
Day 1 Day 2 Day 4 H929 Kappa -- N/A -81.0% OPM2 Lambda -- -4.2%
-52.2% LP1 Lambda surface -9.6% -17.9% -48.7% Lambda intracellular
-31.6% -16.5% -13.5% LP1 IgG surface -7.3% -10.0% -36.4% IgG
intracellular -20.0% -21.3% -13.7% LP1 Kappa intracellular -15.1%
-11.4% -13.4% Ig: Immunoglobulin type
[0432] The results above demonstrating that placental stem cells
reduce the proliferation of multiple myeloma cells were not due to
a general effect of placental stem cell co-culture with other cell
types, but were specific to multiple myeloma cells. For example,
the placental stem cells were found to augment expansion of
CD34.sup.+ hematopoietic cells when co-cultured at three different
ratios (10:1, 1:1, and 1:10) over 7 days.
[0433] Thus, the above studies demonstrate that the placental stem
cells, when co-cultured with multiple myeloma cell lines, reduce
the growth rate of the multiple myeloma cells, downregulate
expression of multiple myeloma cell line genes encoding cell cycle
proteins needed for progression through the cell cycle, and
upregulate genes encoding inhibitors of cell cycle progression. As
such, the placental stem cells would be useful in the reduction of
proliferation of multiple myeloma cells in vivo.
6.5 Example 5
Use of Placental Stem Cells to Suppress Growth of Chondrosarcoma
Cells
[0434] This Example demonstrates that placental stem cells (PDACs)
suppress the proliferation of chondrosarcoma cells in culture.
[0435] TRANSWELL.RTM. culture: To examine the effects of placental
stem cells, isolated as described in Example 1, on tumor cell
growth in a TRANSWELL.RTM. co-culture system, 1.times.10.sup.4 or
5.times.10.sup.4 PDACs were seeded on the bottom chamber of the
TRANSWELL.RTM. system in 600 .mu.L of growth medium and
1.times.10.sup.4 chondrosarcoma cells (ATCC.RTM. No. CRL-7891; 400
.mu.L in growth medium) were seeded on the top chamber of
TRANSWELLs.RTM. (3 .mu.m in diameter). Chondrosarcoma cells were
cultured alone without placental stem cells as a control. All
TRANSWELL.RTM. co-cultures were set up in 24-well plate, and each
condition was set up in triplicate. After 7 days of culture in cell
culture incubator at 37.degree. C. under 5% CO.sub.2,
chondrosarcoma cells on the top chamber were examined using a Leica
microscope.
[0436] Chondrosarcoma is a cancer characterized by the production
of cartilage matrix around the tumor cells. Consistent with this
symptomology, in the TRANSWELL.RTM. experiment, the chondrosarcoma
cells grew as distinct aggregates, clearly visible under the
microscope, in the absence of placental stem cells. In the presence
of placental stem cells both ratios tested, there were visibly
fewer chondrosarcoma cells, and the cells were characterized by a
complete absence of cell aggregates that characterized the growth
of the tumor cells alone. As such, placental stem cells clearly
inhibited the growth of the chondrosarcoma cells.
6.6 Example 6
Inhibition of Osteoclastogenesis Using Lenalidomide
[0437] This Example demonstrates that the small molecule
lenalidomide (sold under the trade name REVLIMID.RTM.;
3-(4-amino-1-oxo 1,3-dihydro-2H-isoindol-2-yl)
piperidine-2,6-dione) can be used to suppress
osteoclastogenesis.
[0438] Lenalidomide has a profound anti-osteoclastogenic effect at
concentrations around 1 .mu.M, generating a steep decline in the
number of osteoclasts formed (FIG. 14). When osteoclast precursors
cultured with the placental stem cells, isolated as in Example 1,
and 0.1 .mu.M or 1 .mu.M lenalidomide, were compared to osteoclasts
grown with placental stem cells or bone marrow-derived mesenchymal
stem cells (BM-MSC), lenalidomide was found to further decrease the
number of osteoclasts that differentiated from the osteoclast
precursors. FIG. 15. Therefore, there is a possible synergistic or
additive anti-ostcoclastogcnic effect of PDACs and lenalidomide at
concentration of between about 0.1 .mu.M to 1 .mu.M.
[0439] Therefore, both lenalidomide alone and lenalidomide in
combination with placental stem cells are effective at reducing the
number of osteoclast precursors, and therefore should be
therapeutic in reducing the number and/or severity of bone lesions
adjunct to a bone-related cancer, such as multiple myeloma.
EQUIVALENTS
[0440] The present disclosure is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the subject matter provided herein, in addition to
those described, will become apparent to those skilled in the art
from the foregoing description and accompanying figures. Such
modifications are intended to fall within the scope of the appended
claims.
[0441] Various publications, patents and patent applications are
cited herein, the disclosures of which are incorporated by
reference in their entireties.
* * * * *
References