U.S. patent application number 16/657428 was filed with the patent office on 2020-02-13 for cd34+,cd45- placental stem cell-enriched cell populations.
This patent application is currently assigned to CELULARITY, INC.. The applicant listed for this patent is CELULARITY, INC.. Invention is credited to Sascha Dawn Abramson, James W. Edinger, Robert J. Hariri, Kristen S. Labazzo, Marian Pereira, Jia-Lun Wang, Qian Ye.
Application Number | 20200048603 16/657428 |
Document ID | / |
Family ID | 39363987 |
Filed Date | 2020-02-13 |
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United States Patent
Application |
20200048603 |
Kind Code |
A1 |
Edinger; James W. ; et
al. |
February 13, 2020 |
CD34+,CD45- PLACENTAL STEM CELL-ENRICHED CELL POPULATIONS
Abstract
Provided herein are methods and compositions for the production
of hepatocytes from placenta stem cells. Further provided herein is
the use of such hepatocytes in the treatment of, and intervention
in, for example, trauma, inflammation, and degenerative disorders
of the liver. Also provided herein are compositions and methods
relating to combinations of nanofibrous scaffolds and adherent
placental stem cells and methods of using the same in cartilage
repair. Finally, provided herein are compositions and methods
relating to nonadherent, CD34.sup.+CD45.sup.- stem cells from
placenta.
Inventors: |
Edinger; James W.; (Belford,
NJ) ; Hariri; Robert J.; (Florham Park, NJ) ;
Wang; Jia-Lun; (Cherry Hill, NJ) ; Ye; Qian;
(Livingston, NJ) ; Pereira; Marian; (Cranford,
NJ) ; Abramson; Sascha Dawn; (Hillsborough, NJ)
; Labazzo; Kristen S.; (Springfield, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CELULARITY, INC. |
Warren |
NJ |
US |
|
|
Assignee: |
CELULARITY, INC.
Warren
NJ
|
Family ID: |
39363987 |
Appl. No.: |
16/657428 |
Filed: |
October 18, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12030170 |
Feb 12, 2008 |
10494607 |
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16657428 |
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60901066 |
Feb 12, 2007 |
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60901076 |
Feb 12, 2007 |
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60905664 |
Mar 7, 2007 |
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60906064 |
Mar 8, 2007 |
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60966577 |
Aug 28, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2506/03 20130101;
C12N 5/067 20130101; C12N 2501/999 20130101; A61P 31/12 20180101;
C12N 2533/32 20130101; C12N 5/0605 20130101; C12N 2506/02 20130101;
C12N 2533/74 20130101; A61K 2035/128 20130101; C12N 2500/62
20130101; A01K 2267/0337 20130101; C12N 5/0607 20130101; C12N
2533/40 20130101; A61P 19/00 20180101; C12N 2501/15 20130101; A01K
67/0271 20130101; A61P 1/16 20180101; C12N 5/0655 20130101; C12N
2500/30 20130101; C12N 2500/36 20130101 |
International
Class: |
C12N 5/071 20060101
C12N005/071; C12N 5/073 20060101 C12N005/073; C12N 5/077 20060101
C12N005/077; C12N 5/074 20060101 C12N005/074; A01K 67/027 20060101
A01K067/027 |
Claims
1. A method of producing a hepatocyte, comprising contacting a
CD10.sup.+, CD34.sup.-, CD105.sup.+ and CD200.sup.+ placental stem
cell with sodium butyrate under conditions and for a time
sufficient for said stem cell to exhibit a characteristic of a
hepatocyte.
2. The method of claim 1, wherein said characteristic is production
of asialogylcoprotein receptor, alpha-1-antitrypsin, albumin,
cytochrome P450 activity, or the increased production of
cytokeratin 18 relative to an undifferentiated placental stem
cell.
3. The method of claim 1, wherein said culturing comprises
encapsulating said stem sell in alginate-poly-L-lysine.
4. A hepatocyte or hepatocytic cell produced by the method of claim
1.
5. A method of treating a subject having a disease, disorder or
condition associated with liver inflammation, comprising
introducing the hepatocyte or hepatocytic cell of claim 4 to said
subject.
6. The method of claim 5, wherein said disease, disorder or
condition is cirrhosis or viral infection.
7. A mouse comprising human placental stem cell-derived hepatocytes
or hepatogenic cells, wherein said mouse is produced by a method
comprising the steps of: a. irradiating said mouse with gamma
radiation sufficient to kill substantially all of the endogenous
bone marrow cells; b. administering to said mouse sufficient bone
marrow or bone marrow-derived cells from a NOD/SCID mouse to
reconstitute the hematopoietic system of the mouse; and c.
transplanting to said mouse a plurality of hepatocytes or
hepatogenic cells, wherein said hepatocytes or hepatogenic cells
are differentiated from a plurality of CD10.sup.+, CD34.sup.-,
CD105.sup.+, CD200.sup.+ placental stem cells.
8. The mouse of claim 7, wherein said placental stem cell is
additionally cytokeratin 18.sup.+.
9. The mouse of claim 7, wherein said hepatocytes or hepatogenic
cells are administered into an ear pinna of the mouse.
10. The mouse of claim 8, wherein said hepatocytes or hepatogenic
cells are infected with a virus.
11. The mouse of claim 10, wherein said virus is hepatitis A virus,
hepatitis B virus, hepatitis C virus, hepatitis D virus, or
hepatitis E virus.
12. A method of identifying an antiviral agent, comprising
contacting the mouse of claim 12 with a compound of interest,
wherein serum from said mouse has detectable levels of virus, and
wherein said compound is an antiviral agent if said contacting
results in a detectable reduction in the amount of said virus in
serum from said mouse, compared to serum from said mouse not
contacted with the compound of interest.
13. The method of claim 12, wherein said virus is hepatitis A
virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, or
hepatitis E virus.
14. The method of claim 13, wherein an antigen of said virus is
detected.
15. The method of claim 13, wherein a nucleic acid of said virus is
detected.
16. The method of claim 14, wherein said virus is hepatitis B
virus.
17. The method of claim 14, wherein said antigen is HBeAg or
HBsAg.
18. A composition comprising a plurality of cells encapsulated in
alginate, wherein said cells are differentiated from placental stem
cells, and wherein said cells express at least one marker of a
hepatocyte not expressed by, or expressed to a detectably different
degree than, an adherent placental stem cell that is CD10.sup.+,
CD34.sup.-, CD105.sup.+ and CD200.sup.+.
19. The composition of claim 18, wherein said alginate is in the
form of beads.
20. The composition of claim 19, wherein said beads are from about
200 .mu.m to about 800 .mu.m in size.
21. The composition of claim 19, wherein said beads average about
500 .mu.m in size.
22. A composition comprising isolated adherent CD10.sup.+,
CD34.sup.-, CD105.sup.+, CD200.sup.+ placental stem cells and an
electrospun nanofibrous scaffold.
23. The composition of claim 22, wherein said nanofibrous scaffold
comprises fibers of poly(L-lactic acid) (PLLA), poly lactic
glycolic acid (PLGA), type I collagen, a copolymer of vinylidene
fluoride and trifluoroethylnee (PVDF-TrFE), poly(-caprolactone),
poly(L-lactide-co-.epsilon.-caprolactone) [P(LLA-CL)] (e.g.,
75:25), and/or a copolymer of
poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and type I
collagen.
24. The composition of claim 22, wherein said nanofibrous scaffold
comprises fibers that average between about 250 nanometers and
about 10 .mu.m in thickness.
25. The composition of claim 22, wherein said composition is
contacted with conditions in which the placental stem cells
differentiate into chondrogenic cells or chondrocytes.
26. A method of making a composition comprising contacting adherent
CD10.sup.+, CD34.sup.-, CD105.sup.+, CD200.sup.+ placental stem
cells with an electrospun nanofibrous scaffold, wherein said
nanofibrous scaffold is made by electrospinning PLLA or PLGA at
about 20 kV at about 30 cm needle to collector distance and about
0.05 mL/min. to about 0.1 mL/min flow rate, wherein said PLLA or
PLGA are in solution at about 10% w/w to about 20% w/w.
27. An isolated cell population enriched for CD34.sup.+, CD45.sup.-
placental stem cells.
28. The cell population of claim 22, wherein at least 50% of cells
in said population are CD34.sup.+ and CD45.sup.-.
29. The cell population of claim 22, wherein at least 70% of cells
in said population are CD34.sup.+ and CD45.sup.-.
30. The cell population of claim 27, wherein at least 90% of cells
in said population are CD34.sup.+ and CD45.sup.-.
31. The cell population of claim 27, wherein said population
comprises a stem cell that is not CD34.sup.+ and CD45.sup.-.
32. The cell population of claim 31, wherein said stem cell that is
not CD34.sup.+ and CD45.sup.- is a CD34.sup.- adherent placental
stem cell.
33. The cell population of claim 32, wherein said adherent
placental stem cell is CD200.sup.+, CD105.sup.+, CD90.sup.+,
CD10.sup.+, CD34 and CD45.sup.-.
34. The cell population of claim 31, wherein said stem cell that is
not CD34.sup.+ and CD45.sup.- is a bone marrow-derived mesenchymal
stem cell.
35. The cell population of claim 31, wherein said stem cell that is
not CD34.sup.+ and CD45.sup.- is a CD34.sup.+, CD45.sup.+
hematopoietic stem cell.
36. The cell population of claim 8, wherein said stem cell that is
not CD34.sup.+ and CD45.sup.- is contained within cord blood or
placental blood.
37. A method of producing a CD34.sup.+, CD45.sup.- placental stem
cell population, comprising selecting CD34.sup.+ cells from a
population of placental cells to form a population of CD34.sup.+
placental cells, and removing from said population of CD34.sup.+
placental cells CD45.sup.+ cells, wherein a CD34.sup.+, CD45.sup.-
placental stem cell population is produced.
Description
[0001] This application claims benefit of U.S. Provisional
Application No. 60/901,066, filed Feb. 12, 2007; U.S. Provisional
Application No. 60/901,076, filed Feb. 12, 2007; U.S. Provisional
Application No. 60/905,664, filed Mar. 7, 2007; U.S. Provisional
Application No. 60/906,064, filed Mar. 8, 2007; and U.S.
Provisional Application No. 60/966,577, filed Aug. 28, 2007, the
disclosures of each of which are incorporated herein in its
entirety.
1. FIELD
[0002] Provided herein are methods and compositions relating to
stem cells from placenta. Provided herein are methods for the
production of hepatocytes from human adherent placental stem cells,
and the use of such hepatocytes in the treatment of, and
intervention in, for example, trauma, inflammatory and degenerative
disorders of the liver. Also provided herein are compositions and
methods relating to combinations of nanofibrous scaffolds and
adherent placental stem cells and methods of using the same in
cartilage repair. Finally, provided herein are compositions and
methods relating to nonadherent, CD34.sup.+CD45.sup.- stem cells
from placenta.
2. BACKGROUND
[0003] Somatic stem cells have been proposed for various
therapeutic applications, including, for example, in animal models
of cell replenishment therapy. The therapeutic potential of grafted
stem cells can only be translated to clinical use if an ethically
acceptable source of autologous stem cells is available, and if
control of self renewal and fate decisions that program stem cell
maturation into specific cell types is achieved.
[0004] A number of studies have described differentiation of
embryonic stem cells down the hepatocyte lineage (see, e.g.,
Sharma, N. S. et al., Biotechnology & Bioengineering, 94 (6):
1053-93 (2006); Maguire, T., et al, Biotechnology &
Bioengineering, 93(3):581-591 (2006) and Chen Y, et al., Cell
Transplant. 2006; 15(10):865-71). In addition, human bone marrow
derived mesenchymal cells were examined for the capacity to
differentiate into functioning hepatocytes with some success (Ong S
Y, Dai H, Leong K W, Tissue Eng. 2006 Oct. 1; Ong S Y, Dai H, Leong
K W Biomaterials (22):4087-97 (2006)(epub Apr. 17, 2006); Sato Y,
Araki I I, Kato J, Nakamura K, Blood. 106(2):756-63 (2005) (epub
Apr. 7, 2005).
[0005] Hepatic disorders increasingly account for significant
morbidity and mortality. Destruction of liver function by
environmental and pathogenic causes presents significant public
health risks to otherwise healthy individuals. Replacement of
damaged or killed hepatocytes in such damaged organs is therefore a
significant clinical goal. However, an ethically acceptable source
for stem cells that can differentiate into hepatocytes remains
unavailable. These and other unmet needs are provided herein.
3. SUMMARY
[0006] In one aspect, provided herein are methods and compositions
for the production of hepatocytes from adherent placental stem
cells, and methods of using such hepatocytes to treat diseases,
disorders or conditions, such as those involving trauma,
inflammation, or systemic disorders of the liver, e.g., diseases,
disorders or conditions associated with hepatic inflammation. In
one embodiment, provided herein is a method of producing a
hepatocyte, comprising culturing a placental stem cell under
conditions and for a time sufficient for said stem cell to exhibit
a characteristic of a hepatocyte. In a specific embodiment, said
characteristic is the production of albumin or expression of a gene
encoding albumin. In another specific embodiment, said
characteristic is the production of urea. In another specific
embodiment, said culturing comprises contacting said stem cell with
sodium butyrate. In another specific embodiment, said culturing
comprises encapsulating said stem cell in alginate-poly-L-lysine.
In another embodiment, provided herein is a hepatocyte produced by
differentiation of a placenta-derived stem cell. Also provided
herein is a method of treating a subject having a disease, disorder
or condition associated with abnormal liver function, comprising
introducing such a hepatocyte into said subject. In a more specific
embodiment, the disease, disorder or condition is cirrhosis of the
liver. In certain embodiments, the disease or conditions results
from liver toxicity caused by, e.g., alcohol or ingestion of toxins
such as, e.g., mushroom toxins. In certain embodiments, the disease
or condition is a viral infection, e.g., a hepatitis A, B, C, D, or
E infection. In certain embodiments, the disease or condition is
fulminant or subfulminant hepatitis. In another aspect, provided
herein is a method for determining whether a compound has liver
toxicity activity, comprising contacting a hepatocyte produced by
differentiation of a placenta-derived stem cell with the compound,
and determining whether the compound is toxic to the
hepatocytes.
[0007] In another embodiment, the placental stem cell is positive
for cytokeratin 18. In another embodiment, provided herein is a
population of placental stem cells, or cells differentiated
therefrom, at least 50%, 70%, 80%, 90%, 95% or 99% of which are
positive for cytokeratin 18. In another embodiment, provided herein
is a population of cells comprising placental stem cells, or cells
differentiated therefrom, wherein at least 50%, 70%, 80%, 90%, 95%
or 99% of the placental stem cells or cells differentiated
therefrom are positive for cytokeratin 18. In another embodiment,
the invention provides a method of isolating a placental stem cell,
or population of placental stem cells, or cells differentiated
therefrom, comprising selecting a cytokeratin 18.sup.+ placental
stem cell, or cytokeratin 18.sup.+ placental stem cells, and
isolating said stem cell or stem cells from other placental
cells.
[0008] In another aspect, provided herein is a composition
comprising a plurality of cells encapsulated in alginate, wherein
said cells are differentiated from placental stem cells. In one
embodiment, said cells express at least one marker of a hepatocyte
not expressed by, or expressed to a detectably different degree
than, a placental stem cell. In another embodiment, said alginate
is in the form of beads. In a specific embodiment, said beads are
from about 200 .mu.m to about 800 .mu.m in size. In another
specific embodiment, said beads average about 500 .mu.m in
size.
[0009] In another aspect, provided herein is a mouse comprising
human placental stem cell-derived hepatocytes or hepatogenic cells,
wherein said mouse is produced by a method comprising the steps of:
(a) irradiating said mouse with gamma radiation sufficient to kill
substantially all of the endogenous bone marrow cells; (b)
administering to said mouse sufficient bone marrow or bone
marrow-derived cells from a NOD/SCID mouse to reconstitute the
hematopoietic system of the mouse; and (c) transplanting to said
mouse a plurality of hepatocytes or hepatogenic cells, wherein said
hepatocytes or hepatogenic cells are differentiated from a
plurality of CD10.sup.+, CD34.sup.-, CD105.sup.+, CD117.sup.-,
CD200.sup.+ placental stem cells. In one embodiment, said placental
stem cell is additionally cytokeratin 18.sup.+ and negative for at
least one other cytokeratin expressed by differentiated
hepatocytes. In another embodiment, said hepatocytes or hepatogenic
cells are administered into an ear pinna of the mouse. In another
embodiment, said hepatocytes or hepatogenic cells are infected with
a virus. In a specific embodiment, said virus is hepatitis A virus,
hepatitis B virus, hepatitis C virus, hepatitis D virus, or
hepatitis E virus. In a more specific embodiment, the virus is
hepatitis B virus.
[0010] In another aspect, provided herein is a method of
identifying an antiviral agent, comprising contacting a mouse with
a compound of interest, wherein serum from said mouse has
detectable levels of virus, and wherein said compound is an
antiviral agent if said contacting results in a detectable
reduction in the amount of said virus in serum from said mouse,
compared to serum from said mouse not contacted with the compound
of interest, and wherein the mouse is produced by a method
comprising the steps of: a. irradiating said mouse with gamma
radiation sufficient to kill substantially all of the endogenous
bone marrow cells; b. administering to said mouse sufficient bone
marrow or bone marrow-derived cells from a NOD/SCID mouse to
reconstitute the hematopoietic system of the mouse; and c.
transplanting to said mouse a plurality of hepatocytes or
hepatogenic cells, wherein said hepatocytes or hepatogenic cells
are differentiated from a plurality of CD10.sup.+, CD34.sup.-,
CD105.sup.+, CD117.sup.+, CD200.sup.+ placental stem cells. In one
embodiment of the method, said virus is hepatitis A virus,
hepatitis B virus, hepatitis C virus, hepatitis D virus, or
hepatitis E virus. In specific embodiments of the method, an
antigen or a nucleic acid of said virus is detected. In a more
specific embodiment, said virus is hepatitis B virus. In a specific
embodiment of the method, wherein a viral antigen is detected, said
antigen is HBeAg or HBsAg. In another specific embodiment, wherein
a viral nucleic acid is detected, said nucleic acid is the
covalently closed circular form of hepatitis B virus. In a more
specific embodiment, said nucleic acid is detected by PCR using
primers specific for the covalently closed circular form of
hepatitis B virus.
[0011] In another aspect, provided herein is a matrix, and
compositions comprising such a matrix, wherein the matrix comprises
placental stem cells that have differentiated to a hepatogenic or
chondrogenic lineage, or to hepatocytes or chondrocytes. In a more
specific embodiment, said matrix is a three-dimensional scaffold.
In another more specific embodiment, said matrix comprises
collagen, gelatin, laminin, fibronectin, pectin, ornithine, or
vitronectin. In another more specific embodiment, said matrix is,
or comprises, a nanofibrous scaffold, e.g., an electrospun
nanofibrous scaffold. In a more specific embodiment, said
nanofibrous scaffold comprises poly(L-lactic acid) (PLLA), type I
collagen, a copolymer of vinylidene fluoride and trifluoroethylnee
(PVDF-TrFE), poly(-caprolactone),
poly(L-lactide-co-.epsilon.-caprolactone) [P(LLA-CL)] (e.g.,
75:25), and/or a copolymer of
poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and type I
collagen. In another more specific embodiment, said electrospun
nanofibrous scaffold promotes the differentiation of placental stem
cells into chondrocytes or hepatocytes. In another specific
embodiment, the electrospun nanofibrous matrix or scaffold
comprises placental stem cells that have differentiated into
chondrocytic cells and/or chondrocytes, or into hepatocytic cells
and/or hepatocytes. In another more specific embodiment, the matrix
is an amniotic membrane or an amniotic membrane-derived
biomaterial. In another more specific embodiment, said matrix
comprises an extracellular membrane protein. In another more
specific embodiment, said matrix comprises a synthetic compound. In
another more specific embodiment, said matrix comprises a bioactive
compound. In another more specific embodiment, said bioactive
compound is a growth factor, cytokine, antibody, or organic
molecule of less than 5,000 daltons.
[0012] In another embodiment, provided herein is a composition
comprising isolated adherent CD10.sup.+, CD34.sup.-, CD105.sup.+,
CD200.sup.+ placental stem cells and an electrospun nanofibrous
scaffold. In a specific embodiment, said nanofibrous scaffold
comprises fibers of poly(L-lactic acid) (PLLA), poly lactic
glycolic acid (PLGA), type I collagen, a copolymer of vinylidene
fluoride and trifluoroethylnee (PVDF-TrFE), poly(-caprolactone),
poly(L-lactide-co-.epsilon.-caprolactone) [P(LLA-CL)] (e.g.,
75:25), and/or a copolymer of
poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and type I
collagen. In another specific embodiment, said nanofibrous scaffold
comprises fibers that average between about 250 nanometers and
about 10 .mu.m in thickness. In another specific embodiment, said
composition is contacted with conditions in which the placental
stem cells differentiate into chondrogenic cells or chondrocytes.
In another embodiment, provided herein is a method of making a
composition comprising contacting adherent CD10.sup.+, CD34.sup.-,
CD105.sup.+, CD200.sup.+ placental stem cells with an electrospun
nanofibrous scaffold, wherein said nanofibrous scaffold is made by
electrospinning PLLA or PLGA at about 20 kV at about 30 cm needle
to collector distance and about 0.05 mL/min. to about 0.1 mL/min
flow rate, wherein said PLLA or PLGA are in solution at about 10%
w/w to about 20% w/w.
[0013] In another aspect, provided herein is an isolated placental
stem cell that is CD34.sup.+ and CD45.sup.-. In a specific
embodiment, said CD34.sup.+, CD45.sup.- stem cell is hematopoietic.
In another specific embodiment, said CD34.sup.+, CD45.sup.- stem
cell is non-adherent when cultured on a tissue culture surface,
e.g., plastic. In a specific embodiment, provided herein is an
isolated cell population enriched in placental stem cells that are
CD34.sup.+ and CD45.sup.-. In specific embodiments, at least 50%,
70%, 90% or 95% of cells in said population are
CD34.sup.+CD45.sup.- placental stem cells. In another specific
embodiment, the isolated cell population comprises proportionately
more CD34.sup.+ and CD45.sup.- placental stem cells than placental
perfusate (e.g., perfusate from perfusion of a placenta with 750 mL
0.9% saline solution). In another specific embodiment, the isolated
cell population comprises a stem cell that is not CD34.sup.+ and
CD45.sup.-. In a more specific embodiment, said stem cell that is
not CD34.sup.+ and CD45.sup.- is a CD34.sup.- adherent placental
stem cell. In a more specific embodiment, said adherent placental
stem cell is CD200.sup.+, CD105.sup.+, CD90.sup.+, CD10.sup.+,
CD34.sup.- and/or CD45. In another specific embodiment, said stem
cell that is not CD34.sup.+ and CD45.sup.- is a bone marrow-derived
mesenchymal stem cell. In another specific embodiment, said stem
cell that is not CD34.sup.+ and CD45.sup.- is a CD34.sup.+,
CD45.sup.+ hematopoietic stem cell. In another specific embodiment,
said stem cell that is not CD34.sup.+ and CD45.sup.+ is contained
within cord blood or placental blood.
[0014] In another specific embodiment, the isolated cell population
is a plurality of total nucleated cells (TNC) from placental
perfusate. In a specific embodiment, the TNC from placental
perfusate comprises placental cells from at least, or at most, 50,
100, 150, 200, 250, 300, 350, 400, 450 or 500 mL placental
perfusate. In another specific embodiment, the TNC from placental
perfusate have been treated to remove at least one type of non-red
blood cell.
[0015] In another embodiment, the CD34.sup.+, CD45.sup.-
hematopoietic placental stem cells are fetal (non-maternal). In
another embodiment, the CD34.sup.+, CD45.sup.- hematopoietic
placental stem cells are maternal. In another embodiment, an
isolated population of hematopoietic placental stem cells comprises
CD34.sup.+, CD45.sup.- hematopoietic placental stem cells that are
fetal (non-maternal). In another embodiment, an isolated population
of hematopoietic placental stem cells comprises CD34.sup.+,
CD45.sup.+ hematopoietic placental stem cells that are
maternal.
[0016] In another aspect, provided herein are methods of isolating
CD34.sup.+, CD45.sup.- hematopoietic placental stem cells. In one
embodiment, the invention provides a method of isolating a
CD34.sup.+, CD45.sup.- placental stem cell population, comprising
selecting CD34.sup.+ cells from a population of placental cells to
form an isolated population of CD34.sup.+ placental cells, and
removing from said population of CD34.sup.+ placental cells
CD45.sup.+ cells, wherein a CD34.sup.+, CD45.sup.- placental stem
cell population is produced. In a specific embodiment, said
selecting CD34.sup.+ cells is done by immunoseparation. In another
specific embodiment, said removing CD45.sup.+ cells is done by
immunoseparation. In another specific embodiment, said selecting or
said removing is done by flow cytometry.
[0017] In another aspect, provided herein is a method of
supplementing a cell population comprising adding a plurality of
CD34.sup.+, CD45.sup.- hematopoietic placental stem cells to create
a supplemented cell population, such that the supplemented cell
population comprises substantially more CD34.sup.+, CD45.sup.-
cells than before said supplementing. In various specific
embodiments in this context, "substantially more" means at least 1,
2, 3, 4, 5, 6, 7, 8, 9 or at least 10% more. In other specific
embodiments, the cell population to be supplemented comprises cord
blood, placental blood, peripheral blood, or a combination thereof.
In more specific embodiments, the cell population to be
supplemented is cord blood, placental blood, peripheral blood, or a
combination thereof. In another more specific embodiment, the cell
population to be supplemented comprises nucleated cells isolated
from cord blood, placental blood, peripheral blood, or a
combination thereof. In other specific embodiments, the stem cell
population to be supplemented comprises a population of
hematopoietic stem cells, a population of adult stem cells, or a
population of embryonic stem cells.
[0018] As used herein, the term "SH2" refers to an antibody that
binds an epitope on the marker CD105. Thus, cells that are referred
to as SH2.sup.+ are CD105.sup.+.
[0019] As used herein, the terms "SH3" and SH4" refer to antibodies
that bind epitopes present on the marker CD73. Thus, cells that are
referred to as SH3.sup.+ and/or SH4.sup.+ are CD73.sup.+.
[0020] As used herein, the term "isolated stem cell" means a stem
cell that is substantially separated from other, non-stem cells of
the tissue, e.g., placenta, from which the stem cell is derived. A
stem cell is "isolated" if at least 50%, 60%, 70%, 80%, 90%, 95%,
or at least 99% of the non-stem cells with which the stem cell is
naturally associated, or stem cells displaying a different marker
profile, are removed from the stem cell, e.g., during collection
and/or culture of the stem cell.
[0021] 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. A stem cell is "isolated" if at
least 50%, 60%, 70%, 80%, 90%, 95%, or at least 99% of the cells
with which the population of cells, or cells from which the
population of cells is derived, is naturally associated, i.e., stem
cells displaying a different marker profile, are removed from the
stem cell, e.g., during collection and/or culture of the stem
cell.
[0022] As used herein, the term "placental stem cell" refers to a
stem cell or progenitor cell, e.g., a multipotent cell, that is
derived from a mammalian placenta, regardless of morphology, cell
surface markers, or the number of passages after a primary culture.
The term "placental stem cell" as used herein does not, however,
refer to a trophoblast, cytotrophoblast, embryonic germ cell or
embryonic stem cell. A cell is considered a "stem cell" if the cell
retains at least one attribute of a stem cell, e.g., 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;
multipotency, e.g., the ability to differentiate, either in vitro,
in vivo or both, into cells of one or more of the three germ
layers; the lack of adult (i.e., differentiated) cell
characteristics, or the like. The terms "placental stem cell" and
"placenta-derived stem cell" may be used interchangeably. Unless
otherwise noted herein, the term "placental" includes the umbilical
cord. The adherent placental stem cells disclosed herein are, in
certain embodiments, multipotent in vitro (that is, the cells
differentiate in vitro under differentiating conditions),
multipotent in vivo (that is, the cells differentiate in vivo), or
both.
[0023] As used herein, a stem cell is "positive" for a particular
marker when that marker is detectable above background. For
example, a placental stem cell is positive for, e.g., CD73 because
CD73 is detectable on placental stem cells, e.g., by flow
cytometry, in an amount detectably greater than background (in
comparison to, e.g., an 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 means that a cell bears that marker in a amount
that produces a signal, e.g., in a cytometer, that is detectably
above background. For example, a cell is "CD200.sup.+" where the
cell is detectably labeled with an antibody specific to CD200, and
the signal from the antibody is detectably higher than a control
(e.g., background). Conversely, "negative" in the same context
means that the cell surface marker is not detectable using an
antibody specific for that marker compared to background. For
example, a cell is "CD34.sup.-" where the cell is not detectably
labeled with an antibody specific to CD34. Unless otherwise noted
herein, cluster of differentiation ("CD") markers are detected
using antibodies. OCT-4 is determined to be present, and a cell is
"OCT-4.sup.+" if OCT-4 is detectable using RT-PCR.
[0024] As used herein, "isolating" placental stem cells, e.g.,
adherent placental stem cells or CD34.sup.+, CD45.sup.- stem cells,
means to remove at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
95% or 99% of the cells with which the stem cells are normally
associated in the intact mammalian placenta. A stem cell from an
organ is "isolated" when it is present in a population of cells
that comprises fewer than 50% of the cells with which the stem cell
is normally associated in the intact organ. "Dim", when associated
with a cell marker, indicates that the marker is present detectably
above background, but within about 5% to about 10% above
background.
[0025] As used herein, "hepatocyte" means a cell that appears
visually, biochemically and/or by gene expression pattern to be a
hepatocyte as that term is normally understood. As used herein,
"hepatogenic cell," referring to a cell differentiated from a
placental stem cell or umbilical cord stem cells, is a cell that
displays one or more characteristics of a terminally-differentiated
hepatocyte, which characteristics are not found in a placental stem
cell or umbilical cord stem cells, or are not found at the same
level in a placental stem cell or umbilical cord stem cell (e.g.,
are detectably higher or lower in a hepatogenic cell when compared
to a placental stem cell or umbilical stem cell assayed for the
characteristic under equivalent conditions), prior to
differentiation into a hepatocyte or hepatogenic cell (e.g., a
placental stem cell or umbilical cord stem cell in an expansion
culture). Thus, the various compositions, methods, and other
embodiments of the present application also encompass cells derived
from placental stem cells that have fully or partially
differentiated into hepatocytes.
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1: Viability of placental stem cells from perfusion
(A), amnion (B), chorion (C), or amnion-chorion plate (D), or
umbilical cord stem cells (E). Numbers on X-axis designate placenta
from which stem cells were obtained.
[0027] FIG. 2: Percent HLA
ABC.sup.-/CD45.sup.-/CD34.sup.-/CD133.sup.+ cells from perfusion
(A), amnion (B), chorion (C), or amnion-chorion plate (D), or
umbilical cord stem cells (E) as determined by FACSCalibur. Numbers
on X-axis designate placenta from which stem cells were
obtained.
[0028] FIG. 3: Percent HLA
ABC.sup.-/CD45.sup.-/CD34.sup.-/CD133.sup.+ cells from perfusion
(A), amnion (B), chorion (C), or amnion-chorion plate (D), or
umbilical cord stem cells (E), as determined by FACS Aria. Numbers
on X-axis designate placenta from which stem cells were
obtained.
[0029] FIG. 4: HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105,
CD117, CD200 expression in stem cells derived from placental
perfusate.
[0030] FIG. 5: HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105,
CD117, CD200 expression in stem cells derived from amnion.
[0031] FIG. 6: HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105,
CD117, CD200 expression in stem cells derived from chorion.
[0032] FIG. 7: HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105,
CD117, CD200 expression in stem cells derived from amnion-chorion
plate.
[0033] FIG. 8: HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105,
CD117, CD200 expression in stem cells derived from umbilical
cord.
[0034] FIG. 9: Average expression of HLA-G, CD10, CD13, CD33, CD38,
CD44, CD90, CD105, CD117, CD200 expression in stem cells derived
from perfusion (A), amnion (B), chorion (C), amnion-chorion plate
(D) or umbilical cord (E).
[0035] FIG. 10: Average percentage of total cells from six matched
human placental perfusate and umbilical cord units. X axis: percent
cells, of total nucleated cells, of the phenotype shown on the
Y-axis.
5. DETAILED DESCRIPTION
5.1 Production of Hepatocytes
[0036] In the sections and discussions that follow, it will be
understood by the skilled artisan that many of the various
compositions and methods can be performed on or using placental
stem cells or umbilical cord stem cells that have differentiated,
or have been differentiated, down the hepatocyte lineage.
[0037] In one aspect, provided herein are methods and compositions
for the production of hepatocytes and/or hepatogenic cells from
placenta-derived cells, particularly placental stem cells or
umbilical cord stem cells. As used herein, "placental stem cells"
or "umbilical cord stem cells" means adherent stem cells unless
otherwise specified. Stem cells may be obtained from a mammalian
placenta or umbilical cord by perfusion (see, e.g., Hariri, U.S.
Pat. Nos. 7,045,148 and 7,255,879, which are hereby incorporated
herein in their entireties. Stem cells may also be obtained from
placenta or umbilical cord by disruption (e.g., maceration) of a
placenta or part thereof (see, e.g., Section 6.2, below). Cells
displaying hepatocyte characteristics, e.g., hepatocytes and/or
hepatogenic cells, may be obtained from placental stem cells. These
cells are useful in the treatment of diseases, disorders or
conditions associated with, for example, cirrhosis of the liver,
including, but not limited to cirrhosis caused by alcohol
ingestion, ingestion of hepatic toxins such as those found in,
e.g., mushrooms of the genus Amanita, or caused by viral
infections, e.g., hepatitis A, B, C, D, or E infection.
[0038] In one embodiment, differentiable cells, such as stem cells,
may be obtained from the placenta or umbilical cord as follows.
Primary cultures of mononuclear cells (MNCs) are isolated from
placentas, e.g., from human placenta perfusates or from physically
and/or enzymatically-disrupted placental tissue. The placentas are
obtained following birth of full-term infants under informed
consent of the donors. Briefly, for perfusion, umbilical vessels
are cannulated then connected to a flow-controlled circuit, and the
placenta is perfused at, e.g., 1 mL/min (room temperature, up to 24
hours) with Dulbecco's modified Eagle's medium (DMEM, Gibco/BRL)
containing high glucose, 1% heparin and penicillin/streptomycin.
Placenta perfusate (750 mL) is then pooled, centrifuged, and the
cell pellet resuspended in PBS containing 1% fetal calf serum (FBS)
then separated by differential gradient density centrifugation
through LYMPHOPREP.TM. (Gibco/BRL). The buffy-coat interface
containing mononucleated cells including adherent placental stem
cells are recovered, resuspended in DMEM/10% FBS, plated on
fibronectin-coated (Sigma) Falcon plates and incubated at
37.degree. C. with 5% humidified CO.sub.2. After a 24-hour
incubation the nonadherent cells are discarded and the adherent
cells are maintained and expanded in fresh culture media;
individual cell colonies develop between 10 and 18 days and are
expanded as placental stem cell lines.
[0039] Human adherent placental stem cells display fibroblast-like
morphology in culture and are HLA-class I positive. Using FACS
analysis these cells do not express the hematopoietic markers CD34
or CD45. However, they do express the multipotential cellular
markers CD10 (CALLA), CD29 (.beta..sub.1 integrin), CD54 (ICAM-1),
CD90 (Thy-1) as well as SH2 (CD105), SH3 (CD73) and CD200. Under
standard growth conditions the doubling time for placental stem
cells is about 18 to 36 hours, and the cells maintain this
phenotype for greater than 40 population doublings in vitro. Human
adherent placental stem cells are distinguishable from human
embryonic stem cells or embryonic germ cells in that human
embryonic stem cells or germ cells are obtained only from the inner
cell mass of the blastula or fetal gonads, not placentas. Human
adherent placental stem cells are also distinguishable from
mesenchymal stem cells from, e.g., bone marrow, cord blood or
peripheral blood, or bone marrow-derived stem cells, in that
placental stem cells form embryoid-like bodies in culture, while
mesenchymal stem cells or bone marrow-derived stem cells do not,
and placental stem cells display unique gene expression pattern
relative to mesenchymal stem cells. See U.S. patent application
Ser. No. 11/648,813, filed Dec. 28, 2006, the disclosure of which
is hereby incorporated herein by reference in its entirety.
[0040] Placental stem cells may be differentiated to hepatocytes by
culturing in culture medium comprising sodium butyrate or by
encapsulating the cells in a suitable microcapsule polymer, e.g.
alginate-poly-L-lysine. Hepatocytes can be produced from
placenta-derived stem cells as described above, and maintained or
cultured as described in below. Hepatocyte differentiation can be
assessed using flow cytometry and monitoring for particular gene
expression or enzymatic activity as described below.
5.2 Placental Stem Cells and Placental Stem Cell Populations
[0041] In one aspect, the methods provided herein use adherent
placental stem cells, that is, stem cells obtainable from a
placenta or part thereof, e.g., amnion, chorion, amnion/chorion
plate, umbilical cord, etc., that (1) adhere to a tissue culture
substrate; and (2) differentiate into one or more non-placental
cell types, and/or cells having tissue-specific cell
characteristics, under the appropriate differentiation conditions.
Placental stem cells are not derived from, nor are they derivable
from, blood, e.g., placental blood or umbilical cord blood, or from
bone marrow.
[0042] Placental stem cells can be either fetal or maternal in
origin (that is, can have the genotype of either the mother or
fetus). Populations of placental stem cells, or populations of
cells comprising placental stem cells, can comprise placental stem
cells that are solely fetal or maternal in origin, or can comprise
a mixed population of placental stem cells of both fetal and
maternal origin. The placental stem cells, and populations of cells
comprising the placental stem cells, can be identified and selected
by the morphological, marker, and culture characteristics discussed
below.
[0043] 5.2.1 Physical and Morphological Characteristics
[0044] The placental stem cells used in the methods disclosed
herein, when cultured in primary culture or in cell culture, adhere
to the tissue culture substrate, e.g., tissue culture container
surface (e.g., tissue culture plastic). Placental stem cells in
culture, e.g., on a tissue culture surface, assume a generally
fibroblastoid appearance, with a number of cyotplasmic processes
extending from the central cell body. The placental stem cells are,
however, morphologically distinguishable from fibroblasts cultured
under the same conditions, as the placental stem cells exhibit a
greater number of such processes than do fibroblasts.
Morphologically, placental stem cells are also distinguishable from
hematopoietic stem cells, which generally assume a more rounded, or
cobblestone, morphology in culture.
[0045] 5.2.2 Cell Surface, Molecular and Genetic Markers
[0046] Adherent placental stem cells, and populations of adherent
placental stem cells, useful in the methods and compositions
described herein, express a plurality of markers that can be used
to identify and/or isolate the stem cells, or populations of cells
that comprise the stem cells. The placental stem cells, and stem
cell populations (that is, two or more placental stem cells)
described herein include stem cells and stem cell-containing cell
populations obtained directly from the placenta, or any part
thereof (e.g., amnion, chorion, placental cotyledons, umbilical
cord, and the like). Placental stem cell populations also includes
populations of (that is, two or more) placental stem cells in
culture, and a population in a container, e.g., a bag. Placental
stem cells are not, however, trophoblasts.
[0047] The placental stem cells described herein are multipotent in
that they can be differentiated in vitro into cells representative
of the three germ layers, e.g., adipocytic cells, chondrocytic
cells, hepatic cells, neurogenic cells, cardiac cells, and the
like. The placental stem cells described herein, however, need not
differentiate in vivo to be considered multipotent, or to be
useful. The term "placental stem cell," therefore, encompasses
cells described herein that differentiate in vitro but not in vivo,
differentiate in vivo but not in vitro, or both in vitro and in
vivo. In one embodiment, the placental stem cells provided herein
can be differentiated in vitro into cells representative of one or
more of the three germ layers, but do not differentiate in vivo,
e.g., in a NOD-SCID mouse.
[0048] Adherent (non-hematopoietic) placental stem cells generally
express the markers CD73, CD105, CD200, HLA-G, and/or OCT-4, and do
not express CD34, CD38, or CD45. Placental stem cells can also
express HLA-ABC (MHC-1), but generally do not express HLA-DR. In a
specific embodiment, adherent placental stem cells are CD10.sup.+,
CD34.sup.-, CD105.sup.+ and CD200.sup.+. These markers can be used
to identify placental stem cells, and to distinguish placental stem
cells from other stem cell types. Because the placental stem cells
can express CD73 and CD105, they can have mesenchymal stem
cell-like characteristics. However, because the placental stem
cells can express CD200 and HLA-G, a fetal-specific marker, they
can be distinguished from mesenchymal stem cells, e.g., bone
marrow-derived mesenchymal stem cells, which express neither CD200
nor HLA-G. In the same manner, the lack of expression of CD34, CD38
and/or CD45 identifies the placental stem cells as
non-hematopoietic stem cells. Such placental stem cells, and
populations of cells comprising such placental stem cells, can be
differentiated into hepatocytes, hepatogenic cells, populations of
hepatocytes, populations of hepatogenic cells, and combinations of
the foregoing.
[0049] In one embodiment, the methods and compositions provided
herein use an isolated placental stem cell that is CD200.sup.+ and
HLA-G.sup.+. In specific embodiments, said stem cell is also
CD73.sup.+ and CD105.sup.+. In another specific embodiment, said
stem cell is also CD34, CD38.sup.- or CD45.sup.-. In a more
specific embodiment, said stem cell is also CD34.sup.-, CD38.sup.-,
CD45.sup.-, CD73.sup.+ and CD105.sup.+. In another specific
embodiment, said stem cell has been expanded, for example, passaged
at least once, at least three times, at least five times, at least
10 times, at least 15 times, or at least 20 times.
[0050] In another embodiment, the methods and compositions provided
herein use an isolated cell population comprising a plurality of
placental stem cells that are CD200.sup.+, HLA-G.sup.+. In various
embodiments, at least 10%, at least 20%, at least 30%, at least
40%, at least 50% at least 60%, at least 70%, at least 80%, at
least 90%, or at least 95% of said placental stem cells in said
population are said CD200.sup.+, HLA-G.sup.+ stem cells. In a
specific embodiment of the isolated populations, said stem cells
are also CD73.sup.+ and CD105'. In another specific embodiment,
said stem cells are also CD34.sup.-, CD38.sup.- or CD45.sup.-. In a
more specific embodiment, said stem cells are also CD34.sup.-,
CD38.sup.-, CD45.sup.-, CD73.sup.+ and CD105.sup.+. In another
embodiment, said isolated population produces one or more
embryoid-like bodies when cultured under conditions that allow the
formation of embryoid-like bodies. In another specific embodiment,
said population has been expanded, for example, passaged at least
once, at least three times, at least five times, at least 10 times,
at least 15 times, or at least 20 times.
[0051] In another embodiment, the methods and compositions provided
herein use an isolated placental stem cell that is CD73.sup.+,
CD105.sup.+, CD200.sup.+. In a specific embodiment of said
populations, said stem cell is also HLA-G.sup.+. In another
specific embodiment, said stem cell is also CD34.sup.-, CD38.sup.-
or CD45.sup.-. In another specific embodiment, said stem cell is
also CD34.sup.-, CD38.sup.- and CD45.sup.-. In a more specific
embodiment, said stem cell is also CD34.sup.-, CD38.sup.-,
CD45.sup.-, and HLA-G.sup.+. In another specific embodiment, said
stem cell has been expanded, for example, passaged at least once,
at least three times, at least five times, at least 10 times, at
least 15 times, or at least 20 times.
[0052] In another embodiment, the methods and compositions provided
herein use an isolated cell population comprising a plurality of
placental stem cells that are CD73.sup.+, CD105.sup.+, CD200.sup.+.
In various embodiments, at least 10%, at least 20%, at least 30%,
at least 40%, at least 50% at least 60%, at least 70%, at least
80%, at least 90%, or at least 95% of said placental stem cells in
said population are said CD73.sup.+, CD105.sup.+, CD200.sup.+
cells. In a specific embodiment of said populations, said stem
cells are HLA-G.sup.+. In another specific embodiment, said stem
cells are CD34.sup.-, CD38.sup.- or CD45.sup.-. In another specific
embodiment, said stem cells are CD34.sup.-, CD38.sup.- and
CD45.sup.-. In a more specific embodiment, said stem cells are
CD34.sup.-, CD38.sup.-, CD45.sup.-, and HLA-G.sup.+. In another
specific embodiment, said population of cells produces one or more
embryoid-like bodies when cultured under conditions that allow the
formation of embryoid-like bodies. In another specific embodiment,
said population has been expanded, for example, passaged at least
once, at least three times, at least five times, at least 10 times,
at least 15 times, or at least 20 times.
[0053] In another embodiment, the methods and compositions provided
herein use an isolated placental stem cell that is CD200.sup.+,
OCT-4.sup.+. In a specific embodiment, said stem cell is also
CD73.sup.+ and CD105.sup.+. In another specific embodiment, said
stem cell is also IILA-G.sup.+. In another specific embodiment,
said stem cell is also CD34.sup.-, CD38.sup.- and CD45.sup.-. In a
more specific embodiment, said stem cell is also CD34.sup.-,
CD38.sup.-, CD45.sup.-, CD73.sup.+, CD105.sup.+ and HLA-G.sup.+. In
another specific embodiment, said stem cell has been expanded, for
example, passaged at least once, at least three times, at least
five times, at least 10 times, at least 15 times, or at least 20
times.
[0054] In another embodiment, the methods and compositions provided
herein use an isolated cell population comprising a plurality of
placental stem cells that are CD200', OCT-4.sup.+. In various
embodiments, at least 10%, at least 20%, at least 30%, at least
40%, at least 50% at least 60%, at least 70%, at least 80%, at
least 90%, or at least 95% of said placental stem cells in said
population are said CD200.sup.+, OCT-4.sup.+ cells. In a specific
embodiment, said stem cells are CD73.sup.+ and CD105.sup.+. In
another specific embodiment, said stem cells are HLA-G.sup.+. In
another specific embodiment, said stem cells are CD34.sup.-,
CD38.sup.- and CD45.sup.-. In a more specific embodiment, said stem
cells are CD34.sup.-, CD38.sup.-, CD45.sup.-, CD73.sup.+,
CD105.sup.+ and HLA-G.sup.+. In another specific embodiment, the
population produces one or more embryoid-like bodies when cultured
under conditions that allow the formation of embryoid-like bodies.
In another specific embodiment, said population has been expanded,
for example, passaged at least once, at least three times, at least
five times, at least 10 times, at least 15 times, or at least 20
times.
[0055] In another embodiment, the methods and compositions provided
herein use an isolated placental stem cell that is CD73.sup.+,
CD105.sup.+ and HLA-G.sup.+. In a specific embodiment of the above
plurality, said stem cell is also CD34.sup.-, CD38.sup.- or
CD45.sup.-. In another specific embodiment, said stem cell is also
CD34.sup.-, CD38.sup.- and CD45.sup.-. In another specific
embodiment, said stem cells are also OCT-4.sup.+. In another
specific embodiment, said stem cell is also CD200.sup.+. In a more
specific embodiment, said stem cell is also CD34.sup.-, CD38.sup.-,
CD45.sup.-, OCT-4.sup.+ and CD200.sup.+. In another specific
embodiment, said stem cell has been expanded, for example, passaged
at least once, at least three times, at least five times, at least
10 times, at least 15 times, or at least 20 times.
[0056] In another embodiment, the methods and compositions provided
herein use an isolated cell population comprising a plurality of
placental stem cells that are CD73.sup.+, CD105.sup.+ and
HLA-G.sup.+. In various embodiments, at least 10%, at least 20%, at
least 30%, at least 40%, at least 50% at least 60%, at least 70%,
at least 80%, at least 90%, or at least 95% of said placental stem
cells in said population are said CD73.sup.+, CD105.sup.+ and
HLA-G.sup.+ cells. In a specific embodiment of the above plurality,
said stem cells are also CD34.sup.-, CD38.sup.- or CD45.sup.-. In
another specific embodiment, said stem cells are also CD34.sup.-,
CD38.sup.- and CD45.sup.-. In another specific embodiment, said
stem cells are also OCT-4.sup.+. In another specific embodiment,
said stem cells are also CD200.sup.+. In a more specific
embodiment, said stem cells are also CD34, CD38.sup.-, CD45.sup.-,
OCT-4.sup.+ and CD200.sup.+. In another specific embodiment, said
population has been expanded, for example, passaged at least once,
at least three times, at least five times, at least 10 times, at
least 15 times, or at least 20 times.
[0057] In another embodiment, the methods and compositions provided
herein use an isolated cell population comprising a plurality of
placental stem cells that are CD73.sup.+, CD105.sup.+ stem cells,
wherein said plurality forms one or more embryoid-like bodies under
conditions that allow formation of embryoid-like bodies. In various
embodiments, at least 10%, at least 20%, at least 30%, at least
40%, at least 50% at least 60%, at least 70%, at least 80%, at
least 90%, or at least 95% of said placental stem cells in said
population are said CD73.sup.+, CD105.sup.+ stem cells. In a
specific embodiment, said stem cells are also CD34.sup.-,
CD38.sup.- or CD45.sup.-. In another specific embodiment, said stem
cells are also CD34.sup.-, CD38.sup.- and CD45.sup.-. In another
specific embodiment, said stem cells are also OCT-4.sup.+. In a
more specific embodiment, said stem cells are also OCT-4.sup.+,
CD34.sup.-, CD38.sup.- and CD45.sup.-. In another specific
embodiment, said population has been expanded, for example,
passaged at least once, at least three times, at least five times,
at least 10 times, at least 15 times, or at least 20 times.
[0058] In another embodiment, the methods and compositions provided
herein use an isolated cell population comprising a plurality of
placental stem cells that are OCT-4.sup.+ stem cells, wherein said
population forms one or more embryoid-like bodies when cultured
under conditions that allow the formation of embryoid-like bodies.
In various embodiments, at least 10%, at least 20%, at least 30%,
at least 40%, at least 50% at least 60%, at least 70%, at least
80%, at least 90%, or at least 95% of said placental cells in said
population are said OCT4.sup.+ stem cells. In a specific embodiment
of the above populations, said stem cells are CD73.sup.+ and
CD105.sup.+. In another specific embodiment, said stem cells are
CD34.sup.-, CD38.sup.-, or CD45.sup.-. In another specific
embodiment, said stem cells are CD200.sup.+. In a more specific
embodiment, said stem cells are CD73.sup.+, CD105.sup.+,
CD200.sup.+, CD34.sup.-, CD38.sup.-, and CD45.sup.-. In another
specific embodiment, said population has been expanded, for
example, passaged at least once, at least three times, at least
five times, at least 10 times, at least 15 times, or at least 20
times.
[0059] In another embodiment, the methods and compositions provided
herein use an isolated placental stem cell that is, or a cell
population comprising a plurality of placental stem cells that are,
CD29.sup.+, CD44.sup.+, CD73.sup.+, CD90.sup.+, CD105.sup.+,
CD200.sup.+, CD34.sup.- and CD133.sup.-.
[0060] In another embodiment, the methods and compositions provided
herein use an isolated placental stem cell that is CD10.sup.+,
CD34.sup.+, CD105.sup.+, and CD200'. Further provided herein is an
isolated population of cells, e.g., 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 said placental stem cells
are CD10.sup.+, CD34.sup.-, CD105.sup.+, CD200.sup.+. In a specific
embodiment of the above embodiments, said stem cells are
additionally CD90.sup.+ and CD45.sup.-. In a specific embodiment,
said stem cell or population of placental stem cells is isolated
away from placental cells that are not stem cells. In another
specific embodiment, said stem cell or population of placental stem
cells is isolated away from placental stem cells that do not
display these characteristics. In another specific embodiment, said
isolated placental stem cell is non-maternal in origin. In another
specific embodiment, at least about 90%, at least about 95%, or at
least about 99% of said cells in said isolated population of
placental stem cells, are non-maternal in origin.
[0061] In another embodiment, the methods and compositions provided
herein use an isolated placental stem cell that is HLA-A,B,C.sup.-,
CD45.sup.-, CD133.sup.- and CD34.sup.-. Further provided herein is
the use of an isolated population of 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 said placental stem cells
are HLA-A,B,C.sup.-, CD45.sup.-, CD133 and CD34.sup.-. In a
specific embodiment, said stem cell or population of placental stem
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 stem cells that do not
display these characteristics. In another specific embodiment, said
isolated placental stem cell is non-maternal in origin. In another
specific embodiment, at least about 90%, at least about 95%, or at
least about 99% of said cells in said isolated population of
placental stem cells, are non-maternal in origin. In another
embodiment, the HLA-A,B,C.sup.-, CD45.sup.-, CD133 and CD34.sup.-
placental stem cell is a stem cell isolated from placental
perfusate. In another embodiment, the HLA-A,B,C.sup.-, CD45.sup.-,
CD133.sup.- and CD34.sup.- placental stem cell is a stem cell
isolated by physical and/or enzymatic disruption of placental
tissue.
[0062] In another embodiment, the methods and compositions provided
herein an isolated placental stem cell that is CD10.sup.+,
CD13.sup.+, CD33.sup.+, CD45.sup.-, CD1 IT and CD133.sup.-. Further
provided herein is an isolated population of 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 said placental stem
cells are CD10.sup.+, CD1.sup.3, CD33.sup.+, CD45.sup.-,
CD117.sup.- and CD133.sup.-. In a specific embodiment, said stem
cell or population of placental stem cells is isolated away from
placental cells that are not stem cells. In another specific
embodiment, said isolated placental stem cell is non-maternal in
origin. In another specific embodiment, at least about 90%, at
least about 95%, or at least about 99% of said cells in said
isolated population of placental stem cells, are non-maternal in
origin. In another specific embodiment, said stem cell or
population of placental stem cells is isolated away from placental
stem cells that do not display these characteristics. In another
embodiment, provided herein is a method of obtaining a placental
stem cell that is CD10.sup.+, CD13.sup.+, CD33.sup.+, CD45.sup.-,
CD117 and CD133.sup.- comprising isolating said cell from placental
perfusate. In another embodiment, the HLA-A,B,C.sup.-, CD45.sup.-,
CD133.sup.- and CD34.sup.- placental stem cell is a stem cell
isolated by physical and/or enzymatic disruption of placental
tissue.
[0063] In another embodiment, the methods and compositions provided
herein an isolated placental stem cell that is CD10.sup.-,
CD33.sup.-, CD44.sup.+, CD45.sup.-, and CD117.sup.- T. Further
provided herein is an isolated population of 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 said placental stem
cells are CD10.sup.-, CD33.sup.-, CD44.sup.+, CD45.sup.-, and
CD117.sup.-. In a specific embodiment, said stem cell or population
of placental stem cells is isolated away from placental cells that
are not stem cells. In another specific embodiment, said isolated
placental stem cell is non-maternal in origin. In another specific
embodiment, at least about 90%, at least about 95%, or at least 99%
of said cells in said isolated population of placental stem cells,
are non-maternal in origin. In another specific embodiment, said
stem cell or population of placental stem cells is isolated away
from placental stem cells that do not display these
characteristics. In another embodiment, provided herein is a method
of obtaining a placental stem cell that is CD10.sup.-, CD33.sup.-,
CD44.sup.+, CD45.sup.-, CD117.sup.- comprising isolating said cell
from placental perfusate. In another embodiment, the
HLA-A,B,C.sup.-, CD45.sup.-, CD133.sup.- and CD34.sup.- placental
stem cell is a stem cell isolated by physical and/or enzymatic
disruption of placental tissue.
[0064] In another embodiment, the methods and compositions provided
herein use an isolated placental stem cell that is CD10.sup.-,
CD13.sup.-, CD33.sup.-, CD45.sup.-, and CD117.sup.-. Further
provided herein an isolated population of 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 said placental stem
cells are CD10.sup.-, CD13.sup.-, CD33.sup.-, CD45.sup.-, and
CD117.sup.-. In a specific embodiment, said stem cell or population
of placental stem cells is isolated away from placental cells that
are not stem cells. In another specific embodiment, said isolated
placental stem cell is non-maternal in origin. In another specific
embodiment, at least about 90%, at least about 95%, or at least 99%
of said cells in said isolated population of placental stem cells,
are non-maternal in origin. In another specific embodiment, said
stem cell or population of placental stem cells is isolated away
from placental stem cells that do not display these
characteristics. In another embodiment, provided herein is a method
of obtaining a placental stem cell that is CD10.sup.-, CD13.sup.-,
CD33.sup.-, CD45.sup.-, and CD117.sup.- comprising isolating said
cell from placental perfusate. In another embodiment, the
HLA-A,B,C.sup.-, CD45.sup.-, CD133.sup.- and CD34.sup.- placental
stem cell is a stem cell isolated by physical and/or enzymatic
disruption of placental tissue.
[0065] In another embodiment, the methods and compositions provided
herein use an isolated placental stem cell that is HLA A,B,C.sup.-,
CD45.sup.-, CD34.sup.-, CD133.sup.-, positive for CD10, CD13, CD38,
CD44, CD90, CD105, CD200 and/or HLA-G, and/or negative for CD117.
In another embodiment, the isolated population of placental stem
cells used in the methods and compositions provided herein are HLA
A,B,C.sup.-, CD45.sup.-, CD34.sup.-, CD133.sup.-, and 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 stem cells in the
population are positive for CD10, CD13, CD38, CD44, CD90, CD105,
CD200 and/or HLA-G, and/or negative for CD117. In a specific
embodiment, said stem cell or population of placental stem cells is
isolated away from placental cells that are not stem cells. In
another specific embodiment, said isolated placental stem cell is
non-maternal in origin. In another specific embodiment, at least
about 90%, at least about 95%, or at least about 99%, of said cells
in said isolated population of placental stem cells, are
non-maternal in origin. In another specific embodiment, said stem
cell or population of placental stem cells is isolated away from
placental stem cells that do not display these characteristics. In
another embodiment, provided herein is a method of obtaining a
placental stem cell that is HLA A,B,C.sup.-, CD45.sup.-,
CD34.sup.-, CD133.sup.- and positive for CD10, CD13, CD38, CD44,
CD90, CD105, CD200 and/or HLA-G, and/or negative for CD117,
comprising isolating said cell from placental perfusate.
[0066] In another embodiment, the methods and compositions provided
herein use a placental stem cell that is CD200.sup.+ and
CD10.sup.+, as determined by antibody binding, and CD117.sup.+, as
determined by both antibody binding and RT-PCR, or a population of
such cells, or a population of cells comprising such isolated
placental stem cells. In another embodiment, the methods and
compositions provided herein use a placental stem cell that is
CD10.sup.+, CD29.sup.-, CD54.sup.+, CD200.sup.+, HLA-G.sup.+, HLA
class F and .beta.-2-microglobulin. In another embodiment, provided
herein are placental stem cells, wherein the expression of at least
one 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 cell is
non-maternal in origin. In another specific embodiment, at least
about 90%, at least about 95%, or at least 99%, of said cells in
said isolated population of placental stem cells, are non-maternal
in origin.
[0067] In another embodiment, placental stem cells used in the
methods and compositions provided herein are positive for
cytokeratin 18. In another embodiment, provided herein is a
population of placental stem cells, or cells differentiated
therefrom, at least 50%, 70%, 80%, 90%, 95% or 99% of which are
positive for cytokeratin 18. In another embodiment, provided herein
is a population of cells comprising placental stem cells, or cells
differentiated therefrom, wherein at least 50%, 70%, 80%, 90%, 95%
or 99% of the placental stem cells or cells differentiated
therefrom are positive for cytokeratin 18. In another embodiment,
the invention provides a method of isolating a placental stem cell,
or population of placental stem cells, or cells differentiated
therefrom, comprising selecting a cytokeratin 18+ placental stem
cell, or cytokeratin 18+ placental stem cells, and isolating said
stem cell or stem cells from other placental cells.
[0068] In another embodiment, the methods and compositions provided
herein use an isolated population of placental stem cells, wherein
a plurality of said 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
another specific embodiment, said plurality is between about 3% and
about 25% of cells in said population of cells. In another
embodiment, the methods and compositions provided herein use a
population of placental stem cells, wherein a plurality of said
placental stem 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 placental stem cells or 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 the same number of cells and cultured
under the same conditions.
[0069] In a specific embodiment of the above-mentioned placental
stem cells, the placental stem cells constitutively secrete IL-6,
IL-8 and monocyte chemoattractant protein (MCP-1).
[0070] Each of the above-referenced placental stem cells, or
pluralities of placental stem cells, can comprise placental stem
cells obtained and isolated directly from a mammalian placenta, or
placental stem cells that have been cultured and passaged at least
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40
or more times, or a combination thereof:
[0071] The pluralities of placental stem cells described above can
comprise about, at least, or no more than, 1.times.10.sup.5,
5.times.10.sup.5, 1.times.10.sup.6, 5.times.10.sup.6,
1.times.10.sup.7, 5.times.10.sup.7, 1.times.10.sup.8,
5.times.10.sup.8, 1.times.10.sup.9, 5.times.10.sup.9,
1.times.10.sup.10, 5.times.10.sup.10, 1.times.10.sup.11 or more
placental stem cells.
[0072] 5.2.3 Selecting and Producing Placental Stem Cell
Populations
[0073] In another embodiment, provided herein is a method of
selecting a plurality of placental stem cells from a plurality of
placental cells, from which hepatocytes and/or hepatogenic cells
can be differentiated, comprising selecting a population of
placental cells wherein at least 10%, at least 20%, at least 30%,
at least 40%, at least 50% at least 60%, at least 70%, at least
80%, at least 90%, or at least 95% of said cells are CD200.sup.+,
HLA-G.sup.+ placental stem cells. In a specific embodiment, said
selecting comprises selecting stem cells that are also CD73.sup.+
and CD105.sup.+. In another specific embodiment, said selecting
comprises selecting stem cells that are also CD34, CD38.sup.- or
CD45.sup.-. In another specific embodiment, said selecting
comprises selecting placental stem cells that are also CD34.sup.-,
CD38.sup.-, CD45.sup.-, CD73.sup.+ and CD105.sup.+. In another
specific embodiment, said selecting also comprises selecting a
plurality of placental stem cells that forms one or more
embryoid-like bodies when cultured under conditions that allow the
formation of embryoid-like bodies.
[0074] In another embodiment, provided herein is a method of
selecting a plurality of placental stem cells from a plurality of
placental cells, comprising selecting a plurality of placental
cells wherein at least 10%, at least 20%, at least 30%, at least
40%, at least 50% at least 60%, at least 70%, at least 80%, at
least 90%, or at least 95% of said cells are CD73.sup.+,
CD105.sup.+, CD200.sup.+ placental stem cells. In a specific
embodiment, said selecting comprises selecting stem cells that are
also HLA-G.sup.+. In another specific embodiment, said selecting
comprises selecting placental stem cells that are also CD34.sup.-,
CD38.sup.- or CD45.sup.-. In another specific embodiment, said
selecting comprises selecting placental stem cells that are also
CD34.sup.-, CD38.sup.- and CD45.sup.-. In another specific
embodiment, said selecting comprises selecting placental stem cells
that are also CD34.sup.-, CD38.sup.-, CD45.sup.-, and HLA-G.sup.+.
In another specific embodiment, said selecting additionally
comprises selecting a population of placental cells that produces
one or more embryoid-like bodies when the population is cultured
under conditions that allow the formation of embryoid-like
bodies.
[0075] In another embodiment, provided herein is a method of
selecting a plurality of placental stem cells from a plurality of
placental cells, comprising selecting a plurality of placental
cells wherein at least 10%, at least 20%, at least 30%, at least
40%, at least 50% at least 60%, at least 70%, at least 80%, at
least 90%, or at least 95% of said cells are CD200.sup.+,
OCT-4.sup.+ placental stem cells. In a specific embodiment, said
selecting comprises selecting placental stem cells that are also
CD73.sup.+ and CD105.sup.+. In another specific embodiment, said
selecting comprises selecting placental stem cells that are also
HLA-G.sup.+. In another specific embodiment, said selecting
comprises selecting placental stem cells that are also CD34.sup.-,
CD38.sup.- and CD45.sup.-. In another specific embodiment, said
selecting comprises selecting placental stem cells that are also
CD34.sup.-, CD38.sup.-, CD45.sup.-, CD73.sup.+, CD105.sup.+ and
HLA-G.sup.+.
[0076] In another embodiment, provided herein is a method of
selecting a plurality of placental stem cells from a plurality of
placental cells, comprising selecting a plurality of placental
cells wherein at least 10%, at least 20%, at least 30%, at least
40%, at least 50% at least 60%, at least 70%, at least 80%, at
least 90%, or at least 95% of said cells are CD73.sup.+,
CD105.sup.+ and HLA-G.sup.+ placental stem cells. In a specific
embodiment, said selecting comprises selecting placental stem cells
that are also CD34.sup.-, CD38.sup.- or CD45.sup.-. In another
specific embodiment, said selecting comprises selecting placental
stem cells that are also CD34.sup.-, CD38.sup.- and CD45.sup.-. In
another specific embodiment, said selecting comprises selecting
placental stem cells that are also CD200.sup.+. In another specific
embodiment, said selecting comprises selecting placental stem cells
that are also CD34.sup.-, CD38.sup.-, CD45.sup.-, OCT-4.sup.+ and
CD200.sup.+.
[0077] In another embodiment, provided herein is a method of
selecting a plurality of placental stem cells from a plurality of
placental cells, comprising selecting a plurality of placental
cells wherein at least 10%, at least 20%, at least 30%, at least
40%, at least 50% at least 60%, at least 70%, at least 80%, at
least 90%, or at least 95% of said cells are CD73.sup.+,
CD105.sup.+ placental stem cells, and wherein said plurality forms
one or more embryoid-like bodies under conditions that allow
formation of embryoid-like bodies. In a specific embodiment, said
selecting comprises selecting placental stem cells that are also
CD34.sup.-, CD38.sup.- or CD45.sup.-. In another specific
embodiment, said selecting comprises selecting placental stein
cells that are also CD34.sup.-, CD38.sup.- and CD45.sup.-. In
another specific embodiment, said selecting comprises selecting
placental stem cells that are also OCT-4.sup.+. In a more specific
embodiment, said selecting comprises selecting placental stem cells
that are also OCT-4.sup.+, CD34.sup.-, CD38.sup.- and
CD45.sup.-.
[0078] In another embodiment, provided herein is a method of
selecting a plurality of placental stem cells from a plurality of
placental cells, comprising selecting a plurality of placental
cells wherein at least 10%, at least 20%, at least 30%, at least
40%, at least 50% at least 60%, at least 70%, at least 80%, at
least 90%, or at least 95% of said isolated placental cells are
OCT4.sup.+ stem cells, and wherein said plurality forms one or more
embryoid-like bodies under conditions that allow formation of
embryoid-like bodies. In a specific embodiment, said selecting
comprises selecting placental stem cells that are also CD73.sup.+
and CD105.sup.+. In another specific embodiment, said selecting
comprises selecting placental stem cells that are also CD34.sup.-,
CD38.sup.-, or CD45.sup.-. In another specific embodiment, said
selecting comprises selecting placental stem cells that are also
CD200.sup.+. In a more specific embodiment, said selecting
comprises selecting placental stem cells that are also CD73.sup.+,
CD105.sup.+, CD200.sup.+, CD34, CD38.sup.-, and CD45.sup.-.
[0079] Also provided herein are methods of producing populations,
or pluralities, of placental stem cells; such cells can be used in
the methods and compositions provided herein. For example, provided
herein is a method of producing a cell population, comprising
selecting any of the pluralities of placental stem cells described
above, and isolating the plurality of placental stem cells from
other cells, e.g., other placental cells. In a specific embodiment,
provided herein is a method of producing a cell population
comprising selecting placental cells, wherein said placental cells
(a) adhere to a substrate, and (b) express CD200 and HLA-G, or
express CD73, CD105, and CD200, or express CD200 and OCT-4, or
express CD73, CD105, and HLA-G, or express CD73 and CD105 and
facilitate the formation of one or more embryoid-like bodies in a
population of placental cells that comprise the stem cell, when
said population is cultured under conditions that allow formation
of embryoid-like bodies, or express OCT-4 and facilitate the
formation of one or more embryoid-like bodies in a population of
placental cells that comprise the stem cell, when said population
is cultured under conditions that allow formation of embryoid-like
bodies.
[0080] In a more specific embodiment, provided herein is a method
of producing a cell population comprising selecting placental stem
cells that (a) adhere to a substrate, and (b) express CD200 and
HLA-G; and isolating said placental stem cells from other cells to
form a cell population. In another specific embodiment, provided
herein is a method of producing a cell population comprising
selecting placental stem cells that (a) adhere to a substrate, and
(b) express CD73, CD105, and CD200; and isolating said placental
stem cells from other cells to form a cell population. In another
specific embodiment, provided herein is a method of producing a
cell population comprising selecting placental stem cells that (a)
adhere to a substrate, and (b) express CD200 and OCT-4; and
isolating said placental stem cells from other cells to form a cell
population. In another specific embodiment, provided herein is a
method of producing a cell population comprising selecting
placental stem cells that (a) adhere to a substrate, (b) express
CD73 and CD105, and (c) form embryoid-like bodies when cultured
under conditions allowing the formation of embryoid-like bodies;
and isolating said placental stem cells from other cells to form a
cell population. In another specific embodiment, provided herein is
a method of producing a cell population comprising selecting
placental stem cells that (a) adhere to a substrate, and (b)
express CD73, CD105, and HLA-G; and isolating said placental stem
cells from other cells to form a cell population. A method of
producing a cell population comprising selecting placental stem
cells that (a) adhere to a substrate, (b) express OCT-4, and (c)
form embryoid-like bodies when cultured under conditions allowing
the formation of embryoid-like bodies; and isolating said placental
stem cells from other cells to form a cell population.
[0081] 5.2.4 Growth in Culture
[0082] The growth of the placental stem cells described herein, as
for any mammalian cell, depends in part upon the particular medium
selected for growth. Under optimum conditions, placental stem cells
typically double in number in 3-5 days. During culture, the
placental stem cells provided 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.
[0083] Populations of isolated placental cells that comprise the
placental stem cells provided herein, when cultured under
appropriate conditions, form embryoid-like bodies, that is,
three-dimensional clusters of cells grow atop the adherent stem
cell layer. Cells within the embryoid-like bodies are expected to
express markers associated with very early stem cells, e.g., OCT-4,
Nanog, SSEA3 and SSEA4. Cells within the embryoid-like bodies are
typically not adherent to the culture substrate, as are the
placental stem cells described herein, but remain attached to the
adherent cells during culture. Embryoid-like body cells are
dependent upon the adherent placental stem cells for viability, as
embryoid-like bodies do not form in the absence of the adherent
stem cells. The adherent placental stem cells thus facilitate the
growth of one or more embryoid-like bodies in a population of
placental cells that comprise the adherent placental stem cells.
Without wishing to be bound by theory, the cells of the
embryoid-like bodies are thought to grow on the adherent placental
stem cells much as embryonic stem cells grow on a feeder layer of
cells. Mesenchymal stem cells, e.g., bone marrow-derived
mesenchymal stem cells, do not develop embryoid-like bodies in
culture.
[0084] 5.2.5 Differentiation
[0085] The placental stem cells, useful in the methods provided
herein, are differentiable into different committed cell lineages.
For example, the placental stem cells can be differentiated into
cells of an hepatocytic lineage. Such differentiation can be
accomplished, for example, by any method known in the art for
differentiating, e.g., bone marrow-derived mesenchymal stem cells
into similar cell lineages. See U.S. patent application Ser. No.
11/648,813, filed Dec. 28, 2006, the disclosure of which is hereby
incorporated herein by reference in its entirety.
[0086] The placental stem cells to be differentiated can be
contained in a polymer carrier, e.g., alginate. In one embodiment,
provided herein is a composition comprising a plurality of cells
encapsulated in alginate. The cells can be placental stem cells,
not contacted with conditions that cause differentiation of a
placental stem cell into a hepatocyte or a hepatogenic cell. The
cells can also be hepatogenic cells or hepatocytes. The cells can
also be a combination of any of the foregoing. Hepatogenic cells
and/or hepatocytes contained within the polymer, e.g., alginate,
are cells that are differentiated from placental stem cells. In one
embodiment, said cells express at least one marker of a hepatocyte
not expressed by, or expressed to a detectably different degree
than, a placental stem cell. Preferably, the polymer, e.g.,
alginate, is in the form of beads that encapsulate a plurality of
placental stem cells, hepatogenic cells, hepatocytes, or
combination thereof. The beads can vary in size. for example, the
beads can vary from about (e.g., .+-.10%) 100 .mu.m to about 1000
.mu.m, between about In a specific embodiment, said beads are from
about 200 .mu.m to about 800 .mu.m in size. In another specific
embodiment, said beads average about 500 .mu.m in size.
5.3 CD34.sup.+CD45.sup.- Placental Stem Cells and Cell Populations
Comprising Them
[0087] In another aspect, provided herein are isolated
hematopoietic CD34.sup.+, CD45.sup.- placental stem cells and/or
CD34.sup.+CD45.sup.dim placental stem cells, and isolated
populations of cells enriched in CD34.sup.+, CD45.sup.- placental
stem cells. As used herein, "CD34.sup.+, CD45.sup.- placental stem
cell" indicates a cell that is capable of differentiating into at
least one type of mature blood cell or progenitor of a mature blood
cell, which is obtained from the placenta but not from placental or
umbilical cord blood, and in certain embodiments includes
CD34.sup.+CD45.sup.dim placental stem cells. The CD34.sup.+,
CD45.sup.- placental stem cell is detectably positive for the
marker CD34, e.g., using a labeled antibody to CD34, and is dim or
negative for CD45, e.g., does not label with a
fluorescently-labeled antibody to CD45 such that the cell is
detectable above background. CD34.sup.+, CD45.sup.- placental stem
cells and CD34.sup.+CD45.sup.dim placental stem cells placental
stem cells are typically non-adherent, that is, they do not adhere
to a tissue culture surface.
[0088] Also provided herein are populations of placental cells
enriched for CD34.sup.+CD45.sup.- placental stem cells and/or
CD34.sup.+CD45.sup.dim placental stem cells. As used herein,
"enriched" indicates that the placental stem cell population
comprises a higher number or higher percentage of
CD34.sup.+CD45.sup.- cells than is found in placental perfusate,
when said placental perfusate comprises about 750 mL of perfusion
solution (e.g., 0.9% NaCl) passed through a human placenta at a
rate of about 50 mL/min. after the placenta has been drained of
cord blood and placental blood and pre-perfused with about 100 mL
of perfusion solution. In a specific embodiment,
CD34.sup.+CD45.sup.- placental stem cells are present in a cell
population at a higher percentage than found in placental
perfusate, e.g., the placental stem cells constitute at least 1%,
2%, 3%, 4%, 5%, 7%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90% or 95% of the cells in the cell population. In another specific
embodiment, the stem cell population comprises about, or at least,
1.times.10.sup.3, 5.times.10.sup.3, 1.times.10.sup.4,
5.times.10.sup.4, 1.times.10.sup.5, 5.times.10.sup.5,
1.times.10.sup.6, 5.times.10.sup.6 or 1.times.10.sup.7
CD34.sup.+CD45.sup.- placental stem cells.
[0089] Typically, from a population of about 100 million total
nucleated cells in placental perfusate, about 5% are CD34.sup.+
cells, and the final yield from a placenta, taking into account
viability, is about 1 million CD34.sup.+ stem cells. CD34.sup.+
stem cells, when collected, appear round, with low cellular
complexity.
[0090] Populations of placental stem cells that comprise
CD34.sup.+CD45.sup.- placental stem cells can also comprise other
populations of CD34.sup.+ cells, for example, CD34.sup.+CD38.sup.+
cells and/or CD34.sup.+CD38.sup.- cells. In a specific embodiment,
said CD34.sup.+CD38.sup.- cells comprise
CD34.sup.+CD38.sup.-lin.sup.- stem cells. In another specific
embodiment, said CD34.sup.+ placental stem cells comprise cells
that are ALDH.sup.+, that is, CD34.sup.+, ALDH.sup.+ placental stem
cells.
[0091] In another embodiment, CD34.sup.+, CD45.sup.- placental stem
cells are combined with stem cells of a second type. In one
embodiment, the stem cells of a second type comprises adherent
placental stem cells that are OCT-4.sup.+ or ABC-p.sup.+. In
another more specific embodiment, said adherent placental stem
cells comprise cells that are OCT4.sup.+ABC-p.sup.+. In another
more specific embodiment, said hematopoietic placental stem cells
are contained within placental perfusate substantially lacking red
blood cells and cellular debris.
[0092] In another embodiment, the adherent placental stem cells,
which are combined with the hematopoietic placental stem cells,
e.g., CD34 CD45.sup.- placental stem cells, comprise cells that
express one or more of markers CD10, CD29, CD44, CD54, CD90, CD73
or CD105, and lack expression of one or more of markers CD34, CD38,
CD45, SSEA3 and SSEA4. In another embodiment, the adherent
placental stem cells comprise cells that are positive for CD10,
CD29, CD44, CD54, CD90, CD73 or CD105, and negative for CD34, CD38,
CD45, SSEA3 and SSEA4. In another embodiment, the adherent
placental stem cells comprise cells that comprise one or more of
markers CD10, CD29, CD44, CD54, CD90, CD73 and CD105, and lack one
or more of markers CD34, CD38, CD45, SSEA3 and SSEA4. In another
embodiment, the adherent placental stem cells comprise cells that
are positive for CD10, CD29, CD44, CD54, CD90, CD73 and CD105, and
negative for CD34, CD38, CD45, SSEA3 and SSEA4. In another
embodiment, the adherent placental stem cells comprise CD34.sup.-
cells. In a specific embodiment, the adherent placental stem cells
are CD34.sup.- CD38 placental stem cells. In another embodiment,
the adherent placental stem cells comprise cells that are positive
for at least one of CD10, CD29, CD33, CD44, CD73, CD105, CD117, and
CD133, and negative for at least one of CD34 or CD45. In another
embodiment, the adherent placental stem cells comprise cells that
are positive for CD10, CD29, CD33, CD44, CD73, CD105, CD117, and
CD133, and negative for CD34 or CD45. In a more specific
embodiment, the adherent placental stem cells comprise cells that
are HLA-ABC.sup.+. In a more specific embodiment, the adherent
placental stem cells comprise cells that are HLA-ABC.sup.-. In a
more specific embodiment, the adherent placental stem cells
comprise cells that are HLA-DR.sup.+. In a more specific
embodiment, the adherent placental stem cells comprise cells that
are HLA-DR.sup.-. In another specific embodiment, the adherent
placental stem cells comprise cells that are CD200.sup.+ or
HLA-G.sup.+. In another specific embodiment, the adherent placental
stem cells comprise cells that are CD200.sup.+ and IILA-G.sup.+. In
another specific embodiment, the adherent placental stem cells
comprise cells that are CD73.sup.+, CD105.sup.+ and CD200.sup.+. In
another specific embodiment, the adherent placental stem cells
comprise cells that are CD200 and OCT-4.sup.+. In another specific
embodiment, the adherent placental stem cells comprise cells that
are CD73.sup.+, CD105.sup.+ and facilitate the formation of
embryoid-like bodies in a population of isolated placental cells
comprising said stem cells, when said population is cultured under
conditions that allow the formation of embryoid-like bodies. In
another specific embodiment, the adherent placental stem cells
comprise cells that are CD73.sup.+, CD105.sup.+ and HLA-G.sup.+. In
another specific embodiment, the adherent placental stem cells
comprise cells that are OCT-4.sup.+ and facilitate the formation of
embryoid-like bodies in a population of isolated placental cells
comprising said stem cells, when said population is cultured under
conditions that allow the formation of embryoid-like bodies.
[0093] In another embodiment, the second population of stem cells
comprises cord blood stem cells and/or bone marrow stem cells.
[0094] CD34.sup.+CD45.sup.- placental stem cells and other stem
cells, to be combined, may be identically HLA-matched, that is,
they may be derived from the same individual. In another
embodiment, the CD34.sup.+CD45.sup.- placental stem cells and other
stem cells may be HLA-mismatched, that is, they may be derived from
different individuals. The combination of CD34.sup.+CD45.sup.-
placental stem cells and other stem cells can comprise stem cells
that are either HLA-matched, partially HLA-matched, and/or
HLA-mismatched to an intended recipient. For combined stem cell
populations comprising non-cord blood stem cells, it is preferred
that at least the stem cells from a second source be HLA-matched or
partially HLA-matched to an intended recipient.
[0095] In various embodiments, the ratio of CD34.sup.+CD45.sup.-
placental stem cells to a second type of stem cell can be 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, or comparing total numbers of stem cells in each
population. In various preferred embodiments, the ratio is about
1:10 to about 10:1, or is about 3:1 to about 1:3.
[0096] Further provided herein are populations of stem cells
comprising CD34.sup.+CD45.sup.- placental stem cells and a second
type of stem cell, wherein the population of stem cells comprises a
therapeutically-effective amount of CD34.sup.+CD45.sup.- placental
stem cells, second type of stem cell, or both. In various
combinations, the populations comprise at least 1.times.10.sup.4,
5.times.10.sup.4, 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, or 1.times.10.sup.11 CD34.sup.+CD45.sup.-
placental stem cells, second type of stem cell, or both, or no more
than 1.times.10.sup.4, 5.times.10.sup.4, 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, or 1.times.10.sup.11
CD34.sup.+CD45.sup.- placental stem cells, second type of stem
cells, or both, or, alternatively, 3, 5, 10, 20, 30, 40, or 50
units or more, of total nucleated cells, from both the placental
stem cell population and the second type of stem cell.
5.4 Methods of Obtaining Placental Stem Cells
[0097] 5.4.1 Stem Cell Collection Composition
[0098] The placental stem cells, e.g., adherent placental stem
cells and/or CD34.sup.+, CD45.sup.- placental stem cells, used in
the methods and compositions provided herein can be collected by
any means known in the art for collecting cells from tissue, e.g.,
by perfusion of a placenta and/or by physical and/or enzymatic
disruption of placental tissue. Generally, stem cells are obtained
from a mammalian placenta using a physiologically-acceptable
solution, e.g., a stem cell collection composition. A stem cell
collection composition is described in detail in related U.S.
Application Publication No. 2007/0190042, entitled "Improved
Composition for Collecting Placental Stem Cells and Preserving
Organs" filed on Dec. 28, 2006.
[0099] The stem cell collection composition can comprise any
physiologically-acceptable solution suitable for the collection
and/or culture of stem cells, for example, a saline solution (e.g.,
phosphate-buffered saline, Kreb's solution, modified Kreb's
solution, Eagle's solution, 0.9% NaCl. etc.), a culture medium
(e.g., DMEM, H.DMEM, etc.), and the like.
[0100] The stem cell collection composition can comprise one or
more components that tend to preserve placental stem cells, that
is, prevent the placental stem cells from dying, or delay the death
of the placental stem cells, reduce the number of placental stem
cells in a population of cells that die, or the like, from the time
of collection to the time of culturing. Such components can be,
e.g., an apoptosis inhibitor (e.g., a caspase inhibitor or JNK
inhibitor); a vasodilator (e.g., magnesium sulfate, an
antihypertensive drug, atrial natriuretic peptide (ANP),
adrenocorticotropin, corticotropin-releasing hormone, sodium
nitroprusside, hydralazine, adenosine triphosphate, adenosine,
indomethacin or magnesium sulfate, a phosphodiesterase inhibitor,
etc.); a necrosis inhibitor (e.g.,
2-(1H-Indol-3-yl)-3-pentylamino-maleimide, pyrrolidine
dithiocarbamate, or clonazepam); a TNF-.alpha. inhibitor; and/or an
oxygen-carrying perfluorocarbon (e.g., perfluorooctyl bromide,
perfluorodecyl bromide, etc.).
[0101] The stem cell collection composition can comprise one or
more tissue-degrading enzymes, e.g., a metalloprotease, a serine
protease, a neutral protease, a hyaluronidase, 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, thennolysin,
elastase, trypsin, LIBERASE, hyaluronidase, and the like.
[0102] The stem cell collection composition can comprise a
bacteriocidally or bacteriostatically effective amount of an
antibiotic. In certain non-limiting embodiments, the antibiotic is
a macrolide (e.g., tobramycin), a cephalosporin (e.g., cephalexin,
cephradine, cefuroxime, cefprozil, cefaclor, cefixime or
cefadroxil), a clarithromycin, an erythromycin, a penicillin (e.g.,
penicillin V) or a quinolone (e.g., ofloxacin, ciprofloxacin or
norfloxacin), a tetracycline, a streptomycin, etc. In a particular
embodiment, the antibiotic is active against Gram(+) and/or Gram(-)
bacteria, e.g., Pseudomonas aeruginosa, Staphylococcus aureus, and
the like.
[0103] The stem cell collection composition can also comprise one
or more of the following compounds: adenosine (about 1 mM to about
50 mM); D-glucose (about 20 mM to about 100 mM); magnesium ions
(about 1 mM to about 50 mM); a macromolecule of molecular weight
greater than 20,000 daltons, in one embodiment, present in an
amount sufficient to maintain endothelial integrity and cellular
viability (e.g., a synthetic or naturally occurring colloid, a
polysaccharide such as dextran or a polyethylene glycol present at
about 25 g/l to about 100 g/l, or about 40 g/l to about 60 g/l); an
antioxidant (e.g., butylated hydroxyanisole, butylated
hydroxytoluene, glutathione, vitamin C or vitamin E present at
about 25 .mu.M to about 100 .mu.M); a reducing agent (e.g.,
N-acetylcysteine present at about 0.1 mM to about 5 mM); an agent
that prevents calcium entry into cells (e.g., verapamil present at
about 2 .mu.M to about 25 .mu.M); nitroglycerin (e.g., about 0.05
g/L to about 0.2 g/L); an anticoagulant, in one embodiment, present
in an amount sufficient to help prevent clotting of residual blood
(e.g., heparin or hirudin present at a concentration of about 1000
units/1 to about 100,000 units/1); or an amiloride containing
compound (e.g., amiloride, ethyl isopropyl amiloride, hexamethylene
amiloride, dimethyl amiloride or isobutyl amiloride present at
about 1.0 .mu.M to about 5 .mu.M).
[0104] 5.4.2 Collection and Handling of Placenta
[0105] Generally, a human placenta is recovered shortly after its
expulsion after birth. In a preferred embodiment, the placenta is
recovered from a patient after informed consent and after a
complete medical history of the patient is taken and is associated
with the placenta. Preferably, the medical history continues after
delivery. Such a medical history can be used to coordinate
subsequent use of the placenta or the stem cells harvested
therefrom. For example, human placental stem cells can be used, in
light of the medical history, for personalized medicine for the
infant associated with the placenta, or for parents, siblings or
other relatives of the infant.
[0106] Prior to recovery of placental stem cells, the umbilical
cord blood and placental blood are removed. In certain embodiments,
after delivery, the cord blood in the placenta is recovered. The
placenta can be subjected to a conventional cord blood recovery
process. Typically a needle or cannula is used, with the aid of
gravity, to exsanguinate the placenta (see, e.g., Anderson, U.S.
Pat. No. 5,372,581; Hessel et al., U.S. Pat. No. 5,415,665). The
needle or cannula is usually placed in the umbilical vein and the
placenta can be gently massaged to aid in draining cord blood from
the placenta. Such cord blood recovery may be performed
commercially, e.g., LifebankUSA, Cedar Knolls, N.J.; ViaCord; Cord
Blood Registry; Cryocell; and the like. Preferably, the placenta is
gravity drained without further manipulation so as to minimize
tissue disruption during cord blood recovery.
[0107] Typically, a placenta is transported from the delivery or
birthing room to another location, e.g., a laboratory, for recovery
of cord blood and collection of stem cells by, e.g., perfusion or
tissue dissociation. The placenta is preferably transported in a
sterile, thermally insulated transport device (maintaining the
temperature of the placenta between 20-28.degree. C.), for example,
by placing the placenta, with clamped proximal umbilical cord, in a
sterile zip-lock plastic bag, which is then placed in an insulated
container. In another embodiment, the placenta is transported in a
cord blood collection kit substantially as described in U.S. Pat.
No. 7,147,626 or in United States Patent Application Publication
No. 2006/0060494. 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.
[0108] The placenta, prior to stem cell collection, can be stored
under sterile conditions and at either room temperature or at a
temperature of 5 to 25.degree. C. (centigrade). The placenta may be
stored for a period of longer than forty eight hours, and
preferably for a period of four to twenty-four hours prior to
perfusing the placenta to remove any residual cord blood. The
placenta is preferably stored in an anticoagulant solution at a
temperature of 5.degree. C. to 25.degree. C. (centigrade). Suitable
anticoagulant solutions are well known in the art. For example, a
solution of heparin or warfarin sodium can be used. In a preferred
embodiment, the anticoagulant solution comprises a solution of
heparin (e.g., 1% w/w in 1:1000 solution). The exsanguinated
placenta is preferably stored for no more than 36 hours before
placental stem cells are collected.
[0109] The mammalian placenta or a part thereof, once collected and
prepared generally as above, can be treated in any art-known
manner, e.g., can be perfused or disrupted, e.g., digested with one
or more tissue-disrupting enzymes, to obtain stem cells.
[0110] 5.4.3 Physical Disruption and Enzymatic Digestion of
Placental Tissue
[0111] In one embodiment, stem cells are collected from a mammalian
placenta by physical disruption, e.g., enzymatic digestion, of the
organ. For example, the placenta, or a portion thereof, may be,
e.g., crushed, sheared, minced, diced, chopped, macerated or the
like, while in contact with the stem cell collection composition
described herein, and the tissue subsequently digested with one or
more enzymes. The placenta, or a portion thereof, may also be
physically disrupted and digested with one or more enzymes, and the
resulting material then immersed in, or mixed into, the stem cell
collection composition described herein. Any method of physical
disruption can be used, provided that the method of disruption
leaves a plurality, more preferably a majority, and more preferably
at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the cells in said
organ viable, as determined by, e.g., trypan blue exclusion.
[0112] The placenta can be dissected into components prior to
physical disruption and/or enzymatic digestion and stem cell
recovery. For example, placental stem cells can be obtained from
all or a portion of the amniotic membrane, chorion, placental
cotyledons, or any combination thereof. Preferably, placental stem
cells are obtained from placental tissue comprising amnion and
chorion. Typically, placental stem cells can be obtained by
disruption of a small block of placental tissue, e.g., a block of
placental tissue that is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,
30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800,
900 or about 1000 cubic millimeters in volume.
[0113] A preferred stem cell collection composition comprises one
or more tissue-disruptive enzyme(s). Enzymatic digestion preferably
uses a combination of enzymes, e.g., a combination of a matrix
metalloprotease and a neutral protease, for example, a combination
of collagenase and dispase. In one embodiment, enzymatic digestion
of placental tissue uses a combination of a matrix metalloprotease,
a neutral protease, and a mucolytic enzyme for digestion of
hyaluronic acid, such as a combination of collagenase, dispase, and
hyaluronidase or a combination of LIBERASE (Boehringer Mannheim
Corp., Indianapolis, Ind.) and hyaluronidase. Other enzymes that
can be used to disrupt placenta tissue include papain,
deoxyribonucleases, serine proteases, such as trypsin,
chymotrypsin, or elastase. Serine proteases may be inhibited by
alpha 2 microglobulin in serum and therefore the medium used for
digestion is usually serum-free. EDTA and DNase are commonly used
in enzyme digestion procedures to increase the efficiency of cell
recovery. The digestate is preferably diluted so as to avoid
trapping stem cells within the viscous digest.
[0114] Any combination of tissue digestion enzymes can be used.
Typical concentrations for tissue digestion enzymes include, e.g.,
50-200 U/mL for collagenase I and collagenase IV, 1-10 U/mL for
dispase, and 10-100 U/mL for elastase. Proteases can be used in
combination, that is, two or more proteases in the same digestion
reaction, or can be used sequentially in order to liberate
placental stem cells. For example, in one embodiment, a placenta,
or part thereof, is digested first with an appropriate amount of
collagenase I at 2 mg/ml for 30 minutes, followed by digestion with
trypsin, 0.25%, for 10 minutes, at 37.degree. C. Scrine proteases
are preferably used consecutively following use of other
enzymes.
[0115] 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 stein cell collection
composition comprising the stem cells, or to a solution in which
the tissue is disrupted and/or digested prior to isolation of the
stem cells with the stem cell collection composition.
[0116] It will be appreciated that where an entire placenta, or
portion of a placenta comprising both fetal and maternal cells (for
example, where the portion of the placenta comprises the chorion or
cotyledons), the placental stem cells collected will comprise a mix
of placental stem cells derived from both fetal and maternal
sources. Where a portion of the placenta that comprises no, or a
negligible number of, maternal cells (for example, amnion), the
placental stem cells collected will comprise almost exclusively
fetal placental stem cells.
[0117] 5.4.4 Placental Perfusion
[0118] Placental stem cells useful in the methods and compositions
provided herein can also be obtained by perfusion of the mammalian
placenta. Methods of perfusing mammalian placenta to obtain stem
cells are disclosed, e.g., in Hariri, U.S. Pat. Nos. 7,045,148 and
7,255,879, and in U.S. Application Publication No. 2007/0190042,
entitled "Improved Composition for Collecting and Preserving
Organs."
[0119] Placental stem cells can be collected by perfusion, e.g.,
through the placental vasculature, using, e.g., a stem cell
collection composition as a perfusion solution. In one embodiment,
a mammalian placenta is perfused by passage of perfusion solution
through either or both of the umbilical artery and umbilical vein.
The flow of perfusion solution through the placenta may be
accomplished using, e.g., gravity flow into the placenta.
Preferably, the perfusion solution is forced through the placenta
using a pump, e.g., a peristaltic pump. The umbilical vein can be,
e.g., cannulated with a cannula, e.g., a TEFLON.RTM. or plastic
cannula, that is connected to a sterile connection apparatus, such
as sterile tubing. The sterile connection apparatus is connected to
a perfusion manifold.
[0120] In preparation for perfusion, the placenta is preferably
oriented (e.g., suspended) in such a manner that the umbilical
artery and umbilical vein are located at the highest point of the
placenta. The placenta can be perfused by passage of a perfusion
fluid, e.g., a stem cell collection composition as provided herein,
through the placental vasculature, or through the placental
vasculature and surrounding tissue. In one embodiment, the
umbilical artery and the umbilical vein are connected
simultaneously to a pipette that is connected via a flexible
connector to a reservoir of the perfusion solution. The perfusion
solution is passed into the umbilical vein and artery. The
perfusion solution exudes from and/or passes through the walls of
the blood vessels into the surrounding tissues of the placenta, and
is collected in a suitable open vessel from the surface of the
placenta that was attached to the uterus of the mother during
gestation. The perfusion solution may also be introduced through
the umbilical cord opening and allowed to flow or percolate out of
openings in the wall of the placenta which interfaced with the
maternal uterine wall. In another embodiment, the perfusion
solution is passed through the umbilical veins and collected from
the umbilical artery, or is passed through the umbilical artery and
collected from the umbilical veins, that is, is passed through only
the placental vasculature (fetal tissue). This can be referred to
as the "closed circuit" method of perfusion. 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. 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.
[0121] 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.
[0122] 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 of perfusion fluid is
adequate to initially exsanguinate the placenta, but more or less
perfusion fluid may be used depending on the observed results.
[0123] The volume of perfusion liquid used to collect placental
stem cells may vary depending upon the number of stem cells to be
collected, the size of the placenta, the number of collections to
be made from a single placenta, etc. In various embodiments, the
volume of perfusion liquid may be from 50 mL to 5000 mL, 50 mL to
4000 mL, 50 mL to 3000 mL, 100 mL to 2000 mL, 250 mL to 2000 mL,
500 mL to 2000 mL, or 750 mL to 2000 mL. Typically, the placenta is
perfused with 700-800 mL of perfusion liquid following
exsanguination.
[0124] The placenta can be perfused a plurality of times over the
course of several hours or several days. Where the placenta is to
be perfused a plurality of times, it may be maintained or cultured
under aseptic conditions in a container or other suitable vessel,
and perfused with the stem cell collection composition, or a
standard perfusion solution (e.g., a normal saline solution such as
phosphate buffered saline ("PBS") with or without an anticoagulant
(e.g., heparin, warfarin sodium, coumarin, bishydroxycotunarin),
and/or with or without an antimicrobial agent (e.g.,
.beta.-mercaptoethanol (0.1 mM); antibiotics such as streptomycin
(e.g., at 40-100 .mu.g/ml), penicillin (e.g., at 40 U/ml),
amphotericin B (e.g., at 0.5 .mu.g/ml). In one embodiment, an
isolated placenta is maintained or cultured for a period of time
without collecting the perfusate, such that the placenta is
maintained or cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or 2 or 3
or more days before perfusion and collection of perfusate. The
perfused placenta can be maintained for one or more additional
time(s), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24 or more hours, and perfused a
second time with, e.g., 700-800 mL perfusion fluid. The placenta
can be perfused 1, 2, 3, 4, 5 or more times, for example, once
every 1, 2, 3, 4, 5 or 6 hours. In a preferred embodiment,
perfusion of the placenta and collection of perfusion solution,
e.g., stem cell collection composition, is repeated until the
number of recovered nucleated cells falls below 100 cells/ml. The
perfusates at different time points can be further processed
individually to recover time-dependent populations of cells, e.g.,
stem cells. Perfusates from different time points can also be
pooled.
[0125] Without wishing to be bound by any theory, after
exsanguination and a sufficient time of perfusion of the placenta,
placental stem cells are believed to migrate into the exsanguinated
and perfused microcirculation of the placenta where, according to
the methods described herein, they are collected, preferably by
washing into a collecting vessel by perfusion. Perfusing the
isolated placenta not only serves to remove residual cord blood but
also provide the placenta with the appropriate nutrients, including
oxygen. The placenta may be cultivated and perfused with a similar
solution which was used to remove the residual cord blood cells,
preferably, without the addition of anticoagulant agents.
[0126] Perfusion according to the methods described herein
typically results in the collection of significantly more placental
stem cells than the number obtainable from a mammalian placenta not
perfused with said solution, and not otherwise treated to obtain
stem cells (e.g., by tissue disruption, e.g., enzymatic digestion).
In this context, "significantly more" means at least 10% more.
Perfusion as described herein yields significantly more placental
stem cells than, e.g., the number of placental stem cells
obtainable from culture medium in which a placenta, or portion
thereof, has been cultured.
[0127] Stem cells can be isolated from placenta by perfusion with a
solution comprising one or more proteases or other
tissue-disruptive enzymes. In a specific embodiment, a placenta or
portion thereof (e.g., amniotic membrane, amnion and chorion,
placental lobule or cotyledon, or combination of any of the
foregoing) is brought to 25-37.degree. C., and is incubated with
one or more tissue-disruptive enzymes in 200 mL of a culture medium
for 30 minutes. Cells from the perfusate are collected, brought to
4.degree. C., and washed with a cold inhibitor mix comprising 5 mM
EDTA, 2 mM dithiothreitol and 2 mM beta-mercaptoethanol. The stem
cells are washed after several minutes with a cold (e.g., 4.degree.
C.) stem cell collection composition.
[0128] It will be appreciated that perfusion using the pan method,
that is, whereby perfusate is collected after it has exuded from
the maternal side of the placenta, results in a mix of fetal and
maternal cells. As a result, the cells collected by this method
comprise a mixed population of placental stem cells of both fetal
and maternal origin. In contrast, perfusion solely through the
placental vasculature, whereby perfusion fluid is passed through
one or two placental vessels and is collected solely through the
remaining vessel(s), results in the collection of a population of
placental stem cells almost exclusively of fetal origin.
[0129] 5.4.5 Isolation, Sorting, and Characterization of Placental
Stem Cells
[0130] 1 Stem cells from mammalian placenta, whether obtained by
perfusion or enyzmatic digestion, can initially be purified from
(i.e., be isolated from) other cells by Ficoll gradient
centrifugation. Such centrifugation can follow any standard
protocol for centrifugation speed, etc. In one embodiment, for
example, cells collected from the placenta are recovered from
perfusate by centrifugation at 5000.times.g for 15 minutes at room
temperature, which separates cells from, e.g., contaminating debris
and platelets. In another embodiment, placental perfusate is
concentrated to about 200 ml, gently layered over Ficoll, and
centrifuged at about 1100.times.g for 20 minutes at 22.degree. C.,
and the low-density interface layer of cells is collected for
further processing.
[0131] Cell pellets can be resuspended in fresh stem cell
collection composition, or a medium suitable for stem cell
maintenance, e.g., IMDM serum-free medium containing 2 U/ml heparin
and 2 mM EDTA (GibcoBRL, NY). The total mononuclear cell fraction
can be isolated, e.g., using LYMPHOPREP.TM. (Nycomed Pharma, Oslo,
Norway) according to the manufacturer's recommended procedure.
[0132] As used herein, "isolating" placental stem cells means to
remove at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%
of the cells with which the stem cells are normally associated in
the intact mammalian placenta. A stem cell from an organ is
"isolated" when it is present in a population of cells that
comprises fewer than 50% of the cells with which the stem cell is
normally associated in the intact organ.
[0133] Placental cells obtained by perfusion or digestion can, for
example, be further, or initially, isolated by differential
trypsinization using, e.g., a solution of 0.05% trypsin with 0.2%
EDTA (Sigma, St. Louis Mo.). Differential trypsinization is
possible because placental stem cells typically detach from plastic
surfaces within about five minutes whereas other adherent
populations typically require more than 20-30 minutes incubation.
The detached placental stem cells can be harvested following
trypsinization and trypsin neutralization, using, e.g., Trypsin
Neutralizing Solution (TNS, Cambrex). In one embodiment of
isolation of adherent cells, aliquots of, for example, about
5-10.times.10.sup.6 cells are placed in each of several T-75
flasks, preferably fibronectin-coated T75 flasks. In such an
embodiment, the cells can be cultured with commercially available
Mesenchymal Stem Cell Growth Medium (MSCGM.TM.) (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.TM.. Flasks
are preferably examined daily for the presence of various adherent
cell types and in particular, for identification and expansion of
clusters of fibroblastoid cells.
[0134] The number and type of cells collected from a mammalian
placenta can be monitored, for example, by measuring changes in
morphology and cell surface markers using standard cell detection
techniques such as flow cytometry, cell sorting,
immunocytochemistry (e.g., staining with tissue specific or
cell-marker specific antibodies) fluorescence activated cell
sorting (FACS), magnetic activated cell sorting (MACS), by
examination of the morphology of cells using light or confocal
microscopy, and/or by measuring changes in gene expression using
techniques well known in the art, such as PCR and gene expression
profiling. These techniques can be used, too, to identify cells
that are positive for one or more particular markers. For example,
using antibodies to CD34, one can determine, using the techniques
above, whether a cell comprises a detectable amount of CD34; if so,
the cell is CD34.sup.+. Likewise, if a cell produces enough OCT-4
RNA to be detectable by RT-PCR, or significantly more OCT-4 RNA
than an adult cell, the cell is OCT-4.sup.+ Antibodies to cell
surface markers (e.g., CD markers such as CD34) and the sequence of
stem cell-specific genes, such as OCT-4, are well-known in the
art.
[0135] Placental cells, particularly cells that have been isolated
by Ficoll separation, differential adherence, or a combination of
both, may be sorted using a fluorescence activated cell sorter
(FACS). Fluorescence activated cell sorting (FACS) is a well-known
method for separating particles, including cells, based on the
fluorescent properties of the particles (Kamarch, 1987, Methods
Enzymol, 151:150-165). Laser excitation of fluorescent moieties in
the individual particles results in a small electrical charge
allowing electromagnetic separation of positive and negative
particles from a mixture. In one embodiment, cell surface
marker-specific antibodies or ligands are labeled with distinct
fluorescent labels. Cells are processed through the cell sorter,
allowing separation of cells based on their ability to bind to the
antibodies used. FACS sorted particles may be directly deposited
into individual wells of 96-well or 384-well plates to facilitate
separation and cloning.
[0136] In one sorting scheme, stem cells from placenta are sorted
on the basis of expression of the markers CD34, CD38, CD44, CD45,
CD73, CD105, CD117, CD200, OCT-4 and/or HLA-G. This can be
accomplished in connection with procedures to select stem cells on
the basis of their adherence properties in culture. For example, an
adherence selection stem can be accomplished before or after
sorting on the basis of marker expression. In one embodiment, for
example, cells are sorted first on the basis of their expression of
CD34; CD34.sup.- cells are retained, and cells that are
CD200.sup.+HLA-G.sup.+ are separated from all other CD34.sup.-
cells. In another embodiment, cells from placenta are based on
their expression of markers CD200 and/or HLA-G; for example, cells
displaying either of these markers are isolated for further use.
Cells that express, e.g., CD200 and/or HLA-G can, in a specific
embodiment, be further sorted based on their expression of CD73
and/or CD105, or epitopes recognized by antibodies SH2, SH3 or SH4,
or lack of expression of CD34, CD38 or CD45. For example, in one
embodiment, placental cells are sorted by expression, or lack
thereof, of CD200, HLA-G, CD73, CD105, CD34, CD38 and CD45, and
placental cells that are CD200.sup.+, HLA-G.sup.+, CD73.sup.+,
CD105.sup.+, CD34.sup.-, CD38.sup.- and CD45.sup.- are isolated
from other placental cells for further use.
[0137] With respect to antibody-mediated detection and sorting of
placental stem 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.sup.- APC (Miltenyi), KDR-Biotin (CD309, Abcam),
CytokeratinK-Fitc (Sigma or Dako), HLA ALC-Fitc (BD), HLA DRDQDP-PE
(BD), .beta.-2-microglobulin-PE (BD), CD80.sup.- PE (BD) and
CD86.sup.- APC (BD).
[0138] Other antibody/label combinations that can be used include,
but are not limited to, CD45.sup.- PerCP (peridin chlorophyll
protein); CD44.sup.- PE; CD19.sup.- PE; CD10.sup.- F (fluorescein);
HLA-G-F and 7-amino-actinomycin-D (7-AAD); HLA-ABC-F; and the
like.
[0139] 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.
[0140] 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.
[0141] In another embodiment, magnetic beads can be used to
separate cells. The cells may be sorted using a magnetic activated
cell sorting (MACS) technique, a method for separating particles
based on their ability to bind magnetic beads (0.5-100 .mu.m
diameter). A variety of useful modifications can be performed on
the magnetic microspheres, including covalent addition of antibody
that specifically recognizes a particular cell surface molecule or
hapten. The beads are then mixed with the cells to allow binding.
Cells are then passed through a magnetic field to separate out
cells having the specific cell surface marker. In one embodiment,
these cells can then isolated and re-mixed with magnetic beads
coupled to an antibody against additional cell surface markers. The
cells are again passed through a magnetic field, isolating cells
that bound both the antibodies. Such cells can then be diluted into
separate dishes, such as microtiter dishes for clonal
isolation.
[0142] Placental stem cells can also be characterized and/or sorted
based on cell morphology and growth characteristics. For example,
placental stem cells can be characterized as having, and/or
selected on the basis of, e.g., a fibroblastoid appearance in
culture. Placental stem cells can also be characterized as having,
and/or be selected, on the basis of their ability to form
embryoid-like bodies. In one embodiment, for example, placental
cells that are fibroblastoid in shape, express CD73 and CD105, and
produce one or more embryoid-like bodies in culture are isolated
from other placental cells. In another embodiment, OCT-4.sup.+
placental cells that produce one or more embryoid-like bodies in
culture are isolated from other placental cells.
[0143] In another embodiment, placental stem cells can be
identified and characterized by a colony forming unit assay. Colony
forming unit assays are commonly known in the art, such as
MESENCULT.RTM. medium (Stem Cell Technologies, Inc., Vancouver
British Columbia)
[0144] Placental stem cells can be assessed for viability,
proliferation potential, and longevity using standard techniques
known in the art, such as trypan blue exclusion assay, fluorescein
diacetate uptake assay, propidium iodide uptake assay (to assess
viability); and thymidine uptake assay, MTT cell proliferation
assay (ATCC; 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.
[0145] Placental stem cells can also be separated from other
placental cells using other techniques known in the art, e.g.,
selective growth of desired cells (positive selection), selective
destruction of unwanted cells (negative selection); separation
based upon differential cell agglutinability in the mixed
population as, for example, with soybean agglutinin; freeze-thaw
procedures; filtration; conventional and zonal centrifugation;
centrifugal elutriation (counter-streaming centrifugation); unit
gravity separation; countercurrent distribution; electrophoresis;
and the like.
5.5 Culture of Placental Stem Cells
[0146] 5.5.1 Culture Media
[0147] Isolated placental stem cells, or placental stem cell
population, or cells or placental tissue from which placental stem
cells grow out, can be used to initiate, or seed, cell cultures.
Cells are generally transferred to sterile tissue culture vessels
either uncoated or coated with extracellular matrix or ligands such
as laminin, collagen (e.g., native or denatured), gelatin,
fibronectin, ornithine, vitronectin, and extracellular membrane
protein (e.g., MATRIGEL (BD Discovery Labware, Bedford,
Mass.)).
[0148] Placental stem cells can be cultured in any medium, and
under any conditions, recognized in the art as acceptable for the
culture of stem cells. Preferably, the culture medium comprises
serum. Placental stem cells can be cultured in, for example,
DMEM-LG (Dulbecco's Modified Essential Medium, low glucose)/MCDB
201 (chick fibroblast basal medium) containing ITS
(insulin-transferrin-selenium), LA+BSA (linoleic acid-bovine serum
albumin), dextrose, L-ascorbic acid, PDGF, EGF, IGF-1, and
penicillin/streptomycin; DMEM-HG (high glucose) comprising 10%
fetal bovine serum (FBS); DMEM-HG comprising 15% FBS; IMDM
(Iscove's modified Dulbecco's medium) comprising 10% FBS, 10% horse
serum, and hydrocortisone; M199 comprising 10% FBS, EGF, and
heparin; .alpha.-MEM (minimal essential medium) comprising 10% FBS,
GLUTAMAX.TM. and gentamicin; DMEM comprising 10% FBS, GLUTAMAX.TM.
and gentamicin, etc. A preferred medium is DMEM-LG/MCDB-201
comprising 2% FBS, ITS, LA+BSA, dextrose, L-ascorbic acid, PDGF,
EGF, and penicillin/streptomycin.
[0149] Other media 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 CELLGRO FREE.TM..
[0150] 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 (HIS)); 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; 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; and one
or more neuroectoderm specification factors, for example, sonic
hedgehog (shh) and/or retinoic acid (ra).
[0151] Placental stem cells can be cultured in standard tissue
culture conditions, e.g., in tissue culture dishes or multiwell
plates. Placental stem cells can also be cultured using a hanging
drop method. In this method, 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 stem cells are cultured.
[0152] In one embodiment, the placental stem cells are cultured in
the presence of a compound that acts to maintain an
undifferentiated phenotype in the placental stem cell. In a
specific embodiment, the compound is a substituted
3,4-dihydropyridimol[4,5-d]pyrimidine. In a more specific
embodiment, the compound is a compound having the following
chemical structure:
##STR00001##
The compound can be contacted with a placental stem cell, or
population of placental stem cells, at a concentration of, for
example, between about 1 .mu.M to about 10 .mu.M.
[0153] 5.5.2 Expansion and Proliferation of Placental Stem
Cells
[0154] Once an isolated placental stem cell, or isolated population
of stem cells (e.g., a stem cell or population of stem cells
separated from at least 50% of the placental cells with which the
stem cell or population of stem cells is normally associated in
vivo), the stem cell or population of stem cells can be
proliferated and expanded in vitro. For example, a population of
placental stem cells can be cultured in tissue culture containers,
e.g., dishes, flasks, multiwell plates, or the like, for a
sufficient time for the stem cells to proliferate to 70-90%
confluence, that is, until the stem cells and their progeny occupy
70-90% of the culturing surface area of the tissue culture
container.
[0155] Placental stem cells can be seeded in culture vessels at a
density that allows cell growth. For example, the cells may be
seeded at low density (e.g., about 1,000 to about 5,000
cells/cm.sup.2) to high density (e.g., about 50,000 or more
cells/cm.sup.2). In a preferred embodiment, the cells are cultured
at about 0 to about 5 percent by volume CO.sub.2 in air. In some
preferred embodiments, the cells are cultured at about 2 to about
25 percent O.sub.2 in air, preferably about 5 to about 20 percent
O.sub.2 in air. The cells preferably are cultured at about
25.degree. C. to about 40.degree. C., preferably 37.degree. C. The
cells are preferably cultured in an incubator. The culture medium
can be static or agitated, for example, using a bioreactor.
Placental stem cells preferably are grown under low oxidative
stress (e.g., with addition of glutathione, ascorbic acid,
catalase, tocopherol, N-acetylcysteine, or the like).
[0156] Once 70%-90% confluence is obtained, the cells may be
passaged. For example, the cells can be enzymatically treated,
e.g., trypsinized, using techniques well-known in the art, to
separate them from the tissue culture surface. After removing the
cells by pipetting and counting the cells, about 20,000-100,000
stem cells, preferably about 50,000 stem cells, are passaged to a
new culture container containing fresh culture medium. Typically,
the new medium is the same type of medium from which the stem cells
were removed. Also provided herein are populations of placental
stem cells that have been passaged at least 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 12, 14, 16, 18, or 20 times, or more.
[0157] 5.5.3 Placental Stem Cell Populations
[0158] In one aspect, placental stem cells and populations of
placental stem cells are used as a source of differentiated cells,
e.g., hepatocytes and/or hepatogenic cells, or chondrocytes and/or
chondrogenic cells. Placental stem cell populations can be isolated
directly from one or more placentas; that is, the placental stem
cell population can be a population of placental cells, comprising
placental stem cells, obtained from, or contained within,
perfusate, or obtained from, or contained within, digestate (that
is, the collection of cells obtained by enzymatic digestion of a
placenta or part thereof). Isolated placental stem cells described
herein can also be cultured and expanded to produce placental stem
cell populations. Populations of placental cells comprising
placental stem cells can also be cultured and expanded to produce
placental stem cell populations.
[0159] Placental stem cell populations provided herein comprise
placental stem cells, for example, placental stem cells as
described herein. In various embodiments, at least 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the cells in an
isolated placental stem cell population are placental stem cells.
That is, a placental stem cell population can comprise, e.g., as
much as 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%
non-stem cells.
[0160] Further provided herein are methods of producing isolated
placental stem cell population by, e.g., selecting placental stem
cells, whether derived from enzymatic digestion or perfusion, that
express particular markers and/or particular culture or
morphological characteristics. Such placental stem cells can be
used to produce, e.g., hepatocytes and/or hepatogenic cells, or
mixed populations of undifferentiated placental stem cells and
hepatogenic cells and/or hepatocytes differentiated from placental
stem cells; or chondrocytes and/or chondrogenic cells, or mixed
populations of undifferentiated placental stem cells and
chondrocytes and/or chondrogenic cells.
[0161] Cell populations comprising placental stem cells can be
produced in a variety of different ways. In one embodiment, for
example, a cell population comprising placental stem cells can be
produced by selecting placental cells that (a) adhere to a
substrate, and (b) express CD200 and HLA-G; and isolating said
cells from other cells to form a cell population. In another
embodiment, the method of producing a cell population comprises
selecting placental cells that (a) adhere to a substrate, and (b)
express CD73, CD105, and CD200; and isolating said cells from other
cells to form a cell population. In another embodiment, the method
of producing a cell population comprises selecting placental cells
that (a) adhere to a substrate and (b) express CD200 and OCT-4; and
isolating said cells from other cells to form a cell population. In
another embodiment, the method of producing a cell population
comprises selecting placental cells that (a) adhere to a substrate,
(b) express CD73 and CD105, and (c) facilitate the formation of one
or more embryoid-like bodies in a population of placental cells
comprising said stem cell when said population is cultured under
conditions that allow for the formation of an embryoid-like body;
and isolating said cells from other cells to form a cell
population. In another embodiment, the method of producing a cell
population comprises selecting placental cells that (a) adhere to a
substrate, and (b) express CD73, CD105 and HLA-G; and isolating
said cells from other cells to form a cell population. In another
embodiment, the method of producing a cell population comprises
selecting placental cells that (a) adhere to a substrate, (b)
express OCT-4, and (c) facilitate the formation of one or more
embryoid-like bodies in a population of placental cells comprising
said stem cell when said population is cultured under conditions
that allow for the formation of an embryoid-like body; and
isolating said cells from other cells to form a cell population. In
any of the above embodiments, the method can additionally comprise
selecting placental cells that express ABC-p (a placenta-specific
ABC transporter protein; see, e.g., Allikmets et al., Cancer Res.
58(23):5337-9 (1998)). The method can also comprise selecting cells
exhibiting at least one characteristic specific to, e.g., a
mesenchymal stem cell, for example, expression of CD29, expression
of CD44, expression of CD90, or expression of a combination of the
foregoing.
[0162] In any of the above embodiments of producing a cell
population, the resulting population of cells can be additionally
assessed for the ability of the cells, or of the population, to
produce, e.g., hepatocytes, hepatogenic cells, chondrocytes, or
chondrocytic cells.
[0163] In the above embodiments, the substrate can be any surface
on which culture and/or selection of cells, e.g., placental stem
cells, can be accomplished. Typically, the substrate is plastic,
e.g., tissue culture dish or multiwell plate plastic. Tissue
culture plastic can be coated with a biomolecule, e.g., laminin or
fibronectin.
[0164] Cells, e.g., placental stem cells, can be selected for a
placental stem cell population by any means known in the art of
cell selection. For example, cells can be selected using an
antibody or antibodies to one or more cell surface markers, for
example, in flow cytometry or FACS. Selection can be accomplished
using antibodies in conjunction with magnetic beads. Antibodies
that are specific for certain stem cell-related markers are known
in the art. For example, antibodies to OCT-4 (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.
[0165] The isolated placental stem cell population can comprise
placental cells that are not stem cells, or cells that are not
placental cells.
[0166] Isolated placental stem cell populations can be combined
with one or more populations of non-stem cells or non-placental
cells. For example, an isolated population of placental stem cells
can be combined with blood (e.g., placental blood or umbilical cord
blood), blood-derived stem cells (e.g., stem cells derived from
placental blood or umbilical cord blood), populations of
blood-derived nucleated cells, bone marrow-derived mesenchymal
cells, bone-derived stem cell populations, crude bone marrow, adult
(somatic) stem cells, populations of stem cells contained within
tissue, cultured stem cells, populations of fully-differentiated
cells (e.g., chondrocytes, fibroblasts, amniotic cells,
osteoblasts, muscle cells, cardiac cells, etc.) and the like. Cells
in an isolated placental stem cell population can be combined with
a plurality of cells of another type in ratios of about
100,000,000:1, 50,000,000:1, 20,000,000:1, 10,000,000:1,
5,000,000:1, 2,000,000:1, 1,000,000:1, 500,000:1, 200,000:1,
100,000:1, 50,000:1, 20,000:1, 10,000:1, 5,000:1, 2,000:1, 1,000:1,
500:1, 200:1, 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1; 1:2; 1:5;
1:10; 1:100; 1:200; 1:500; 1:1,000; 1:2,000; 1:5,000; 1:10,000;
1:20,000; 1:50,000; 1:100,000; 1:500,000; 1:1,000,000; 1:2,000,000;
1:5,000,000; 1:10,000,000; 1:20,000,000; 1:50,000,000; or about
1:100,000,000, comparing numbers of total nucleated cells in each
population. Cells in an isolated placental stem cell population can
be combined with a plurality of cells of a plurality of cell types,
as well.
[0167] In one, an isolated population of placental stem cells is
combined with a plurality of hematopoietic stem cells. Such
hematopoietic stem cells can be, for example, contained within
unprocessed placental, umbilical cord blood or peripheral blood; in
total nucleated cells from placental blood, umbilical cord blood or
peripheral blood; in an isolated population of CD34.sup.+ cells
from placental blood, umbilical cord blood or peripheral blood; in
unprocessed bone marrow; in total nucleated cells from bone marrow;
in an isolated population of CD34.sup.+ cells from bone marrow, or
the like.
[0168] Additional placental stem cells and placental stem cell
populations that can be used in connection with the compositions
and methods provided herein are described, for example, in U.S.
patent application Ser. No. 11/648,813, and in U.S. Provisional
Application No. 60/846,641, each of which is hereby incorporated by
reference in its entirety.
[0169] 5.5.4 Induction of Differentiation Into Hepatic Cells
[0170] Differentiation of placental stem cells into hepatic cells
can be accomplished, for example, by placing placental stem cells
in cell culture conditions that induce differentiation into hepatic
cells. In a specific embodiment, the placental stem cells are
contacted with sodium butyrate for a time sufficient for the
placental stem cells to exhibit one or more characteristics of a
hepatocyte or hepatogenic cell.
[0171] An example hepatogenic medium comprises DMEM supplemented
with sodium butyrate. Cells are cultured, e.g., for 14-28 days,
refeeding every 3-4 days. Differentiation can be confirmed by
assaying for, e.g., increased production of cytokeratin 18
(relative to an undifferentiated placental stem cell). Typically,
placental stem cells express cytokeratin 18, but do not express at
least one other cytokeratin expressed by hepatocytes.
Differentiation can also be affirmed by the presence of one or more
of asialogylcoprotein receptor, alpha-1-antitrypsin, albumin and
cytochrome P450 activity. A placental stem cell is considered to
have differentiated into a hepatic cell when the cell displays one
or more of these characteristics.
[0172] In one aspect, provided herein are methods of producing and
isolated population of hepatocytes and/or hepatogenic cells by,
e.g., selecting a plurality of placental stem cells, whether
derived from enzymatic digestion or perfusion, that express
particular markers and/or particular culture or morphological
characteristics, and exposing such cells to conditions that cause
the differentiation of at least some of said placental stem cells
into hepatocytes and/or hepatogenic cells. In one embodiment, for
example, provided herein is a method of producing hepatocytes
and/or hepatogenic cells comprising (1) selecting placental cells
that (a) adhere to a substrate, and (b) express CD200 and HLA-G, or
express CD73, CD105, and CD200, or express CD200 and OCT-4, or
express CD73, CD105 and HLA-G, or express CD73 and CD105 and
facilitate the formation of one or more embryoid-like bodies in a
population of placental cells comprising said stem cell when said
population is cultured under conditions that allow for the
formation of an embryoid-like body, or express OCT-4 and (c)
facilitate the formation of one or more embryoid-like bodies in a
population of placental cells comprising said stem cell when said
population is cultured under conditions that allow for the
formation of an embryoid-like body; (2) isolating said placental
cells from other placental cells; and exposing said cells to sodium
butyrate for a time sufficient to produce a detectable number of
said hepatocytes and/or hepatogenic cells. In a specific
embodiment, the placental stem cells are CD10.sup.+, CD34.sup.-,
CD105.sup.+ and CD200.sup.+.
[0173] In various specific embodiments of the above methods, at
least, or about, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95% or 98% of said placental stem cells differentiate into
said hepatocytes and/or hepatogenic cells. In another specific
embodiment of any of the above embodiments of the method, said
placental cells are exposed to sodium butyrate for a time
sufficient for a plurality of said cells to exhibit a detectable
increase in the production of cytokeratin 18 relative to an
undifferentiated placental stem cell, a detectable amount of
asialogylcoprotein receptor, or a detectable amount of cytochrome
P450 7A1 activity, a detectable amount of albumin, or a detectable
amount of expression of a gene encoding albumin. In another
specific embodiment,
[0174] 5.5.5 Induction of Differentiation into Chondrocytic
Cells
[0175] Chondrogenic differentiation of adherent placental stem
cells can be accomplished, for example, by placing the placental
stem cells in cell culture conditions that induce differentiation
into chondrocytes. A preferred chondrocytic medium comprises MSCGM
(Cambrex) or Dulbecco's Modified Eagle's Medium (DMEM) supplemented
with 15% cord blood serum. In one embodiment, placental stem cells
are aliquoted into a sterile polypropylene tube, centrifuged (e.g.,
at 150.times.g for 5 minutes), and washed twice in Incomplete
Chondrogenesis Medium (Cambrex). The cells are resuspended in
Complete Chondrogenesis Medium (Cambrex) containing 0.01 .mu.g/ml
transforming growth factor beta-3 (TGF-.beta.3) at a concentration
of about 1-20.times.10.sup.5 cells/ml. In other embodiments,
placental stem cells are contacted with exogenous growth factors,
e.g., GDF-5 or TGF-.beta.3, with or without ascorbate.
[0176] Another example chondrogenic medium comprises DMEM, 1% FBS,
insulin, ascorbate 2-phosphate, and TGF-.beta.1. A similar
chondrogenic medium comprises, in 1 L, DMEM, 1% FBS, 1%
penicillin-streptomycin, 37.5 .mu.g/ml ascorbate-2-phosphate, ITS
premix (comprising, e.g., 100 mg of insulin, 100 mg of transferrin,
and 100 pg of sodium selenate), and 10 ng/ml TGF-1 Another example
chondrogenic medium is a defined medium (any standard defined
medium suitable for mammalian cell culture) that includes 100 nM
dexamethasone and 10 ng/ml transforming growth factor-33
(TGF-.beta.3).
[0177] Chondrogenic medium can be supplemented with amino acids
including proline and glutamine, sodium pyruvate, dexamethasone,
ascorbic acid, and insulin/transferrin/selenium. Chondrogenic
medium can be supplemented with sodium hydroxide and/or
collagen.
[0178] The adherent placental stem cells may be cultured at high or
low density. Cells are preferably cultured in the absence of serum.
Placental stem cells can be cultured under chondrogenic conditions
either statically or dynamically, e.g., under conditions in which
medium is circulated around cells. Cell culture can proceed for a
detectable amount of differentiation occurs. In specific
embodiments, adherent placental stem cells are cultured for about
28 days to about 56 days under chondrogenic conditions.
[0179] Adherent placental stem cells can be induced to
differentiate into chondrocytes or chondrocytic cells by seeding
the cells onto an electrospun nonwoven microfibrous or nanofibrous
mat and culturing the cells under chondrogenic conditions. Such
mats, and their production, are described in Section 5.7.1.4
herein.
[0180] Chondrogenesis can be assessed by e.g., observation of
production of esoinophilic ground substance, safranin-O staining
for glycosaminoglycan expression; methylene blue dye binding for
determination of glycosaminoglycan expression; hematoxylin/eosin
staining, assessing cell morphology, and/or RT/PCR confirmation of,
or staining for, collagen 2 and collagen 9 gene expression.
Chondrogenesis can also be observed by growing the stem cells in a
pellet, formed, e.g., by gently centrifuging stem cells in
suspension (e.g., at about 800 g for about 5 minutes). After about
1-28 days, the pellet of stem cells begins to form a tough matrix
and demonstrates a structural integrity not found in non-induced,
or non-chondrogenic, cell lines, pellets of which tend to fall
apart when challenged. Chondrogenesis can also be demonstrated,
e.g., in such cell pellets, by staining with a stain that stains
collagen, e.g., Sirius Red, and/or a stain that stains
glycosaminoglycans (GAGs), such as, e.g., Alcian Blue (also called
Alcian blue 8GX, Ingrain blue 1, or C.I. 74240). A placental stem
cell is considered to have differentiated into a chondrocytic cell
when the cell displays one or more of these characteristics.
Chondrogenesis can also be assessed by determination of gene
expression, e.g., by real-time PCR, for early stage chondrogenesis
markers fibromodulin and cartilage oligomeric matrix protein; gene
expression for mid-stage chondrogenesis markers aggrecan, versican,
decorin and biglycan; and gene expression for types II and X
collagens and chondroadherein, which are markers of mature
chondrocytes.
5.6 Preservation of Placental Stem Cells
[0181] Placental stem cells, and cells differentiated therefrom,
e.g., hepatocytes and/or hepatogenic cells, chondrocytes and/or
chondrocytic cells, can be preserved, that is, placed under
conditions that allow for long-term storage, or conditions that
inhibit cell death by, e.g., apoptosis or necrosis.
[0182] Placental stem cells, and cells differentiated therefrom,
e.g., hepatocytes and/or hepatogenic cells, chondrocytes and/or
chondrocytic 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 Ser. No. 11/648,812, entitled "Improved Composition for
Collecting Placental Stem Cells and Preserving Organs" filed on
Dec. 28, 2006. In one embodiment, provided herein is a method of
preserving a population of cells, e.g., placental stem cells,
and/or hepatocytes and/or hepatogenic cells, chondrocytes and/or
chondrocytic cells differentiated therefrom, comprising contacting
said cells with a stem cell collection composition comprising an
inhibitor of apoptosis and an oxygen-carrying perfluorocarbon,
wherein said inhibitor of apoptosis is present in an amount and for
a time sufficient to reduce or prevent apoptosis in the population
of stem cells, as compared to a population of stem cells not
contacted with the inhibitor of apoptosis. In a specific
embodiment, said inhibitor of apoptosis is a caspase inhibitor. In
another specific embodiment, said inhibitor of apoptosis is a JNK
inhibitor. In a more specific embodiment, said JNK inhibitor does
not modulate differentiation or proliferation of said cells. In
another embodiment, said stem cell collection composition comprises
said inhibitor of apoptosis and said oxygen-carrying
perfluorocarbon in separate phases. In another embodiment, said
stem cell collection composition comprises said inhibitor of
apoptosis and said oxygen-carrying perfluorocarbon in an emulsion.
In another embodiment, the stem cell collection composition
additionally comprises an emulsifier, e.g., lecithin. In another
embodiment, said apoptosis inhibitor and said perfluorocarbon are
between about 0.degree. C. and about 25.degree. C. at the time of
contacting the cells. In another more specific embodiment, said
apoptosis inhibitor and said perfluorocarbon are between about
2.degree. C. and 10.degree. C., or between about 2.degree. C. and
about 5.degree. C., at the time of contacting the cells. In another
more specific embodiment, said contacting is performed during
transport of said population of stem cells. In another more
specific embodiment, said contacting is performed during freezing
and thawing of said population of stem cells.
[0183] In another embodiment, provided herein is a method of
preserving a population of placental stem cells, and/or hepatocytes
and/or hepatogenic cells, chondrocytes and/or chondrocytic cells
differentiated therefrom, 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 (described
in U.S. Pat. No. 4,798,824; also known as VIASPAN.RTM.; 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. In
another embodiment, said organ-preserving compound is hydroxyethyl
starch, lactobionic acid, raffinose, or a combination thereof. In
another embodiment, the stem cell collection composition
additionally comprises an oxygen-carrying perfluorocarbon, either
in two phases or as an emulsion.
[0184] In another embodiment of the method, placental stem cells
are contacted with a stem cell collection composition comprising an
apoptosis inhibitor and oxygen-carrying perfluorocarbon,
organ-preserving compound, or combination thereof, during
perfusion. In another embodiment, said cells are contacted during a
process of tissue disruption, e.g., enzymatic digestion. In another
embodiment, placental stem cells are contacted with said stem cell
collection compound after collection by perfusion, or after
collection by tissue disruption, e.g., enzymatic digestion.
[0185] Typically, during placental cell collection, enrichment and
isolation, it is preferable to minimize or eliminate cell stress
due to hypoxia and mechanical stress. In another embodiment of the
method, therefore, a stem cell, or population of stem cells, is
exposed to a hypoxic condition during collection, enrichment or
isolation for less than six hours during said preservation, wherein
a hypoxic condition is a concentration of oxygen that is less than
normal blood oxygen concentration. In a more specific embodiment,
said population of stem cells is exposed to said hypoxic condition
for less than two hours during said preservation. In another more
specific embodiment, said population of stem cells is exposed to
said hypoxic condition for less than one hour, or less than thirty
minutes, or is not exposed to a hypoxic condition, during
collection, enrichment or isolation. In another specific
embodiment, said population of stem cells is not exposed to shear
stress during collection, enrichment or isolation.
[0186] The placental stem cells used in the compositions and
methods provided herein can be cryopreserved, e.g., in
cryopreservation medium in small containers, e.g., ampoules.
Suitable cryoprescrvation medium includes, but is not limited to,
culture medium including, e.g., growth medium, or cell freezing
medium, for example commercially available cell freezing medium,
e.g., C2695, C2639 or C6039 (Sigma). Cryopreservation medium
preferably comprises DMSO (dimethylsulfoxide), at a concentration
of, e.g., about 10% (v/v). Cryopreservation medium may comprise
additional agents, for example, methylcellulose and/or glycerol.
Placental stem cells are preferably cooled at about 1.degree.
C./min during cryopreservation. A preferred cryopreservation
temperature is about -80.degree. C. to about -180.degree. C.,
preferably about -125.degree. C. to about -140.degree. C.
Cryopreserved cells can be transferred to liquid nitrogen prior to
thawing for use. In some embodiments, for example, once the
ampoules have reached about -90.degree. C., they are transferred to
a liquid nitrogen storage area. Cryopreserved cells preferably are
thawed at a temperature of about 25.degree. C. to about 40.degree.
C., preferably to a temperature of about 37.degree. C.
5.7 Uses of Placental Stem Cells
[0187] 5.7.1 Compositions Comprising Placental Stem Cells
[0188] The methods described herein can use compositions comprising
placental stem cells, or biomolecules therefrom. In the same
manner, the pluralities and populations of placental stem cells
described herein can be combined with any
physiologically-acceptable or medically-acceptable compound,
composition or device for use in, e.g., research or
therapeutics.
[0189] 5.7.1.1 Cryopreserved Placental Stem Cells
[0190] The placental stem cell populations 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. Placental stem cell populations can be prepared in a form
that is easily administrable to an individual. For example,
provided herein is a placental stem cell population that is
contained within a container that is suitable for medical use. Such
a container can be, for example, a sterile plastic bag, flask, jar,
or other container from which the placental stem cell population
can be easily dispensed. For example, the container can be a blood
bag or other plastic, medically-acceptable bag suitable for the
intravenous administration of a liquid to a recipient. The
container is preferably one that allows for cryopreservation of the
combined stem cell population.
[0191] Cryopreserved placental stem cell populations can comprise
placental stem cells derived from a single donor, or from multiple
donors. The placental stem cell population can be completely
HLA-matched to an intended recipient, or partially or completely
HLA-mismatched.
[0192] Cryopreserved placental stem cells can be, for example, in
the form of a composition comprising an placental stem cell
population in a container. In a specific embodiment, the stem cell
population is cryopreserved. In another specific embodiment, the
container is a bag, flask, or jar. In a more specific embodiment,
said bag is a sterile plastic bag. In a more specific embodiment,
said bag is suitable for, allows or facilitates intravenous
administration of said placental stem cell population. The bag can
comprise multiple lumens or compartments that are interconnected to
allow mixing of the placental stem cells and one or more other
solutions, e.g., a drug, prior to, or during, administration. In
another specific embodiment, the composition comprises one or more
compounds that facilitate cryopreservation of the combined stem
cell population. In another specific embodiment, said placental
stem cell population is contained within a
physiologically-acceptable aqueous solution. In a more specific
embodiment, said physiologically-acceptable aqueous solution is a
0.9% NaCl solution. In another specific embodiment, said placental
stem cell population comprises placental cells that are HLA-matched
to a recipient of said stem cell population. In another specific
embodiment, said combined stem cell population comprises placental
cells that are at least partially HLA-mismatched to a recipient of
said stem cell population. In another specific embodiment, said
placental stem cells are derived from a plurality of donors.
[0193] 5.7.1.2 Pharmaceutical Compositions
[0194] Populations of placental stem cells, or populations of cells
comprising placental stem cells, can be formulated into
pharmaceutical compositions for use in vivo. Such pharmaceutical
compositions comprise a population of placental stem cells, or a
population of cells comprising placental stem cells, in a
pharmaceutically-acceptable carrier, e.g., a saline solution or
other accepted physiologically-acceptable solution for in vivo
administration. Pharmaceutical compositions provided herein,
comprising placental stem cells, can comprise any of the placental
stem cell populations, or placental stem cell types, described
elsewhere herein. The pharmaceutical compositions can comprise
fetal, maternal, or both fetal and maternal placental stem cells.
The pharmaceutical compositions can further comprise placental stem
cells obtained from a single individual or placenta, or from a
plurality of individuals or placentae.
[0195] The pharmaceutical compositions can comprise any number of
placental stem cells. For example, a single unit dose of placental
stem cells can comprise, in various embodiments, about, at least,
or no more than 1.times.10.sup.5, 5.times.10.sup.5,
1.times.10.sup.6, 5.times.10.sup.6, 1.times.10.sup.7,
5.times.10.sup.7, 1.times.10.sup.8, 5.times.10.sup.8,
1.times.10.sup.9, 5.times.10.sup.9, 1.times.10.sup.10,
5.times.10.sup.10, 1.times.10.sup.11 or more placental stem
cells.
[0196] The pharmaceutical compositions may comprise populations of
cells that comprise 50% viable cells or more (that is, at least 50%
of the cells in the population are functional or living).
Preferably, at least 60% of the cells in the population are viable.
More preferably, at least 70%, 80%, 90%, 95%, or 99% of the cells
in the population in the pharmaceutical composition are viable.
[0197] 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, and the like.
[0198] 5.7.1.3 Placental Stem Cell Conditioned Media
[0199] The placental stem cells provided herein can be used to
produce conditioned medium, that is, medium comprising one or more
biomolecules secreted or excreted by the stem cells. In various
embodiments, the conditioned medium comprises medium in which
placental stem cells have grown for at least 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14 or more days. In other embodiments, the
conditioned medium comprises medium in which placental stem cells
have grown to at least 30%, 40%, 50%, 60%, 70%, 80%, 90%
confluence, or up to 100% confluence. Such conditioned medium can
be used to support the culture of a separate population of
placental stem cells, or stem cells of another kind. In another
embodiment, the conditioned medium comprises medium in which
placental stem cells have been differentiated into an adult cell
type. In another embodiment, conditioned medium comprises medium in
which placental stem cells and non-placental stem cells have been
cultured.
[0200] Thus, in one embodiment, provided herein is a composition
comprising culture medium, e.g., conditioned medium, from a culture
of adherent placental stem cells, wherein said adherent placental
stem cells (a) adhere to a substrate; (b) express CD200 and HLA-G,
or express CD73, CD105, and CD200, or express CD200 and OCT-4, or
express CD73, CD105, and HLA-G, or express CD73 and CD105 and
facilitate the formation of one or more embryoid-like bodies in a
population of placental cells that comprise the placental stem
cells, when said population is cultured under conditions that allow
formation of embryoid-like bodies, or express OCT-4 and facilitate
the formation of one or more embryoid-like bodies in a population
of placental cells that comprise the placental stem cells when said
population is cultured under conditions that allow formation of
embryoid-like bodies, wherein said culture of placental stem cells
has been cultured in said medium for 24 hours or more. In a
specific embodiment, said composition comprises medium conditioned
by CD34.sup.+, CD45.sup.- placental stem cells. In another specific
embodiment, the composition further comprises a plurality of said
placental stem cells, e.g., a plurality of adherent placental stem
cells and/or a plurality of non-adherent, CD34.sup.+, CD45.sup.-
placental stem cells. In another specific embodiment, the
composition comprises a plurality of non-placental cells. In a more
specific embodiment, said non-placental cells comprise CD34.sup.+
cells, e.g., hematopoietic progenitor cells, derived from a
non-placental source such as peripheral blood hematopoietic
progenitor cells, cord blood hematopoietic progenitor cells, or
placental blood hematopoietic progenitor cells. The non-placental
cells can also comprise other stem cells, such as mesenchymal stem
cells, e.g., bone marrow-derived mesenchymal stem cells. The
non-placental cells can also be one ore more types of adult cells
or cell lines. In another specific embodiment, the composition
comprises an anti-proliferative agent, e.g., an anti-MIP-1.alpha.
or anti-MIP-1.beta. antibody.
[0201] In another embodiment, conditioned medium is, or comprises,
medium conditioned by a population of hepatocytes, hepatogenic
cells, chondrocytes and/or chondrocytic cells differentiated from
placental stem cells. Such a population can comprise placental stem
cells, hepatogenic cells or chondrogenic cells differentiated from
placental stem cells, hepatocytes or chondrocytes differentiated
from placental stem cells, or any combination of the foregoing.
Thus, in one embodiment, provided herein is a composition
comprising culture medium from a culture of hepatocytes,
hepatogenic cells, chondrocytes and/or chondrocytic cells
differentiated from placental stem cells, wherein said placental
stem cells (a) adhere to a substrate; (b) express CD200 and HLA-G,
or express CD73, CD105, and CD200, or express CD200 and OCT-4, or
express CD73, CD105, and HLA-G, or express CD73 and CD105 and
facilitate the formation of one or more embryoid-like bodies in a
population of placental cells that comprise the placental stem
cells, when said population is cultured under conditions that allow
formation of embryoid-like bodies, or express OCT-4 and facilitate
the formation of one or more embryoid-like bodies in a population
of placental cells that comprise the placental stem cells when said
population is cultured under conditions that allow formation of
embryoid-like bodies, wherein said culture of hepatocytes,
hepatogenic cells, chondrocytes and/or chondrocytic cells has been
cultured in said medium for 24 hours or more. In a specific
embodiment, the composition further comprises a plurality of said
placental stem cells. In another specific embodiment, the
composition comprises a plurality of non-placental cells, e.g.,
hepatocytes from primary culture; hepatocyte cell line cells;
hepatoma cells, and the like. In a more specific embodiment, said
non-placental cells comprise CD34.sup.+ cells, e.g., hematopoietic
progenitor cells, such as peripheral blood hematopoietic progenitor
cells, cord blood hematopoietic progenitor cells, or placental
blood hematopoietic progenitor cells. The non-placental cells can
also comprise other stem cells, such as mesenchymal stem cells,
e.g., bone marrow-derived mesenchymal stem cells. The non-placental
cells can also be one ore more types of adult cells or cell lines.
In another specific embodiment, the composition comprises an
anti-proliferative agent, e.g., an anti-MIP-1.alpha. or
anti-MIP-1.beta. antibody.
[0202] 5.7.1.4 Matrices Comprising Placental Stem Cells
[0203] In another aspect, provided herein are matrices, hydrogels,
scaffolds, and the like that comprise a population of hepatocytes,
hepatogenic cells, chondrocytes and/or chondrocytic cells,
differentiated from the adherent placental stem cells described
herein.
[0204] Placental stem cells, or hepatocytes, hepatogenic cells,
chondrocytes and/or chondrocytic cells differentiated from the
placental stem cells, can be seeded onto a natural matrix, e.g., a
placental biomaterial such as an amniotic membrane material. Such
an amniotic membrane material can be, e.g., amniotic membrane
dissected directly from a mammalian placenta; fixed or heat-treated
amniotic membrane, substantially dry (i.e., <20% H.sub.2O)
amniotic membrane, chorionic membrane, substantially dry chorionic
membrane, substantially dry amniotic and chorionic membrane, and
the like. Preferred placental biomaterials on which placental stem
cells can be seeded are described in Hariri, U.S. Application
Publication No. 2004/0048796.
[0205] Placental stem cells, or cells differentiated therefrom,
e.g., hepatocytes, hepatogenic cells, chondrocytes and/or
chondrocytic cells, 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. Cells in such a matrix
can also be cultured so that the cells are mitotically expanded
prior to implantation. The hydrogel can be, 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.
[0206] In some embodiments, the formulation comprises an in situ
polymerizable gel (see, e.g., U.S. Patent Application Publication
2002/0022676; Anseth et al., J. Control Release, 78(1-3): 199-209
(2002); Wang et al., Biomaterials, 24(22):3969-80 (2003).
[0207] In some embodiments, the polymers are at least partially
soluble in aqueous solutions, such as water, buffered salt
solutions, or aqueous alcohol solutions, that have charged side
groups, or a monovalent ionic salt thereof. Examples of polymers
having acidic side groups that can be reacted with cations are
poly(phosphazenes), poly(acrylic acids), poly(methacrylic acids),
copolymers of acrylic acid and methacrylic acid, poly(vinyl
acetate), and sulfonated polymers, such as sulfonated polystyrene.
Copolymers having acidic side groups formed by reaction of acrylic
or methacrylic acid and vinyl ether monomers or polymers can also
be used. Examples of acidic groups are carboxylic acid groups,
sulfonic acid groups, halogenated (preferably fluorinated) alcohol
groups, phenolic OH groups, and acidic OH groups.
[0208] The placental stem cells, or hepatocytes, hepatogenic cells,
chondrocytes and/or chondrocytic cells differentiated from the
placental stem cells, or co-cultures thereof can be seeded onto a
three-dimensional framework or scaffold and implanted in vivo. Such
a framework can be implanted in combination with any one or more
growth factors, cells, drugs or other components that stimulate
tissue formation.
[0209] 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(s-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.
[0210] In another embodiment, the scaffold is, or comprises, a
nanofibrous scaffold, e.g., an electrospun nanofibrous scaffold. In
a more specific embodiment, said nanofibrous scaffold comprises
poly(L-lactic acid) (PLLA), poly lactic glycolic acid (PLGA), type
I collagen, a copolymer of vinylidene fluoride and
trifluoroethylnee (PVDF-TrFE), poly(-caprolactone),
poly(L-lactide-co-.epsilon.-caprolactone) [P(LLA-CL)] (e.g.,
75:25), and/or a copolymer of
poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and type I
collagen. In another more specific embodiment, said scaffold
promotes the differentiation of placental stem cells into
chondrocytes. Methods of producing nanofibrous scaffolds, e.g.,
electrospun nanofibrous scaffolds, are known in the art. See, e.g.,
Xu et al., Tissue Engineering 10(7): 1160-1168 (2004); Xu et al.,
Biomaterials 25:877-886 (20040; Meng et al., J. Biomaterials Sci.,
Polymer Edition 18(1):81-94 (2007).
[0211] The compositions listed above can be electrospun into a
nonwoven mat comprising nanoscale fiber meshes with controllable
porosity. In electrospinning, a high voltage is used to create an
electrically charged jet of polymer solution, which dries or
solidifies to leave a polymer fiber. One electrode is placed into
the polymer solution in, e.g., a tube or capillary, e.g., a needle,
and the other attached to a collector. An electric field is passed
to the end of the tube or capillary, inducing a charge on the
surface of the liquid. Mutual charge repulsion causes a force
directly opposite to the surface tension of the solution. As the
intensity of the electric field is increased, the repulsive
electrostatic force overcomes the surface tension, and a charged
jet of fluid is ejected from the solution at the tip of the tube or
capillary. Solvent in the discharged polymer solution jet
evaporates, leaving behind a charged polymer fiber, which lays
itself randomly on a grounded collecting metal screen. The
thickness of the mat, and the thickness of the fibers in the mat,
can be adjusted by increasing or decreasing the distance between
tube and collection screen, with increasing distance generally
resulting in finer fibers and a less-dense mat; by increasing or
decreasing the electric potential, in kilovolts, with increasing
potential generally resulting in decreased fiber thickness; by
increasing or decreasing the flow rate, with increasing flow rate
generally resulting in thicker fibers; or increasing or decreasing
the polymer concentration in solution, with increasing polymer
concentration generally resulting in increased fiber thickness. The
diameter of the tube at the tip can also be varied. In specific
embodiments, any polymer, e.g., any of the polymers disclosed
herein, suitable for electrospinning to create nanoscale nonwoven
fibrous meshes or mats, e.g., PLLA or PLGA, can be electrospun at a
voltage of, e.g., about 5 kV, 10 kV, 15 kV, 20 kV, 25 kV, 30 kV, 35
kV or about 40 kV; and the needle distance can be varied, e.g.,
from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 0, 12, 14, 16, 18, 20, 22,
24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 or about 50 cm
between needle tip and collection screen. The needle gauge can be
varied, e.g., from about 8 G to about 24 G, e.g., 12 G or 22 G. The
flow rate can be varied, e.g., from about 0.01 to about 1.0 mL/min,
e.g., about 0.05 to about 0.1 mL/min. The solution concentration,
using any of the polymers, can range from about 5% to about 50% w/w
in solution, e.g., about 10% to about 25% w/w. In specific
embodiments, PLLA or PLGA at about 10% to about 25% w/w in solution
can be electrospun using a 12 G needle or 22 G needle using a tip
to collection screen distance of 30 cm and a flow rate of about
0.05 mL/min to about 0.1 mL/min to produce electrospun mats
comprising fibers of an average diameter of about 250 nm to about
10 .mu.m.
[0212] Placental stem cells of the invention can also be seeded
onto, or contacted with, a physiologically-acceptable ceramic
material including, but not limited to, mono-, di-, tri-,
alpha-tri-, beta-tri-, and tetra-calcium phosphate, hydroxyapatite,
fluoroapatites, calcium sulfates, calcium fluorides, calcium
oxides, calcium carbonates, magnesium calcium phosphates,
biologically active glasses such as BIOGLASS.RTM., and mixtures
thereof. Porous biocompatible ceramic materials currently
commercially available include SURGIBONE.RTM. (CanMedica Corp.,
Canada), ENDOBON.RTM. (Merck Biomaterial France, France),
CEROS.RTM. (Mathys, AG, Bettlach, Switzerland), and mineralized
collagen bone grafting products such as HEALOS.TM. (DePuy, Inc.,
Raynham, Mass.) and VITOSS.RTM., RHAKOSS.TM., and CORTOSS.RTM.
(Orthovita, Malvern, Pa.). The framework can be a mixture, blend or
composite of natural and/or synthetic materials.
[0213] In another embodiment, placental stem cells, or hepatocytes
and/or hepatogenic cells differentiated from the 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.
[0214] The placental stem cells, or hepatocytes, hepatogenic cells,
chondrocytes and/or chondrocytic cells differentiated from the
placental stem cells, can, in another embodiment, be seeded onto
foam scaffolds that may be composite structures. Such foam
scaffolds can be molded into a useful shape, such as that of a
portion of a specific structure in the body to be repaired,
replaced or augmented. In some embodiments, the framework is
treated, e.g., with 0.1M acetic acid followed by incubation in
polylysine, PBS, and/or collagen, prior to inoculation of 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.
[0215] In some embodiments, the scaffold comprises, or is treated
with, materials that render it non-thrombogenic. These treatments
and materials may also promote and sustain endothelial growth,
migration, and extracellular matrix deposition. Examples of these
materials and treatments include but are not limited to natural
materials such as basement membrane proteins such as laminin and
Type IV collagen, synthetic materials such as EPTFE, and segmented
polyurethaneurea silicones, such as PURSPAN.TM. (The Polymer
Technology Group, Inc., Berkeley, Calif.). The scaffold can also
comprise anti-thrombotic agents such as heparin; the scaffolds can
also be treated to alter the surface charge (e.g., coating with
plasma) prior to seeding with placental stem cells.
[0216] Thus, in another aspect, provided herein is a composition
comprising isolated adherent CD10.sup.+, CD34.sup.-, CD105.sup.+,
CD200.sup.+ placental stem cells and an electrospun nanofibrous
scaffold. In a specific embodiment, said nanofibrous scaffold
comprises fibers of poly(L-lactic acid) (PLLA), poly lactic
glycolic acid (PLGA), type I collagen, a copolymer of vinylidene
fluoride and trifluoroethylnee (PVDF-TrFE), poly(-caprolactone),
poly(L-lactide-co-.epsilon.-caprolactone) [P(LLA-CL)] (e.g.,
75:25), and/or a copolymer of
poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and type I
collagen. In another specific embodiment, said nanofibrous scaffold
comprises fibers that average between about 250 nanometers and
about 10 m in thickness. In another specific embodiment, said
composition is contacted with conditions in which the placental
stem cells differentiate into chondrogenic cells or chondrocytes.
In another embodiment, provided herein is a method of making a
composition comprising contacting adherent CD10.sup.+, CD34.sup.-,
CD105.sup.+, CD200.sup.+ placental stem cells with an electrospun
nanofibrous scaffold, wherein said nanofibrous scaffold is made by
electrospinning PLLA or PLGA at about 20 kV at about 30 cm needle
to collector distance and about 0.05 mL/min. to about 0.1 mL/min
flow rate, wherein said PLLA or PLGA are in solution at about 10%
w/w to about 20% w/w.
[0217] 5.7.2 Genetically Modified Placental Stem Cells
[0218] In another aspect, the placental stem cells and umbilical
cord stem cells described herein can be genetically modified, e.g.,
to produce a nucleic acid or polypeptide of interest, or to produce
a differentiated cell, e.g., a hepatocyte, hepatogenic cell,
chondrocyte and/or chondrocytic cell, that produces a nucleic acid
or polypeptide of interest. Genetic modification can be
accomplished, e.g., using virus-based vectors including, but not
limited to, non-integrating replicating vectors, e.g., papilloma
virus vectors, SV40 vectors, adenoviral vectors; integrating viral
vectors, e.g., retrovirus vector or adeno-associated viral vectors;
or replication-defective viral vectors. Other methods of
introducing DNA into cells include the use of liposomes,
electroporation, a particle gun, direct DNA injection, or the
like.
[0219] Stem cells can be, e.g., transformed or transfected with DNA
controlled by or in operative association with, one or more
appropriate expression control elements, for example, promoter or
enhancer sequences, transcription terminators, polyadenylation
sites, internal ribosomal entry sites. Preferably, such a DNA
incorporates a selectable marker. Following the introduction of the
foreign DNA, engineered stem cells can be, e.g., grown in enriched
media and then switched to selective media. In one embodiment, the
DNA used to engineer a placental stem cell comprises a nucleotide
sequence encoding a polypeptide of interest, e.g., a cytokine,
growth factor, differentiation agent, or therapeutic
polypeptide.
[0220] The DNA used to engineer the stem cell can comprise any
promoter known in the art to drive expression of a nucleotide
sequence in mammalian cells, e.g., human cells. For example,
promoters include, but are not limited to, CMV promoter/enhancer,
SV40 promoter, papillomavirus promoter, Epstein-Barr virus
promoter, elastin gene promoter, and the like. In a specific
embodiment, the promoter is regulatable so that the nucleotide
sequence is expressed only when desired. Promoters can be either
inducible (e.g., those associated with metallothionein and heat
shock proteins) or constitutive.
[0221] In another specific embodiment, the promoter is
tissue-specific or exhibits tissue specificity. Examples of such
promoters include but are not limited to: myelin basic protein gene
control region (Readhead et al., 1987, Cell 48:703)
(oligodendrocyte cells); elastase I gene control region (Swit et
al., 1984, Cell 38:639; Ornitz et al., 1986, Cold Spring Harbor
Symp. Quant. Biol. 50:399; MacDonald, 1987, Hepatology 7:425)
(pancreatic acinar cells); insulin gene control region (Hanahan,
1985, Nature 315:115) (pancreatic beta cells); myosin light chain-2
gene control region (Shani, 1985, Nature 314:283) (skeletal
muscle).
[0222] The cells of the invention may be engineered to "knock out"
or "knock down" expression of one or more genes. The expression of
a gene native to a cell can be diminished by, for example,
inhibition of expression by inactivating the gene completely by,
e.g., homologous recombination. In one embodiment, for example, an
exon encoding an important region of the protein, or an exon 5' to
that region, is interrupted by a positive selectable marker, e.g.,
neo, preventing the production of normal mRNA from the target gene
and resulting in inactivation of the gene. A gene may also be
inactivated by creating a deletion in part of a gene or by deleting
the entire gene. By using a construct with two regions of homology
to the target gene that are far apart in the genome, the sequences
intervening the two regions can be deleted (Mombaerts et al., 1991,
Proc. Nat. Acad. Sci. U.S.A. 88:3084). Antisense, DNAzymes, small
interfering RNA, and ribozyme molecules that inhibit expression of
the target gene can also be used to reduce the level of target gene
activity in the stem cells. For example, antisense RNA molecules
which inhibit the expression of major histocompatibility gene
complexes (HLA) have been shown to be most versatile with respect
to immune responses. Triple helix molecules can be utilized in
reducing the level of target gene activity. See, e.g., L. G. Davis
et al. (eds), 1994, BASIC METHODS IN MOLECULAR BIOLOGY, 2nd ed.,
Appleton & Lange, Norwalk, Conn., which is incorporated herein
by reference.
[0223] In a specific embodiment, the placental or umbilical cord
stem cells of the invention can be genetically modified with a
nucleic acid molecule comprising a nucleotide sequence encoding a
polypeptide of interest, wherein expression of the polypeptide of
interest is controllable by an exogenous factor, e.g., polypeptide,
small organic molecule, or the like. Such a polypeptide can be a
therapeutic polypeptide. In a more specific embodiment, the
polypeptide of interest is IL-12 or interleukin-1 receptor
antagonist (IL-1Ra). In another more specific embodiment, the
polypeptide of interest is a fusion of interleukin-1 receptor
antagonist and dihydrofolate reductase (DHFR), and the exogenous
factor is an antifolate, e.g., methotrexate. Such a construct is
useful in the engineering of placental or umbilical cord stem cells
that express IL-1Ra, or a fusion of IL-1Ra and DHFR, upon contact
with methotrexate. Such a construct can be used, e.g., in the
treatment of rheumatoid arthritis. In this embodiment, the fusion
of IL-1Ra and DHFR is translationally upregulated upon exposure to
an antifolate such as methotrexate. Therefore, in another specific
embodiment, the nucleic acid used to genetically engineer a
placental stem cell or umbilical cord stem cell can comprise
nucleotide sequences encoding a first polypeptide and a second
polypeptide, wherein said first and second polypeptides are
expressed as a fusion protein that is translationally upregulated
in the presence of an exogenous factor. The polypeptide can be
expressed transiently or long-term (e.g., over the course of weeks
or months).
[0224] Such a nucleic acid molecule can additionally comprise a
nucleotide sequence encoding a polypeptide that allows for positive
selection of engineered stem cells, or allows for visualization of
the engineered stem cells. In another more specific embodiment, the
nucleotide sequence encodes a polypeptide that is, e.g.,
fluorescent under appropriate visualization conditions, e.g.,
luciferase (Luc). In a more specific embodiment, such a nucleic
acid molecule can comprise IL-1Ra-DHFR-IRES-Luc, where IL-1Ra is
interleukin-1 receptor antagonist, IRES is an internal ribosomal
entry site, and DHFR is dihydrofolate reductase.
[0225] 5.7.3 Immortalized Placental Stem Cell Lines
[0226] Mammalian placental cells can be conditionally immortalized
by transfection with any suitable vector containing a
growth-promoting gene, that is, a gene encoding a protein that,
under appropriate conditions, promotes growth of the transfected
cell, such that the production and/or activity of the
growth-promoting protein is regulatable by an external factor. In a
preferred embodiment the growth-promoting gene is an oncogene such
as, but not limited to, v-myc, N-myc, c-myc, p53, SV40 large T
antigen, polyoma large T antigen, E1a adenovirus or E7 protein of
human papillomavirus.
[0227] 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. Nall. 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 VP 16 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.
[0228] 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. 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.
[0229] 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/FI2
containing N2 supplements. For example, a placental cell culture
prepared as described above may be infected after, e.g., five days
in vitro by incubation for about 20 hours in one volume of
conditioned medium and two volumes of DMEM/F12 containing N2
supplements. Transfected cells carrying a selectable marker may
then be selected as described above.
[0230] Following transfection, cultures are passaged onto a surface
that permits proliferation, e.g., allows at least 30% of the cells
to double in a 24 hour period. Preferably, the substrate is a
polyornithine/laminin substrate, consisting of tissue culture
plastic coated with polyornithine (10 .mu.g/mL) and/or laminin (10
.mu.g/mL), a polylysine/laminin substrate or a surface treated with
fibronectin. Cultures are then fed every 3-4 days with growth
medium, which may or may not be supplemented with one or more
proliferation-enhancing factors. Proliferation-enhancing factors
may be added to the growth medium when cultures are less than 50%
confluent.
[0231] 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.
[0232] 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.
[0233] 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.
[0234] 5.7.4 Assays
[0235] The placental stem cells, or hepatocytes and/or hepatogenic
cells, described herein can be used in assays to determine the
influence of culture conditions, environmental factors, molecules
(e.g., biomolecules, small inorganic molecules. etc.) and the like
on the proliferation, expansion, and/or differentiation of such
cells, compared to cells not exposed to such conditions.
[0236] In one embodiment, the hepatocytes and/or hepatogenic cells
described herein are assayed for changes in proliferation,
expansion or differentiation upon contact with a molecule. In one
embodiment, for example, provided herein is a method of identifying
a compound that modulates the proliferation of a plurality of
hepatocytes and/or hepatogenic cells differentiated from placental
stem cells, comprising contacting said cells with said compound
under conditions that allow proliferation, wherein if said compound
causes a detectable change in proliferation of said cells compared
to such cells not contacted with said compound, said compound is
identified as a compound that modulates proliferation of
hepatocytes and/or hepatogenic cells. In a specific embodiment,
said compound is identified as an inhibitor of proliferation. In
another specific embodiment, said compound is identified as an
enhancer of proliferation.
[0237] In another embodiment, provided herein is a method of
identifying a compound that modulates the expansion of a plurality
of hepatocytes and/or hepatogenic cells differentiated from
placental stem cells, comprising contacting said hepatocytes and/or
hepatogenic cells with said compound under conditions that allow
expansion, wherein if said compound causes a detectable change in
expansion of said hepatocytes and/or hepatogenic cells compared to
a plurality of hepatocytes and/or hepatogenic cells not contacted
with said compound, said compound is identified as a compound that
modulates expansion of hepatocytes and/or hepatogenic cells. In a
specific embodiment, said compound is identified as an inhibitor of
expansion. In another specific embodiment, said compound is
identified as an enhancer of expansion.
[0238] In another embodiment, provided herein is a method of
identifying a compound that modulates the differentiation of a
placental stem cell, e.g., differentiation to a hepatocyte and/or a
hepatogenic cell, comprising contacting said stem cells with said
compound under conditions that allow differentiation to a
hepatocyte or a hepatogenic cell, wherein if said compound causes a
detectable change in differentiation of said stem cells compared to
a stem cell not contacted with said compound, said compound is
identified as a compound that modulates proliferation of placental
stem cells. In a specific embodiment, said compound is identified
as an inhibitor of differentiation. In another specific embodiment,
said compound is identified as an enhancer of differentiation.
[0239] 5.7.5 Cell Banks
[0240] Stem cells from postpartum placentas can be cultured in a
number of different ways to produce a set of lots, e.g., a set of
individually-administrable doses, of placental stem cells. Such
lots can, for example, be obtained from stem cells from placental
perfusate or from enzyme-digested placental tissue. Sets of lots of
placental stem cells, obtained from a plurality of placentas, can
be arranged in a bank of placental stem cells for, e.g., long-term
storage. Generally, adherent stem cells are obtained from an
initial culture of placental material to form a seed culture, which
is expanded under controlled conditions to form populations of
cells from approximately equivalent numbers of doublings. Lots are
preferably derived from the tissue of a single placenta, but can be
derived from the tissue of a plurality of placentas.
[0241] In one embodiment, stem cell lots are obtained as follows.
Placental tissue is first disrupted, e.g., by mincing, digested
with a suitable enzyme, e.g., collagenase (see Section 5.2.3,
above). The placental tissue preferably comprises, e.g., the entire
amnion, entire chorion, or both, from a single placenta, but can
comprise only a part of either the amnion or chorion. The digested
tissue is cultured, e.g., for about 1-3 weeks, preferably about 2
weeks. After removal of non-adherent cells, high-density colonies
that form are collected, e.g., by trypsinization. These cells are
collected and resuspended in a convenient volume of culture medium,
and defined as Passage 0 cells.
[0242] Passage 0 cells are then used to seed expansion cultures.
Expansion cultures can be any arrangement of separate cell culture
apparatuses, e.g., a Cell Factory by NUNC.TM.. Cells in the Passage
0 culture can be subdivided to any degree so as to seed expansion
cultures with, e.g., 1.times.10.sup.3, 2.times.10.sup.3,
3.times.10.sup.3, 4.times.10.sup.3, 5.times.10.sup.3,
6.times.10.sup.3, 7.times.10.sup.3, 8.times.10.sup.3,
9.times.10.sup.3, 1.times.10.sup.4, 1.times.10.sup.4,
2.times.10.sup.4, 3.times.10.sup.4, 4.times.10.sup.4,
5.times.10.sup.4, 6.times.10.sup.4, 7.times.10.sup.4,
8.times.10.sup.4, 9.times.10.sup.4, or 10.times.10.sup.4 stem
cells. Preferably, from about 2.times.10.sup.4 to about
3.times.10.sup.4 Passage 0 cells are used to seed each expansion
culture. The number of expansion cultures can depend upon the
number of Passage 0 cells, and may be greater or fewer in number
depending upon the particular placenta(s) from which the stem cells
are obtained.
[0243] Expansion cultures are grown until the density of cells in
culture reaches a certain value, e.g., about 1.times.10.sup.5
cells/cm.sup.2. Cells can either be collected and cryopreserved at
this point, or passaged into new expansion cultures as described
above. Cells can be passaged, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19 or 20 times prior to use. A record
of the cumulative number of population doublings is preferably
maintained during expansion culture(s). The cells from a Passage 0
culture can be expanded for 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16,
18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 or 40 doublings, or up
to 60 doublings. Preferably, however, the number of population
doublings, prior to dividing the population of cells into
individual doses, is between about 15 and about 30, preferably
about 20 doublings. The cells can be culture continuously
throughout the expansion process, or can be frozen at one or more
points during expansion.
[0244] Cells to be used for individual doses can be frozen, e.g.,
cryopreserved for later use. Individual doses can comprise, e.g.,
about 1 million to about 100 million cells per ml, and can comprise
between about 10.sup.6 and about 10.sup.9 cells in total.
[0245] In a specific embodiment, of the method, Passage 0 cells are
cultured for approximately 4 doublings, then frozen in a first cell
bank. Cells from the first cell bank are frozen and used to seed a
second cell bank, the cells of which are expanded for about another
eight doublings. Cells at this stage are collected and frozen and
used to seed new expansion cultures that are allowed to proceed for
about eight additional doublings, bringing the cumulative number of
cell doublings to about 20. Cells at the intermediate points in
passaging can be frozen in units of about 100,000 to about 10
million cells per ml, preferably about 1 million cells per ml for
use in subsequent expansion culture. Cells at about 20 doublings
can be frozen in individual doses of between about 1 million to
about 100 million cells per ml for administration or use in making
a stem cell-containing composition.
[0246] In a preferred embodiment, the donor from which the placenta
is obtained (e.g., the mother) is tested for at least one pathogen.
If the mother tests positive for a tested pathogen, the entire lot
from the placenta is discarded. Such testing can be performed at
any time during production of placental stem cell lots, including
before or after establishment of Passage 0 cells, or during
expansion culture. Pathogens for which the presence is tested can
include, without limitation, hepatitis A, hepatitis B, hepatitis C,
hepatitis D, hepatitis E, human immunodeficiency virus (types I and
II), cytomegalovirus, herpesvirus, and the like.
[0247] In a modification of the above banks, a plurality, or all,
of the placental stem cells in a bank, e.g., placental stem cells
from a single placenta, or from multiple placentas, are exposed to
conditions that cause differentiation of the cells into hepatocytes
and/or hepatogenic cells. Such cells can be selected based on the
expression of one or more hepatocyte markers not present in, or
present at a detectably different level in, placental stem cells.
In such an embodiment, a bank of cells can comprise populations of
hepatocytes and/or hepatogenic cells, alone or in combination with
placental stem cells not differentiated to hepatocytes or
hepatogenic cells.
[0248] 5.7.6 Treatment of Liver Disease
[0249] In another aspect, provided herein is a method of treating a
subject having a disease, disorder or condition associated with
abnormal liver function, comprising introducing a hepatocyte, or
population of hepatocytes, produced according to the methods of
differentiating placental stem cells into hepatocytes disclosed
herein, into said subject. In a more specific embodiment, the
disease, disorder or condition is cirrhosis of the liver. In
certain embodiments, the disease or conditions results from liver
toxicity caused by, e.g., alcohol or ingestion of toxins such as,
e.g., mushroom toxins. In certain embodiments, the disease or
condition is a viral infection, e.g., a hepatitis A, B, C, D, or E
infection.
[0250] An individual having a disease associated with abnormal
liver function, e.g., an individual diagnosed with cirrhosis, can
be treated with a plurality of placental stem cells, and,
optionally, one or more therapeutic agents, at any time during the
progression of the disease. For example, the individual can be
treated immediately after diagnosis, or within 1, 2, 3, 4, 5, 6
days of diagnosis, or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,
25, 30, 35, 40, 45, 50 or more weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9,
10 or more years after diagnosis. The individual can be treated
once, or multiple times during the clinical course of the disease.
In one embodiment, the individual is administered a dose of about
300 million placental stem cells. Dosage, however, can vary
according to the individual's physical characteristics, e.g.,
weight, and can range from 1 million to 10 billion placental stem
cells per does, preferably between 10 million and 1 billion per
dose, or between 100 million and 50 million placental stem cells
per dose.
[0251] The administration is preferably intravenous, but can be by
any art-accepted route for the administration of live cells.
Placental stem cells, e.g., placental stem cells that have been
differentiated into cells that express one or more characteristics
of a hepatocyte, can be transplanted directly into one or more
sites of a liver, e.g., in a buffer or medium solution, hydrogel,
alginate. In one embodiment, the placental stem cells are from a
cell bank.
[0252] In another embodiment, the plurality of placental stem cells
has been contacted with one or more agents that promote
differentiation of the placental stem cells into hepatocytes or
into hepatogenic cells. In such a plurality of cells, some of the
cells can be undifferentiated placental stem cells (i.e., placental
stem cells that have not begun to differentiate into hepatocytes);
some of the cells can be placental stem cells that have begun to
express one or more characteristics of hepatocytes; and some can be
cells, differentiated from placental stem cells, that have begun to
express a plurality, a majority, or all of the characteristics of a
terminally-differentiated hepatocyte.
[0253] 5.7.7 Use of Placental Stem Cell-Derived Hepatocytes to
Identify Antiviral Agents
[0254] The hepatocytes and hepatocyte cultures described herein can
be used in in vitro or in vivo assays to determine whether a
compound is an antiviral agent.
[0255] In vitro assays. A plurality (e.g., population) of placental
stem cells can be used to identify antiviral agents in one
embodiment as follows. A population of placental stem cells is
established as described elsewhere herein. The population of
placental stem cells is contacted with one or more compounds or
agents that promote the differentiation of the placental stem cells
into hepatocytes. The placental stem cells are then cultured until
at least a plurality of the placental stem cells express one or
more markers characteristic of hepatocytes, or, in the case of,
e.g., cytokeratin 18, at a level characteristic of hepatocytes.
[0256] Placenta-derived hepatocytes, or hepatogenic cells, are then
infected with virus. The virus is preferably a virus that
specifically infects hepatic tissue, e.g., hepatitis A, B, C, D or
E. Virus stocks can be obtained from the serum of one or more
individuals infected with the virus who have detectable level of
the virus in their serum. Virus can also be obtained from infected
animals, e.g., from infected rodents, rabbits, or the like; from
commercial sources, or from cell lines infected with the virus.
[0257] Regardless of source, the virus is contacted with the
placenta-derived hepatocytes, or population of placental stem cells
comprising hepatocytes and/or hepatogenic cells, and is given
sufficient time to infect. The hepatocytes or population of
placental cells can be about, at least or at most 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90% or 95% confluent at the time of
infection, e.g., in a tissue culture dish or flask. In various
embodiments, the hepatocytes or hepatogenic cells, or population of
hepatocytes or hepatogenic cells, differentiated from placental
stem cells, are infected with a virus, e.g., a hepatotrophic virus,
e.g., with about 1.times.10.sup.8 to 1.times.10.sup.9 virions.
[0258] In a specific embodiment, placenta-derived hepatocytes or
populations of placental stem cells comprising hepatocytes and/or
hepatogenic cells are collected from cell culture by trypsinization
and centrifugation, washed, an resuspended in medium. Aliquots of
cells are placed on coverslips in a 12-well plate, and treated with
2% dimethylsulfoxide (DMSO) and optionally for 6-10 days. Cells are
then incubated with virions, e.g., in infected serum, at 37.degree.
C. for 10-20 hours. In another embodiment, placental stem
cell-derived hepatocytes or populations of placental stem cells
comprising hepatocytes and/or hepatogenic cells are infected by
co-culture with an virus-infected cells line that sheds virus,
e.g., hepatoma cell line HIB 611, which is infected with HBV (see
Ochiya et al., "An In Vitro System for Infection with Hepatitis B
Virus That Uses Primary Human Fetal Hepatocytes," Proc. Natl. Acad.
Sci. U.S.A. 86:1875-1879 (1989).
[0259] At 2-5 days post-infection, the cells are collected, and the
level of virus present is determined. Viral levels can be
determined using, e.g., one or more antibodies that recognize a
viral antigen, e.g., hepatitis A virus surface antigen (antibodies
available from AbD Serotec); hepatitis B virus HBsAg of HBeAg;
hepatitis C virus core antigen; hepatitis D core antigen or
hepatitis E core antigen. In another embodiment, the level of virus
in the infected cells is determined by quantitative or
semi-quantitative PCR. In a specific embodiment, the primers used
detect or are specific for a replicating form of the virus, e.g.,
are specific for the covalently closed circular form of HBV
(cccHBV). In another specific embodiment, the primers used can
amplify both cccHBV and the relaxed circular form of HBV.
[0260] A compound or agent can be tested for antiviral activity at
several points in the above-outlined procedure. For example, the
compound can be contacted with the hepatocytes or hepatogenic cells
provided herein at the time of collection from culture; after
centrifugation but before contact with the virus; at the same time
as contact with the virus (e.g., simultaneously or within minutes);
or after contact with the virus. For example, in one embodiment,
contact between the compound and hepatocytes or hepatogenic cells
provided herein can be accomplished prior to infection with the
virus, to determine the effect the compound has on initial
infection. In another embodiment, the compound can be contacted
with infected hepatocytes or hepatogenic cells provided herein from
about 1 to about 5 days after infection to determine, e.g., if the
compound has an effect on production of virus compared to cells
that are not contacted with the compound. In another embodiment,
infected cells can be contacted with the compound of interest and
cocultured with uninfected hepatocytes or hepatogenic cells
provided herein, or primary hepatocyte cultures, to determine if
the compound has any effect on infection of uninfected cells.
[0261] In a specific embodiment, a compound is an antiviral
compound if the compound causes a detectable reduction in the
amount of virus produced by infected hepatocytes or hepatogenic
cells provided herein at any time, e.g., within 1, 2, 3, 4, 5, 6 or
7 days after contact with the compound, compared to hepatocytes or
hepatogenic cells not contacted with the compound. In another
specific embodiment, a compound is an antiviral compound if the
compound causes a detectable reduction in the number of infected
hepatocytes in a coculture of infected hepatocytes and uninfected
hepatocytes in the presence of the compound, compared to a
coculture in the absence of the compound.
[0262] In vivo assays. Hepatocytes or hepatogenic cells,
differentiated from placental stem cells, can be used as part of an
in vivo assay to identify antiviral compounds. In the assay,
hepatocytes or hepatogenic cells are infected with a virus, e.g.,
hepatitis B virus, and implanted into a specific mouse host to
cause an initiation of viremia within the mouse. A compound is then
administered to the mouse, and the effects of the compound on the
resulting viral load or viral replication is determined. Such an
assay is described in detail in Example 10.
[0263] In one embodiment, the assays uses a normal host mouse that
is irradiated with an otherwise-lethal dose of gamma irradiation,
and protected by the administration of bone marrow from an
immune-restricted mouse, e.g., a NOD/SCID mouse. At the same time
as the bone marrow is administered to the host mouse, or within 6-7
days after administration of bone marrow, a plurality of infected
placental stem cell-derived hepatocytes or hepatogenic cells is
administered to the host mouse, e.g., intraperitoneally, under the
kidney capsule, into the host mouse liver, into the ear pinnae,
etc. Within 6-20 days post-transplantation, a compound of interest
is administered to the mouse. Such administration can be by any
medically-acceptable route, but administration is preferably
intraperitoneal or topical.
[0264] The host mouse can be assessed an any time after
administration of the compound to determine, e.g., viral load in
the serum, or for other indicia of viral presence or replication.
In various embodiments, a tissue from the mouse is assayed for the
presence of virions, e.g replicating forms of the virus. As above,
viral levels can be determined using, e.g., one or more antibodies
that recognize a viral antigen, e.g., hepatitis A virus surface
antigen (antibodies available from AbD Serotec); hepatitis B virus
HBsAg of HBeAg; hepatitis C virus core antigen; hepatitis D core
antigen or hepatitis E core antigen. In a specific embodiment, for
example, the presence of hepatitis B virus in a serum sample from a
host mouse is detected using one or more antibodies to a hepatitis
B virus envelope protein, surface antigen, or core antigen. In
another embodiment, the level of virus in the infected cells is
determined by quantitative or semi-quantitative PCR. In a specific
embodiment, the primers used detect or are specific for a
replicating form of the virus, e.g., are specific for the
covalently closed circular form of HBV (cccHBV). In another
specific embodiment, the primers used can amplify both cccHBV and
the relaxed circular form of HBV.
[0265] 5.7.8 Treatment of Cartilage Damage
[0266] In other embodiments, isolated placental stem cells,
isolated populations of placental stem cells, and/or chondrocytic
cells or chondrocytes differentiated therefrom, may be used in
autologous or allogeneic tissue regeneration or replacement
therapies or protocols, including, but not limited to repair of
cartilage tissue. In a specific embodiment, placental stem cells
can be used to heal or repair a disease, disorder or condition
affecting cartilage, including trauma to cartilage (e.g., breaks,
tears, etc.). In a more specific embodiment, the cartilage is
articular cartilage. Placental stem cells can be administered to
the cartilage directly, e.g., in a cell suspension, or can be
administered to the cartilage in combination with a matrix, e.g.,
an electrospun nanofibrous scaffold, such as electrospun
nanofibrous scaffolds described in Section 5.7.1.4, above.
Placental stem cells contacted with (e.g., seeded onto) to the
cartilage, with or without a scaffold, are preferably CD200.sup.+,
CD105', CD90.sup.+, CD34.sup.-, CD45.sup.- placental stem cells,
but can be any of the placental stem cells described herein. All or
a plurality of the placental stem cells used to treat a disease,
disorder or condition in cartilage can be differentiated to
chondrocytic cells prior to administration to the cartilage, or can
be administered in an undifferentiated state. The chondrocytic
cells or chondrocytes can be administered alone, or in combination
with placental stem cells and/or another type of stem cell, in a
cell suspension, or can be administered to the cartilage in
combination with a matrix, e.g., an electrospun nanofibrous
scaffold.
[0267] The effectiveness of a particular population of placental
stem cells, alone or in combination with a scaffold, e.g., an
electrospun nanofibrous scaffold, can be evaluated in an animal
model that does not spontaneously heal a cartilage injury, e.g., a
rabbit osteochondral defect model, e.g., as described in Example
14, below.
[0268] 5.7.9 Uses of CD34.sup.+, CD45.sup.- Placental Stem
Cells
[0269] CD34.sup.+, CD45.sup.- placental stem cells, and cell
populations enriched for CD34.sup.+, CD45.sup.- placental stem
cells, provided herein can be used to treat an individual in need
of hematopoietic stem cells, e.g., an individual in need of
hematopoietic reconstitution, for example, after chemotherapy or
myeloablation. In one embodiment, placental CD34.sup.+CD45.sup.-
stem cells alone are used to treat such an individual. In another
embodiment, placental CD34.sup.+CD45.sup.- stem cells are used in
combination with, or to supplement, a second type of stem cell, or
a second population of stem cells. Stem cells in such a second
population can comprise hematopoietic stem cells, non-hematopoietic
stem cells, or both. In one embodiment, the second population of
stem cells comprises stem cells in cord blood. In another
embodiment, the second population of stem cells comprises stem
cells in bone marrow. In a specific embodiment, the stem cells are
transplanted into the individual.
[0270] Typically, a patient receiving a stem cell infusion, for
example for a bone marrow transplantation, receives one unit of
nucleated cells, where a unit is approximately 1.times.10.sup.9
nucleated cells (corresponding to 1-2.times.10.sup.6 CD34.sup.+
stem cells). Thus, in one embodiment, the number of nucleated
cells, comprising CD34.sup.+CD45.sup.- placental stem cells,
administered to an individual, is at least five times the number of
cells normally administered in a bone marrow replacement. In
another specific embodiment of the method, the number of nucleated
cells administered to an individual is at least ten times the
number of cells normally administered in a bone marrow replacement.
In another specific embodiment, the number of nucleated cells
administered to an individual is at least fifteen times the number
of cells normally administered in a bone marrow replacement. In
another embodiment of the method, the total number of nucleated
cells, which includes CD34.sup.+CD45.sup.- placental stem cells,
administered to an individual is between 1-1000.times.10.sup.8 per
kilogram of body weight.
[0271] In other embodiments, CD34.sup.+CD45.sup.- placental stem
cells, e.g., a cell population enriched in CD34.sup.+CD45.sup.-
placental stem cells, improves engraftment in an individual in need
of stem cells, e.g., hematopoietic stem cells, compared to
engraftment in an individual not receiving a population of
hematopoietic stem cells enriched in CD34.sup.+CD45.sup.- placental
stem cells. In various embodiments, the engraftment is improved at
least, or at, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20 or 21 days post-transplant. In another more
specific embodiment, CD34.sup.+CD45.sup.- placental stem cells
improve engraftment in an individual in need of stem cells at
least, or at, more than 21 days post-transplant. In specific
embodiments, CD34.sup.+CD45.sup.- placental stem cells improves
engraftment in an individual in need of stem cells at least, or at,
more than 25, 30, 35, 40, 45, 50, 55 weeks, or 1 year or longer
post-transplant.
[0272] The CD34.sup.+CD45.sup.- placental stem cells, and
populations of stem cells comprising such stem cells, can be
prepared in a form that is easily administrable to an individual.
For example, the cells or cell population can be contained within a
container suitable for medical use. Such a container can be, for
example, a sterile plastic bag, flask, jar, or other container from
which the combined stem cell population can be easily dispensed.
Preferably, the container is a container that allows, or
facilitates, intravenous administration of a combined stem cell
population. The container, e.g., bag, can hold the placenta-derived
stem cells and stem cells from a second source together, e.g., as a
mixed cell population, or can hold the two stem cell populations
separately. In the latter embodiment, the bag preferably comprises
multiple lumens or compartments that are interconnected to allow
mixing of the placenta-derived stem cells and stem cells from a
second source prior to, or during, administration. The container is
preferably one that allows for cryopreservation of the combined
stem cell population.
[0273] Thus, in one embodiment, provided herein is a composition
comprising a CD34.sup.+CD45.sup.- placental stem cell-enriched cell
population in a container. In another embodiment, provided herein
is a composition comprising a stem cell population, wherein said
stem cell population comprises CD34.sup.+CD45.sup.-
placenta-derived stem cells and second type of stem cells in a
container. In a specific embodiment, the container is a bag, flask,
or jar. In a more specific embodiment, said placenta-derived stem
cells and said second type of stem cells are contained together in
said bag. In another more specific embodiment, said
placenta-derived stem cells and said second type of stem cells from
a second source are contained separately within said bag. In
another specific embodiment, the composition comprises one or more
compounds that facilitate cryopreservation of the combined stem
cell population. In another specific embodiment, said combined stem
cell population is contained within a physiologically-acceptable
aqueous solution. In a more specific embodiment, said
physiologically-acceptable aqueous solution is a 0.9% NaCl
solution. In another more specific embodiment, said bag is a
sterile plastic bag. In a more specific embodiment, said bag allows
or facilitates intravenous administration of said stem cells. In
another specific embodiment, the stem cells comprise
CD34.sup.+CD45.sup.- placental cells that are HLA-matched to said
stem cells from a second source. In another specific embodiment,
stem cells comprise CD34.sup.+CD45.sup.- placental stem cells that
are at least partially HLA-mismatched to the second type of stem
cell. In another specific embodiment, said placenta-derived stem
cells are derived from a plurality of donors. In another specific
embodiment, said stem cells from a second source are derived from a
plurality of donors.
[0274] CD34.sup.+CD45.sup.- stem cells and cell populations
comprising CD34.sup.+CD45.sup.- placental stem cells can be
cultured for a period of time prior to administration to an
individual. For example, in one embodiment, the stem cells can be
cultured in medium comprising Notch agonist, e.g., a deletion form
of a Notch protein consisting essentially of the intracellular
domain of the Notch protein, or a Delta protein. See U.S.
2004/0067583.
[0275] In another embodiment, a population of CD34.sup.+CD45.sup.-
placental stem cells provided herein and a population of umbilical
cord blood cells are administered sequentially to a patient in need
thereof. In one embodiment, the population of placental stem cells
is administered first and the population of stem cells of a second
type is administered second. In another embodiment, the stem cells
of a second type are administered first and the
CD34.sup.+CD45.sup.- placental stem cells are administered
second.
[0276] Combined populations of CD34.sup.+CD45.sup.- placental stem
cells, and stem cells of a second type, e.g., cord blood-derived
stem or progenitor cells, or cord blood, including banked or
cryopreserved cord blood, can be mixed, prior to transplantation,
by any medically-acceptable means. In one embodiment, the two
populations are physically mixed. In another embodiment of the
method, populations are mixed immediately prior to (i.e., within 1,
2, 3, 4, 5, 7, 10 minutes of) administration to said individual. In
another embodiment, populations are mixed at a point in time more
than five minutes prior to administration to said individual. In
another embodiment of the method, the CD34.sup.+CD45.sup.-
placental stem cells, and/or stem cells of a second type, are
cryopreserved and thawed prior to administration to said
individual. In another embodiment, stem cells are mixed at a point
in time more than 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 prior to
administration to said individual, wherein either or both of the
populations of stem cells have been cryopreserved and thawed prior
to said administration. In another embodiment, the stem cell
populations may be administered more than once.
[0277] In another embodiment, the CD34.sup.+CD45.sup.- placental
stem cells and/or stem cells of a second type are preconditioned
prior to transplantation. In a preferred embodiment,
preconditioning comprises storing the cells in a gas-permeable
container generally for a period of time at about -5.degree. C. to
about 23.degree. C., about 0.degree. C. to about 10.degree. C., or
preferably about 4.degree. C. to about 5.degree. C. The cells may
be stored between 18 hours and 21 days, between 48 hours and 10
days, preferably between 3-5 days. The cells may be cryopreserved
prior to preconditioning or, may be preconditioned immediately
prior to administration.
[0278] Either or both of the CD34 CD45.sup.- placental stem cells,
or stem cells of a second type, may be differentiated prior to
contacting an individual in need of stem cells. For example, for
contacting for the purpose of hematopoietic engraftment, the stem
cells may be differentiated to cells in the hematopoietic lineage.
In certain embodiments, the method of transplantation of stem cell
populations comprises (a) induction of differentiation of the
CD34.sup.+CD45.sup.- placental stem cells, (b) mixing the placental
stem cells with a population of stem cells of a second type, e.g.,
cord blood stem cells, to form a combined cell population, and (c)
administration of the combined cell population to an individual in
need thereof. In another embodiment the method of transplantation
comprises (a) induction of differentiation of stem cells of a
second type; (b) mixing the differentiated cells with
CD34.sup.+CD45.sup.- placental stem cells to form a combined cell
population; and (c) administration of the combined cell population
to an individual in need thereof. In another embodiment, the method
of transplantation of combined stem cell populations comprises (a)
mixing CD34.sup.+CD45.sup.- placental stem cells with a population
of cord blood cells; (b) induction of differentiation of the
mixture of the cord blood cells and CD34'CD45.sup.- placental stem
cells and (c) administration of the mixture to a patient in need
thereof.
[0279] The stem cell populations provided herein, enriched for
CD34.sup.+CD45.sup.- placental stem cells, may be transplanted into
a patient in any pharmaceutically or medically acceptable manner,
including by injection, e.g., intravenous injection, intramuscular
injection, intraperitoneal injection, intraocular injection, direct
injection into a particular tissue, transfusion, etc. For example,
combined stem cell populations, e.g., placental stem cells in
combination with cord blood-derived stem cells) may be transplanted
by intravenous infusion. In another embodiment, a combined stem
cell population comprising placental stem cells and cardiac stem
cells, in suspension, may be injected directly into cardiac tissue,
e.g., an ischemic area in a heart. The combined stem cell
populations may comprise, or be suspended in, any
pharmaceutically-acceptable carrier. The combined stem cell
populations may be carried, stored, or transported in any
pharmaceutically or medically acceptable container, for example, a
blood bag, transfer bag, plastic tube or vial.
[0280] After transplantation, engraftment in a human recipient may
be assessed using, e.g., nucleic acid or protein detection or
analytical methods. For example, the polymerase chain reaction
(PCR), STR, SSCP, RFLP analysis, AFLP analysis, and the like, may
be used to identify engrafted cell-specific nucleotide sequences in
a tissue sample from the recipient. Such nucleic acid detection and
analysis methods are well-known in the art. In one embodiment,
engraftment may be determined by the appearance of engrafted
cell-specific nucleic acids in a tissue sample from a recipient,
which are distinguishable from background. The tissue sample
analyzed may be, for example, a biopsy (e.g., bone marrow aspirate)
or a blood sample.
[0281] In one embodiment, a sample of peripheral blood is taken
from a patient immediately prior to a medical procedure, e.g.,
myeloablation. After the procedure, a combined stem cell as
provided herein is administered to the patient. At least once
post-administration, a second sample of peripheral blood is taken.
An STR profile is obtained for both samples, e.g., using PCR
primers for markers (alleles) available from, e.g., LabCorp
(Laboratory Corporation of America). A difference in the number or
characteristics of the markers (alleles) post-administration
indicates that engraftment has taken place.
[0282] Engraftment can also be demonstrated by detection of
re-emergence of neutrophils.
[0283] In another example, engrafted cell-specific markers may be
detected in a tissue sample from the recipient using antibodies
directed to markers specific to either the transplanted stem cells,
or cells into which the transplanted stem cells would be expected
to differentiate. In one embodiment, engraftment of a combination
of placental stem cells and cord blood-derived stem cells may be
assessed by FACS analysis to determine the presence of CD45.sup.+,
CD19.sup.+, CD33', CD7.sup.+ and/or CD3.sup.+ cells by adding the
appropriate antibody and allowing binding; washing (e.g., with
PBS); fixing the cells (e.g., with 1% paraformaldehyde); and
analyzing on an appropriate FACS apparatus (e.g., a FACSCalibur
flow cytometer (Becton Dickinson)). Where placental stem cells
and/or stein cells from a second source are from an individual of a
different sex than a recipient, e.g., male donor and female
recipient, engraftment can be determined by detection of
sex-specific markers, e.g., Y-chromosome-specific markers.
Placental stem cells and/or stem cells from a second source may
also be genetically modified to express a unique marker or nucleic
acid sequence that facilitates identification, e.g., an RFLP
marker, expression of .beta.-galactosidase or green fluorescent
protein, or the like.
[0284] The degree of engraftment may be assessed by any means known
in the art. In one embodiment, the degree of engraftment is
assessed by a grading system as follows, which uses a thin section
of fixed and antibody-bound tissue from the transplant recipient.
In this example grading system, engraftment is graded as follows:
0=no positive cells (that is, no cells bound by an antibody
specific to an engrafted cell); 0.5=one or two positive cells,
perhaps positive, but difficult to differentiate from background or
non-specific staining; 1=2-20 scattered positive cells;
2=approximately 20-100 scattered or clustered positive cells
throughout the tissue; 3=more than 100 positive cells comprising
less than 50% of the tissue; 4=more than 50% of cells are positive.
In specific embodiments, engraftment is determined where greater
than 0.5%, 1%, 2%, 3%, 4%, 5%, 7.5%, 10%, 15%, 20% or greater of
the cells are positively stained.
[0285] In another embodiment, the degree of engraftment is
determined by analysis of the gain of one or more biological
functions carried out by the engrafted cells. For example, where a
recipient, who has undergone myeloablative therapy, receives a
transplant of a combined stem cell population comprising placental
stem cells and cord blood-derived stem cells, the degree of
engraftment may be determined by the degree to which normal
hematopoiesis, blood cell populations and blood function return to
normal.
[0286] Where the combined stem cell population in whole or in part
is HLA-mismatched to an intended recipient, it may be necessary to
treat the recipient to reduce immunological rejection of the donor
cells. Methods for reducing immunological rejection are disclosed
in, e.g., U.S. Pat. Nos. 5,800,539 and 5,806,529, both of which are
incorporated herein by reference.
[0287] In one embodiment, therefore, combined stem cell populations
comprising hematopoietic stem cells can be used to treat patients
having a blood cancer, such as a lymphoma, leukemia (such as
chronic or acute myelogenous leukemia, acute lymphocytic leukemia,
Hodgkin's disease, etc.), myelodysplasia, myelodysplastic syndrome,
and the like. In another embodiment, the disease, disorder or
condition is chronic granulomatous disease.
[0288] Because hematopoietic reconstitution can be used in the
treatment of anemias, further provided herein is the treatment of
an individual with CD34.sup.+CD45.sup.- placental stem cells of the
invention, wherein the individual has an anemia or disorder of the
blood hemoglobin. The anemia or disorder may be natural (e.g.,
caused by genetics or disease), or may be artificially-induced
(e.g., by accidental or deliberate poisoning, chemotherapy, and the
like). In another embodiment, the disease or disorder is a marrow
failure syndrome (e.g., aplastic anemia, Kostmann syndrome,
Diamond-Blackfan anemia, amegakaryocytic thrombocytopenia, and the
like), a bone marrow disorder or a hematopoietic disease or
disorder. In a specific embodiment, the CD34.sup.+CD45.sup.-
placental stem cells are administered with a plurality of
mesenchymal stem cells and/or a plurality of adherent placental
stem cells.
[0289] In another embodiment, the CD34.sup.+CD45.sup.- placental
stem cells provided herein can be introduced, alone or in
combination with a second type of stem cell, e.g., a mesenchymal
stem cell or an adherent placental stem cell, into a damaged organ
for organ neogenesis and repair of injury in vivo. Such injury may
be due to conditions and disorders including, but not limited to,
myocardial infarction, seizure disorder, multiple sclerosis,
stroke, hypotension, cardiac arrest, ischemia, inflammation,
age-related loss of cognitive function, cerebral palsy,
neurodegenerative disease, Alzheimer's disease, Parkinson's
disease, Leigh disease, AIDS dementia, memory loss, amyotrophic
lateral sclerosis, ischemic renal disease, brain or spinal cord
trauma, heart-lung bypass, glaucoma, retinal ischemia, or retinal
trauma.
[0290] In other embodiments, the disease, disorder or condition
includes, but is not limited to lysosomal storage diseases, such as
Tay-Sachs, Niemann-Pick, Fabry's, Gaucher's disease (e.g.,
glucocerebrosidase deficiency), Hunter's, and Hurler's syndromes,
Maroteaux-Lamy syndrome, fucosidosis (fucosidase deficiency),
Batten disease (CLN3), as well as other gangliosidoses,
mucopolysaccharidoses, and glycogenoses.
[0291] In other embodiments, the CD34.sup.+CD45.sup.- placental
stem cells provided herein can be used as autologous or
heterologous transgene carriers in gene therapy to correct, for
example, inborn errors of metabolism, adrenoleukodystrophy (e.g.,
co-A ligase deficiency), metachromatic leukodystrophy
(arylsulfatase A deficiency) (e.g., symptomatic, or presymptomatic
late infantile or juvenile forms), globoid cell leukodystrophy
(Krabbe's disease; galactocerebrosidase deficiency), acid lipase
deficiency (Wolman disease), cystic fibrosis, glycogen storage
disease, hypothyroidism, sickle cell anemia, thalassemia (e.g.,
beta thalassemia), Pearson syndrome, Pompe's disease,
phenylketonuria (PKU), porphyrias, maple syrup urine disease,
homocystinuria, mucoplysaccharidosis, chronic granulomatous disease
and tyrosinemia and Tay-Sachs disease or to treat solid tumors or
other pathological conditions.
[0292] In other embodiments, the disease, disorder or condition is
a disease, disorder or condition requiring replacement or repair of
one or more tissues. For example, the CD34.sup.+ CD45.sup.-
placental stem cells provided herein, alone or in combination with
a second type of stem cell, e.g., a mesenchymal stein cell or an
adherent placental stem cell, can be used in therapeutic
transplantation protocols, e.g., to augment or replace stem or
progenitor cells of the liver, pancreas, kidney, lung, nervous
system, muscular system, bone, bone marrow, thymus, spleen, mucosal
tissue, gonads, or hair. The combined stem cell populations
provided herein can also be used for augmentation, repair or
replacement of, e.g., cartilage, tendon, or ligaments. For example,
in certain embodiments, prostheses (e.g., hip prostheses) are
coated with replacement cartilage tissue constructs grown from
combined stem cell populations provided herein. In other
embodiments, joints (e.g., knee) are reconstructed with cartilage
tissue constructs grown from combined stem cell populations.
Cartilage tissue constructs can also be employed in major
reconstructive surgery for different types of joints (for
protocols, see e.g., Resnick, D., and Niwayama, G., eds., 1988,
DIAGNOSIS OF BONE AND JOINT DISORDERS, 2D ED., W. B. Saunders Co.).
The combined stem cell populations can be used to repair damage of
tissues and organs resulting from trauma, metabolic disorders, or
disease. In one embodiment, a patient can be administered a
combined stem cell population to regenerate or restore tissues or
organs which have been damaged as a consequence of disease, e.g.,
to repair heart tissue following myocardial infarction.
[0293] In another embodiment, the CD34.sup.+CD45.sup.- placental
stem cells provided herein, alone or in combination with a second
type of stem cell, e.g., a mesenchymal stem cell or an adherent
placental stem cell, may be used to treat an individual who has
received a lethal or sub-lethal dose of radiation. Such radiation
may be accidentally received, for example in a nuclear incident,
whether work- or aggression-related, or therapeutic, for example,
as part of a medical procedure. The particular type of radiation
(e.g., alpha, beta, gamma) is not critical. The combined stem cell
populations provided herein may be used to ameliorate one or more
symptoms of radiation sickness, for example, nausea, loss of
appetite, lethargy, dyspnea, decreased white blood cell count,
chronic anemia, fatigue, weakness, paleness, difficulty breathing,
feelings of malaise, and the like, whether such symptoms are
indicative of recoverable or fatal radiation sickness. In another
embodiment, the individual has one or more symptoms associated with
acute radiation syndrome (ARS). The combined stem cell populations
provided herein may also be used to partially or fully reconstitute
the hematopoietic system of an individual that has received a
lethal or sub-lethal dose of radiation, such that the individual
becomes partially or fully chimeric. Such chimerism may be
temporary or permanent (e.g., may persist for 1, 2, 3 weeks, or 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11 months or longer). In a preferred
embodiment, a combined stem cell population provided herein is
provided to the individual within the first 24 hours after
exposure. The individual may be administered a combined stem cell
population within the first hour, 2 hours, 3 hours, 4 hours, 5
hours, 6 hours, 9 hours, 12 hours, 15 hours, 18 hours, or 21 hours
after exposure to radiation. A combined stem cell population as
provided herein may also be administered within 2 days, 3 days, 4
days, 5 days, 6 days, one week, 2 weeks, 3 weeks, 4 weeks or 5
weeks after exposure to radiation.
[0294] Therapeutic or prophylactic treatment of an individual with
the CD34.sup.+CD45.sup.- placental stem cells provided herein may
be considered effective if the disease, disorder or condition is
measurably improved in any way. Such improvement may be shown by a
number of indicators. Measurable indicators include, for example,
detectable changes in a physiological condition or set of
physiological conditions associated with a particular disease,
disorder or condition (including, but not limited to, blood
pressure, heart rate, respiratory rate, counts of various blood
cell types, levels in the blood of certain proteins, carbohydrates,
lipids or cytokines or modulation expression of genetic markers
associated with the disease, disorder or condition). Treatment of
an individual with the CD34.sup.+CD45.sup.- placental stem cells
provided herein would be considered effective if any one of such
indicators responds to such treatment by changing to a value that
is within, or closer to, the normal value. The normal value may be
established by normal ranges that are known in the art for various
indicators, or by comparison to such values in a control.
Introduction of a combined stem cell population as provided herein
for the purposes of engraftment, e.g., hematopoietic engraftment,
would be considered successful if the individual to whom the
combined stem cell population is introduced exhibits any
indications of engraftment (e.g., markers of engrafted cells
appearing in biopsy or tissue samples, or blood sample; detection
of one or more biochemical functions performed by the engrafted
cells, etc.). In medical science, the efficacy of a treatment is
also often characterized in terms of an individual's impressions
and subjective feeling of the individual's state of health.
Improvement therefore may also be characterized by subjective
indicators, such as the individual's subjective feeling of
improvement, increased well-being, increased state of health,
improved level of energy, or the like, after administration of the
stem cells or supplemented cell populations provided herein.
6. EXAMPLES
6.1 Example 1: Obtaining Placental Stem Cells
[0295] 6.1.1 Tissue Disruption/Enzymatic Digestion
[0296] An exemplary protocol for obtaining stem cells from
placental tissue by enzymatic digestion is as follows. Frozen
placental tissue (three pieces of approximately
-1.times.1.times.0.5 cm each) is obtained. The tissue is umbilical
cord, maternal surface of the placenta, or amniotic membrane.
Digestive enzymes used include trypsin-EDTA (0.25%, GIBCO BRL);
collagenase IA (Sigma), collagenase I (Worthington), collagenase IA
(Sigma)+Trypsin-EDTA, collagenase 1 (Worthington)+Trypsin-EDTA, or
Elastase+Collagenase I+Collagenase IV+Daspase (Worthington).
Digestion of placental tissue is as follows. Tissue is minced in
the presence of enzymes (1 g in 10 ml in 50 ml tube) at 37.degree.
C., 250 rpm shaking, tube position at 45.degree. angle for 1 hr
(C25 Incubator Shaker, New Brunswick Scientific, Edison, N.J.,
USA). The supernatant is then discarded. The pellet is washed with
20 ml Hank's+5% FCS (3 times), and re-suspended in 12 ml culture
medium. 3 ml of the resulting suspension are aliquoted into T-75
flasks containing 10 ml culture medium each (four flasks per
digestion). Optionally, 10 ml Trypsin/EDTA is added for 30 min at
37.degree. C., with shaking at 250 rpm, with recentrifugation and
an additional wash with 10 ml Hank's+5% FCS. Cells are plated and
cultured, selecting for adherent cells.
[0297] The following method may also be used. A placenta is
obtained less than 24 hours after expulsion. After cleaning the
placenta, a hemostat is clamped to the distal end of the umbilical
cord. The umbilical cord is cut at the junction with the placenta
and transferred to a sterile dish. After cutting the cord below the
hemostat, the cord is massaged to remove blood clots, and
transferred to 500 ml PBS containing gentamicin and amphotericin B.
5 g of this cord is used. A scalpel is used to trim the remaining
placental material by cutting in a radius of about 3 inches from
the umbilical cord attachment point. Blood clots are forced from
the remaining material, and 5 g of the amnion-chorion, centered at
the umbilical cord root, is transferred to the same container as
the umbilical cord. The umbilical cord and amnion-chorion tissue is
sliced, then minced to pieces about 1 mm.sup.3 in size. The tissue
is then digested with 1 mg/ml Collagenase 1A (20 ml/g tissue) for 1
hour at 37.degree. C. followed by Trypsin-EDTA (10 ml/g tissue) for
30 minutes at 37.degree. C. After three washes in 5% FBS in PBS,
the tissue is resuspended in culture medium (20 ml/g tissue) and
transferred to T flasks at about 0.22 ml/cm.sup.2.
[0298] 6.1.2 Perfusion
[0299] A post-partum placenta is obtained within 24 hours after
birth. The umbilical cord is clamped with an umbilical cord clamp
approximately 3 to 4 inches about the placental disk, and the cord
is cut above the clamp. The umbilical cord is either discarded, or
processed to recover, e.g., umbilical cord stem cells, and/or to
process the umbilical cord membrane for the production of a
biomaterial. Excess amniotic membrane and chorion is cut from the
placenta, leaving approximately 1/4 inch around the edge of the
placenta. The trimmed material is discarded.
[0300] Starting from the edge of the placental membrane, the
amniotic membrane is separated from the chorion using blunt
dissection with the fingers. When the amniotic membrane is entirely
separated from the chorion, the amniotic membrane is cut around the
base of the umbilical cord with scissors, and detached from the
placental disk. The amniotic membrane can be discarded, or
processed, e.g., to obtain stem cells by enzymatic digestion, or to
produce, e.g., an amniotic membrane biomaterial.
[0301] The fetal side of the remaining placental material is
cleaned of all visible blood clots and residual blood using sterile
gauze, and is then sterilized by wiping with an iodine swab than
with an alcohol swab. The umbilical cord is then clamped crosswise
with a sterile hemostat beneath the umbilical cord clamp, and the
hemostat is rotated away, pulling the cord over the clamp to create
a fold. The cord is then partially cut below the hemostat to expose
a cross-section of the cord supported by the clamp. Alternatively,
the cord is clamped with a sterile hemostat. The cord is then
placed on sterile gauze and held with the hemostat to provide
tension. The cord is then cut straight across directly below the
hemostat, and the edge of the cord near the vessel is
re-clamped.
[0302] The vessels exposed as described above, usually a vein and
two arteries, are identified, and opened as follows. A closed
alligator clamp is advanced through the cut end of each vessel,
taking care not to puncture the clamp through the vessel wall.
Insertion is halted when the tip of the clamp is slightly above the
base of the umbilical cord. The clamp is then slightly opened, and
slowly withdrawn from the vessel to dilate the vessel.
[0303] Plastic tubing, connected to a perfusion device or
peristaltic pump, is inserted into each of the placental arteries.
Plastic tubing, connected to a 250 mL collection bag, is inserted
into the placental vein. The tubing is taped into place.
[0304] A small volume of sterile injection grade 0.9% NaCl solution
to check for leaks. If no leaks are present, the pump speed is
increased, and about 750 mL of the injection grade 0.9% NaCl
solution is pumped through the placental vasculature. Perfusion can
be aided by gently massaging the placental disk from the outer
edges to the cord. When a collection bag is full, the bag is
removed from the coupler connecting the tubing to the bag, and a
new bag is connected to the tube.
[0305] When collection is finished, the collection bags are weighed
and balanced for centrifugation. After centrifugation. each bag is
placed inside a plasma extractor without disturbing the pellet of
cells. The supernatant within the bags is then removed and
discarded. The bag is then gently massaged to resuspend the cells
in the remaining supernatant. Using a sterile 1 mL syringe, about
300-500 .mu.L of cells is withdrawn from the collection bag, via a
sampling site coupler, and transferred to a 1.5 mL centrifuge tube.
The weight and volume of the remaining perfusate are determined,
and 1/3 volume of hetastarch is added to the perfusate and mixed
thoroughly. The number of cells per mL is determined. Red blood
cells are removed from the perfusate using a plasma extractor.
[0306] Placental cells are then immediately cultured to isolate
placental stem cells, or are cryopreserved for later use.
[0307] 6.1.3 Culture of Isolated Stem Cells
[0308] Primary Culture:
[0309] The purpose of primary culture is to establish cells from
digested placental tissue. The digested tissue is suspended in
culture medium and placed into Corning T-flasks, which are
incubated in a humidified chamber maintained at 37.degree. C. with
5% CO.sub.2. Half of the medium is replenished after 5 days of
culture. High-density colonies of cells form by 2 weeks of culture.
Colonies are harvested with Trypsin-EDTA, which is then quenched
with 2% FBS in PBS. Cells are centrifuged and resuspended in
culture medium for seeding expansion cultures. These cells are
defined as Passage 0 cells having doubled 0 times.
[0310] Expansion Culture:
[0311] Cells harvested from primary culture, harvested from
expansion culture, or thawed from the cell bank are used to seed
expansion cultures. Cell Factories (NUNC.TM.) are treated with 5%
CO.sub.2 in air at 50 ml/min/tray for 10 min through a sterile
filter and warmed in a humidified incubator maintained at
37.degree. C. with 5% CO.sub.2. Cell seeds are counted on a
hemacytometer with trypan blue, and cell number, viability, passage
number, and the cumulative number of doublings are recorded. Cells
are suspended in culture medium to about 2.3.times.10.sup.4
cells/ml and 110 ml/tray are seeded in the Cell Factories. After
3-4 days and again at 5-6 days of culture, culture medium is
removed and replaced with fresh medium, followed by another
treatment with 5% CO.sub.2 in air. When cells reach approximately
10.sup.5 cells/cm.sup.2, cells are harvested with Trypsin-EDTA,
followed by quenching with 2% FBS in PBS. Cell are then centrifuged
and resuspended in culture medium.
6.2 Example 2: Isolation and Characterization of Placental Stem
Cells from Perfusate
[0312] This Example demonstrates the collection and
characterization of placental stem cells from several different
perfusion experiments.
Materials and Methods
[0313] Placenta donors were recruited from expectant mothers that
enrolled in private umbilical cord blood banking programs and
provided informed consent permitting the use of the exsanguinated
placenta following recovery of cord blood for research purposes.
These donors also permitted use of blinded data generated from the
normal processing of their umbilical cord blood specimens for
cryopreservation. This allowed comparison between the composition
of the collected cord blood and the effluent perfusate recovered
using this experimental method described below. All donor data was
kept confidential.
[0314] Following exsanguination of the umbilical cord and placenta,
the placenta was placed in a sterile, insulated container at room
temperature and delivered to the laboratory within 4 hours of
birth. Placentas were discarded if, on inspection, they had
evidence of physical damage such as fragmentation of the organ or
avulsion of umbilical vessels. Placentas were maintained at room
temperature (23.+-.2.degree. C.) or refrigerated (4.degree. C.) in
sterile containers for 2 to 20 hours. Periodically, the placentas
were immersed and washed in sterile saline at 25.+-.3.degree. C. to
remove any visible surface blood or debris. The umbilical cord was
transected approximately 5 cm from its insertion into the placenta
and the umbilical vessels were cannulated with Teflon or
polypropylene catheters connected to a sterile fluid path allowing
bidirectional perfusion of the placenta and recovery of the
effluent fluid.
Placental Conditioning
[0315] Placentas were obtained from delivery rooms along with cord
blood after obtaining written parental consent, and were processed
at room temperature within 12 to 24 hours after delivery. Before
processing, the membranes were removed and the maternal site washed
clean of residual blood. The placenta was maintained under varying
conditions in an attempt to simulate and sustain a physiologically
compatible environment for the proliferation and recruitment of
residual cells. The umbilical vessels were cannulated with
catheters made from 20 gauge Butterfly needles use for blood sample
collection. The cannula was flushed with IMDM serum-free medium
(GibcoBRL, NY) containing 2 U/ml heparin (EJkins-Sinn, N.J.).
Placentas were then perfused with heparinized (2 U/mL) Dulbecco's
modified Eagle Medium (DMEM) at the rate of 15 mL/minute for 10
minutes and the perfusates were collected from the maternal sites
within one hour and the nucleated cells counted. Perfusion of the
placenta continued at a rate of 50 mL per minute until
approximately 150 mL of perfusate was collected. This volume of
perfusate was labeled "early fraction". The perfusion and
collection procedures were repeated once or twice until the number
of recovered nucleated cells fell below 100/microL. Continued
perfusion of the placenta at the same rate resulted in the
collection of a second fraction of approximately 150 mL and was
labeled "late fraction". During the course of the procedure, the
placenta was gently massaged to aid in the perfusion process and
assist in the recovery of cellular material. Effluent fluid was
collected from the perfusion circuit by both gravity drainage and
aspiration through the arterial cannula.
[0316] The perfusates were pooled and subjected to light
centrifugation to remove platelets, debris and denucleated cell
membranes. The nucleated cells were then isolated by Ficoll-Hypaque
density gradient centrifugation and after washing, resuspended in
DMEM. For isolation of the adherent cells, aliquots of
5-10.times.10.sup.6 cells were placed in each of several T-75
flasks and cultured with commercially available Mesenchymal Stem
Cell Growth Medium (MSCGM) obtained from BioWhittaker, and placed
in a tissue culture incubator (37.degree. C., 5% CO.sub.2). After
10 to 15 days, non-adherent cells were removed by washing with PBS,
which was then replaced by MSCGM. The flasks were examined daily
for the presence of various adherent cell types and in particular,
for identification and expansion of clusters of adherent
fibroblastoid cells.
Cell Recovery and Isolation
[0317] Cells were recovered from the perfusates by centrifugation
at 400.times.g for 15 minutes at room temperature. The cell pellets
were resuspended in IMDM serum-free medium containing 2 U/ml
heparin and 2 mM EDTA (GibcoBRL, NY). The total mononuclear cell
fraction was isolated using LYMPHOPREP.TM. (Nycomed Pharma, Oslo,
Norway) according to the manufacturer's recommended procedure and
the mononuclear cell fraction was resuspended. Cells were counted
using a hemocytometer. Viability was evaluated by trypan blue
exclusion. Isolation of mesenchymal cells was achieved by
differential trypsinization using a solution of 0.05% trypsin with
0.2% EDTA (Sigma). Differential trypsinization was possible because
fibroblastoid cells, including adherent placental stem cells,
detached from plastic surfaces within about five minutes whereas
other adherent populations required more than 20-30 minutes
incubation.
[0318] The detached fibroblastoid cells were harvested following
trypsinization and trypsin neutralization, using Trypsin
Neutralyzing Solution (TNS, BioWhitaker). The cells were washed in
DMEM and resuspended in MSCGM. Flow cytometry was carried out using
a Becton-Dickinson FACSCalibur instrument and FITC and PE labeled
monoclonal antibodies, selected on the basis of known markers for
bone marrow-derived MSC (mesenchymal stem cells), including
antibodies to CD10, CD34, CD44, CD45 and CD90. Antibodies were
purchased from Becton-Dickinson and Caltag laboratories (South San
Francisco, Calif.), and SH2, SH3 and SH4 antibody-producing
hybridomas were obtained from the American Type Culture Collection.
Reactivities of the MoAbs in their cultured supernatants were
detected by FITC or PE labeled F(ab)'.sub.2 goat anti-mouse
antibodies. Lineage differentiation was carried out using the
commercially available induction and maintenance culture media
(BioWhittaker), used as per manufacturer's instructions.
Isolation of Placental Stem Cells
[0319] Microscopic examination of the adherent cells in the culture
flasks revealed morphologically different cell types, including
spindle-shaped cells; round cells with large nuclei and numerous
perinuclear small vacuoles; and star-shaped cells with several
projections, through one of which the cells were attached to the
flask. Although no attempts were made to further characterize these
adherent cells, similar cells were observed in the culture of bone
marrow, cord and peripheral blood, and therefore considered to be
non-stem cell in nature.
[0320] Fibroblastoid adherent cells, appearing as clusters, were
similar in appearance to mesenchymal stem cells (MSC), and were
isolated by differential trypsinization and subcultured in
secondary flasks. The cells appeared rounded after trypsinization.
Phase microscopy of the rounded cells, after trypsinization, showed
the cells to be highly granulated and similar to bone
marrow-derived MSC produced in the laboratory or purchased from
commercial sources, e.g., BioWhittaker. When subcultured, the
adherent placental cells, in contrast to their earlier phase,
adhered within hours, assumed the characteristic fibroblastoid
shape, and formed a growth pattern similar to the reference bone
marrow-derived MSC. Moreover, during subculturing and refeeding,
loosely bound mononuclear cells were washed out and the cultures
remained homogeneous and devoid of any visible non-fibroblastoid
cell contaminants.
Flow Cytometry
[0321] The expression of CD34, CD38, SH2, SH3, SH4 and other stem
cell-associated surface markers on early and late fraction purified
mononuclear cells was assessed by flow cytometry. In a specific
case, cells were washed in PBS and then double-stained with
anti-CD34 phycoerythrin and anti-CD38 fluorescein isothiocyanate
(Becton Dickinson, Mountain View, Calif.).
[0322] In separate experiments, placental stem cells obtained from
separate perfusions, designated PLSC-1 through PLSC-29, were
assessed for the expression of CD10, CD29, CD34, CD44, CD45, CD54,
CD90, SH2 (CD105), SH3 (CD73), SH4 (CD73) and HLA-1 by flow
cytometry. Adherent cells designated PLSC-1 to PLSC-3, PLSC-5 to
PLSC-10, PLSC-15 to PLSC-21, PLSC-23, PLSC-26 and PLSC-27 were
found to be positive for CD10, CD29, CD54, SH2, SH3 and SH4, and
negative for CD34 and CD45. Adherent cells designated PLSC-15 to
PLSC-21, PLSC-23, PLSC-26 and PLSC-27 were additionally found to be
positive for CD44, CD90 and HLA1. See Table 1, below.
[0323] mRNA was collected from adherent cells from PLSC-3 and
PLCS-6 to PLSC-10 cells, and rtPCR was performed using primers
specific to OCT-4 (POU5F). All of the cell populations tested were
positive for OCT-4 mRNA.
TABLE-US-00001 TABLE 1 Characterization of placental stem cells
(PLSC) collected from separate perfusion experiments. Frozen ID #
Medium (Vials) CD34 CD45 CD10 CD29 CD54 SH2 SH3 SH4 SSEA4 CD44 HLA1
CD90 Oct4 PLSC-1 BW Y (2) - - + + + + + + PLSC-2 BW Y (6) - - + + +
+ + + PLSC-3 BW Y (2) - - + + + + + + + PLSC-4 BW None PLSC-5 BW Y
(9) - - + + + + + + PLSC-6 BW Y (26) - - +/low + + + + + + PLSC-7
BW Y (2) - - + + + + + + + PLSC-8 BW Y (10) - + + + + + + + PLSC-9
BW Y (11) - - + + + + + + + PLSC-10 BW Y (10) - - + + + + + + +
PLSC-11 D-5% FCS Y (9) PLSC-12 D-5% FCS Y (7) PLSC-13 D-5% FCS Y
(5) PLSC-14 D-5% FCS Y (9) PLSC-15 Anthro-1 Y (7) - - + + + + + + +
+ + PLSC-16 Anthro-1 Y (8) - - + + + + + + + + + PLSC-17 Anthro-1 Y
(8) - - + + + + + + + + + PLSC-18 Anthro-1 Y (8) - - + + + + + + +
+ + PLSC-19 BWtoA Y (17) - - + + + + + + + + + PLSC-20 BWtoA Y (40)
- - + + + + + + + + + PLSC-21 BWtoA Y (9) - - + + + + + + +/- + + +
PLSC-22 BWtoA FTE PLSC-23 Anthro-1 Y (10) - - + + + + + + + + +
PLSC-24 Anthro-1 FTE PLSC-25 Anthro-1 FTE PLSC-26 Anthro-1 Y (15) -
- + + + + + + + + + PLSC-27 Anthro-1 Y (25) - - + + + + + + + + +
+: Detected by flow cytometry, or, for OCT-4, gene expression
detected by RT-PCR. -: Not detected. Blank: Presence of marker was
not tested. FTE: Failed to expand. BW--BioWhittaker complete medium
(RPM1 1640 + 10% FBS). D-5% FCS: DMEM-5% FCS. BWtoA: BW to Anthro-1
medium
Differentiation
[0324] In separate experiments, the adherent fibroblastoid cells
were demonstrated to be stem cells. The cells were differentiated
in vitro into cells of adipocytic lineage, as evidenced by the
formation of oil droplets detectable by the stain Oil Red. The
cells were also differentiated in vitro into cells of a neurogenic
lineage as evidenced by the development of dendrite-like spindles
characteristic of neural cells, and the appearance of glial acid
fibrillary protein and neurofilament proteins, both markers of
neural cells. The cells were also differentiated in vitro into
cells of a chondrogenic lineage, as evidenced by the appearance of
glycosaminoglycans, produced by cartilage-producing cells, that
were detectable by Periodic Acid Schiff reagent. In a separate
experiment, it was determined that placental stem cells did not
differentiate in a NOD-SCID mouse model.
6.3 Example 3: Isolation of Placental Stem Cells from Placental
Structures
[0325] 6.3.1 Materials & Methods
[0326] 6.3.1.1 Isolation of Populations of Placental Cells
Comprising Placental Stem Cells
[0327] Distinct populations of placental cells were obtained from
the placentas of normal, full-term pregnancies. All donors provided
full written consent for the use of their placentas for research
purposes. Placental stem cells were obtained from the following
sources: (1) placental perfusate (from perfusion of the placental
vasculature); and enzymatic digestions of (2) amnion, (3) chorion,
(4) amnion-chorion plate, and (5) umbilical cord. The various
placental tissues were cleaned in sterile PBS (Gibco-Invitrogen
Corporation, Carlsbad, Calif.) and placed on separate sterile Petri
dishes. The various tissues were minced using a sterile surgical
scalpel and placed into 50 mL Falcon Conical tubes. The minced
tissues were digested with IX Collagenase (Sigma-Aldrich, St.
Louis, Mo.) for 20 minutes in a 37.degree. C. water bath,
centrifuged, and then digested with 0.25% Trypsin-EDTA
(Gibco-Invitrogen Corp) for 10 minutes in a 37.degree. C. water
bath. The various tissues were centrifuged after digestion and
rinsed once with sterile PBS (Gibco-Invitrogen Corp). The
reconstituted cells were then filtered twice, once with 100 .mu.m
cell strainers and once with 30 .mu.m separation filters, to remove
any residual extracellular matrix or cellular debris.
[0328] 6.3.1.2 Cellular Viability Assessment and Cell Counts
[0329] The manual trypan blue exclusion method was employed post
digestion to calculate cell counts and assess cellular viability.
Cells were mixed with Trypan Blue Dye (Sigma-Aldrich) at a ratio of
1:1, and the cells were read on hemacytometer.
[0330] 6.3.1.3 Cell Surface Marker Characterization
[0331] Cells that were HLA
ABC.sup.-/CD45.sup.-/CD34.sup.-/CD133.sup.+ were selected for
characterization. Cells having this phenotype were identified,
quantified, and characterized by two of Becton-Dickinson flow
cytometers, the FACSCalibur and the FACS Aria (Becton-Dickinson,
San Jose, Calif., USA). The various placental cells were stained,
at a ratio of about 10 .mu.L of antibody per 1 million cells, for
30 minutes at room temperature on a shaker. The following
anti-human antibodies were used: Fluorescein Isothiocyanate (FITC)
conjugated monoclonal antibodies against HLA-G (Serotec, Raleigh,
N.C.), CD10 (BD Immunocytometry Systems, San Jose, Calif.), CD44
(BD Biosciences Pharmingen, San Jose, Calif.), and CD105 (R&D
Systems Inc., Minneapolis, Minn.); Phycoerythrin (PE) conjugated
monoclonal antibodies against CD44, CD200, CD117, and CD13 (BD
Biosciences Pharmingen); Phycoerythrin-Cy5 (PE Cy5) conjugated
Streptavidin and monoclonal antibodies against CD117 (BD
Biosciences Pharmingen); Phycoerythrin-Cy7 (PE Cy7) conjugated
monoclonal antibodies against CD33 and CD10 (BD Biosciences);
Allophycocyanin (APC) conjugated streptavidin and monoclonal
antibodies against CD38 (BD Biosciences Pharmingen); and
Biotinylated CD90 (BD Biosciences Pharmingen). After incubation,
the cells were rinsed once to remove unbound antibodies and were
fixed overnight with 4% paraformaldehyde (USB, Cleveland, Ohio) at
4.degree. C. The following day, the cells were rinsed twice,
filtered through a 30 .mu.m separation filter, and were run on the
flow cytometer(s).
[0332] Samples that were stained with anti-mouse IgG antibodies (BD
Biosciences Pharmingen) were used as negative controls and were
used to adjust the Photo Multiplier Tubes (PMTs). Samples that were
single stained with anti-human antibodies were used as positive
controls and were used to adjust spectral
overlaps/compensations.
[0333] 6.3.1.4 Cell Sorting and Culture
[0334] One set of placental cells (from perfusate, amnion, or
chorion), prior to any culture, was stained with
7-Amino-Actinomycin D (7AAD; BD Biosciences Pharmingen) and
monoclonal antibodies specific for the phenotype of interest. The
cells were stained at a ratio of 10 .mu.L of antibody per 1 million
cells, and were incubated for 30 minutes at room temperature on a
shaker. These cells were then positively sorted for live cells
expressing the phenotype of interest on the BD FACS Aria and plated
into culture. Sorted (population of interest) and "All"
(non-sorted) placental cell populations were plated for
comparisons. The cells were plated onto a fibronectin
(Sigma-Aldrich) coated 96 well plate at the cell densities listed
in Table 2 (cells/cm.sup.2). The cell density, and whether the cell
type was plated in duplicate or triplicate, was determined and
governed by the number of cells expressing the phenotype of
interest.
TABLE-US-00002 TABLE 2 Cell plating densities 96 Well Plate Culture
Density of Plated Cells Conditions Cell Source Sorted All All Max.
Density Perfusate Set #1: 40.6 K/cm.sup.2 40.6 K/cm.sup.2 93.8
K/cm.sup.2 Set #2 40.6 K/cm.sup.2 40.6 K/cm.sup.2 93.8 K/cm.sup.2
Set #3: 40.6 K/cm.sup.2 40.6 K/cm.sup.2 93.8 K/cm.sup.2 Amnion Set
#1: 6.3 K/cm.sup.2 6.3 K/cm.sup.2 62.5 K/cm.sup.2 Set #2 6.3
K/cm.sup.2 6.3 K/cm.sup.2 62.5 K/cm.sup.2 Chorion Set #1: 6.3
K/cm.sup.2 6.3 K/cm.sup.2 62.5 K/cm.sup.2 Set #2 6.3 K/cm.sup.2 6.3
K/cm.sup.2 62.5 K/cm.sup.2
[0335] Complete medium (60% DMEM-LG (Gibco) and 40% MCDB-201
(Sigma); 2% fetal calf serum (Hyclone Labs.); lx
insulin-transferrin-selenium (ITS); lx linoleic acid-bovine serum
albumin (LA-BSA); 10.sup.-9 M dexamethasone (Sigma); 10.sup.-4 M
ascorbic acid 2-phosphate (Sigma); epidermal growth factor 10 ng/mL
(R&D Systems); and platelet-derived growth factor (PDGF-BB) 10
ng/mL (R&D Systems)) was added to each well of the 96 well
plate and the plate was placed in a 5% CO.sub.2/37.degree. C.
incubator. On day 7, 100 .mu.L of complete medium was added to each
of the wells. The 96 well plate was monitored for about two weeks
and a final assessment of the culture was completed on day 12. This
is very early in the placental stem cell culture, and represents
passage 0 cells.
[0336] 6.3.1.5 Data Analysis
[0337] FACSCalibur data was analyzed in FlowJo (Tree star, Inc)
using standard gating techniques. The BD FACS Aria data was
analyzed using the FACSDiva software (Becton-Dickinson). The FACS
Aria data was analyzed using doublet discrimination gating to
minimize doublets, as well as, standard gating techniques. All
results were compiled in Microsoft Excel and all values, herein,
are represented as average.+-.standard deviation (number, standard
error of mean).
[0338] 6.3.2 Results
[0339] 6.3.2.1 Cellular Viability
[0340] Post-digestion viability was assessed using the manual
trypan blue exclusion method (FIG. 1). The average viability of
cells obtained from the majority of the digested tissue (from
amnion, chorion or amnion-chorion plate) was around 70%. Amnion had
an average viability of 74.35%.+-.10.31% (n=6, SEM=4.21), chorion
had an average viability of 78.18%.+-.12.65% (n=4, SEM=6.32),
amnion-chorion plate had an average viability of 69.05%.+-.10.80%
(n=4, SEM=5.40), and umbilical cord had an average viability of
63.30%.+-.20.13% (n=4, SEM=10.06). Cells from perfusion, which did
not undergo digestion, retained the highest average viability,
89.98.+-.6.39% (n=5, SEM=2.86).
[0341] 6.3.2.2 Cell Quantification
[0342] The populations of placental cells and umbilical cord cells
were analyzed to determine the numbers of HLA
ABC.sup.-/CD45.sup.-/CD34.sup.-/CD133.sup.+ cells. From the
analysis of the BD FACSCalibur data, it was observed that the
amnion, perfusate, and chorion contained the greatest total number
of these cells, 30.72.+-.21.80 cells (n=4, SEM=10.90),
26.92.+-.22.56 cells (n=3, SEM=13.02), and 18.39.+-.6.44 cells
(n=2, SEM=4.55) respectively (data not shown). The amnion-chorion
plate and umbilical cord contained the least total number of cells
expressing the phenotype of interest, 4.72.+-.4.16 cells (n=3,
SEM=2.40) and 3.94.+-.2.58 cells (n=3, SEM=1.49) respectively (data
not shown).
[0343] Similarly, when the percent of total cells expressing the
phenotype of interest was analyzed, it was observed that amnion and
placental perfusate contained the highest percentages of cells
expressing this phenotype (0.0319%.+-.0.0202% (n=4, SEM=0.0101) and
0.0269%.+-.0.0226% (n=3, SEM=0.0130) respectively (FIG. 2).
Although umbilical cord contained a small number of cells
expressing the phenotype of interest (FIG. 2), it contained the
third highest percentage of cells expressing the phenotype of
interest, 0.020.+-.0.0226% (n=3, SEM=0.0131) (FIG. 2). The chorion
and amnion-chorion plate contained the lowest percentages of cells
expressing the phenotype of interest, 0.0184.+-.0.0064% (n=2,
SEM-0.0046) and 0.0177.+-.0.0173% (n=3, SEM=0.010) respectively
(FIG. 2).
[0344] Consistent with the results of the BD FACSCalibur analysis,
the BD FACS Aria data also identified amnion, perfusate, and
chorion as providing higher numbers of HLA
ABC.sup.-/CD45.sup.-/CD34.sup.-/CD133.sup.+ cells than the
remaining sources. The average total number of cells expressing the
phenotype of interest among amnion, perfusate, and chorion was
126.47.+-.55.61 cells (n=15, SEM=14.36), 81.65.+-.34.64 cells
(n=20, SEM=7.75), and 51.47.+-.32.41 cells (n=15, SEM=8.37),
respectively (data not shown). The amnion-chorion plate and
umbilical cord contained the least total number of cells expressing
the phenotype of interest, 44.89.+-.37.43 cells (n=9, SEM=12.48)
and 11.00.+-.4.03 cells (n=9, SEM=1.34) respectively (data not
shown).
[0345] BD FACS Aria data revealed that the perfusate and amnion
produced the highest percentages of HLA
ABC.sup.-/CD45/CD34.sup.-/CD133.sup.+ cells, 0.1523.+-.0.0227%
(n=15, SEM=0.0059) and 0.0929.+-.0.0419% (n=20, SEM=0.0094)
respectively (FIG. 3). The amnion-chorion plate contained the third
highest percentage of cells expressing the phenotype of interest,
0.0632.+-.0.0333% (n=9, SEM=0.0111) (FIG. 3). The chorion and
umbilical cord contained the lowest percentages of cells expressing
the phenotype of interest, 0.0623.+-.0.0249% (n=15, SEM=0.0064) and
0.0457.+-.0.0055% (n=9, SEM=0.0018) respectively (FIG. 3).
[0346] After HLA ABC.sup.-/CD45.sup.-/CD34.sup.-/CD133.sup.+ cells
were identified and quantified from each cell source, its cells
were further analyzed and characterized for their expression of
cell surface markers HLA-G, CD10, CD13, CD33, CD38, CD44, CD90,
CD105, CD117, CD200, and CD105.
[0347] 6.3.2.3 Placental Perfusate-Derived Cells
[0348] Perfusate-derived cells appeared generally positive for
HLA-G, CD33, CD117, CD10, CD44, CD200, CD90, CD38, CD105, and CD13
(FIG. 4). The average expression of each marker for
perfusate-derived cells was the following: 37.15%.+-.38.55% (n=4,
SEM=19.28) of the cells expressed HLA-G; 36.37%.+-.21.98% (n=7,
SEM=8.31) of the cells expressed CD33; 39.39%.+-.39.91% (n=4,
SEM=19.96) of the cells expressed CD117; 54.97%.+-.33.08% (n=4,
SEM=16.54) of the cells expressed CD10; 36.79%.+-.11.42% (n=4,
SEM-5.71) of the cells expressed CD44; 41.83%.+-.19.42% (n=3,
SEM=11.21) of the cells expressed CD200; 74.25%.+-.26.74% (n=3,
SEM=15.44) of the cells expressed CD90; 35.10%.+-.23.10% (n=3,
SEM=13.34) of the cells expressed CD38; 22.87%.+-.6.87% (n=3,
SEM=3.97) of the cells expressed CD105; and 25.49%.+-.9.84% (n=3,
SEM=5.68) of the cells expressed CD13.
[0349] 6.3.2.4 Amnion-Derived Cells
[0350] Amnion-derived cells were consistently positive for HLA-G,
CD33, CD117, CD10, CD44, CD200, CD90, CD38, CD105, and CD13 (FIG.
5). The average expression of each marker for amnion-derived was
the following: 57.27%.+-.41.11% (n=3, SEM=23.73) of the cells
expressed HLA-G; 16.23%.+-.15.81% (n=6, SEM=6.46) of the cells
expressed CD33; 62.32%.+-.37.89% (n=3, SEM=21.87) of the cells
expressed CD117; 9.71%.+-.13.73% (n=3, SEM=7.92) of the cells
expressed CD10; 27.03%.+-.22.65% (n=3, SEM=13.08) of the cells
expressed CD44; 6.42%.+-.0.88% (n=2, SEM=0.62) of the cells
expressed CD200; 57.61%.+-.22.10% (n=2, SEM=15.63) of the cells
expressed CD90; 63.76%.+-.4.40% (n=2, SEM=3.11) of the cells
expressed CD38; 20.27%.+-.5.88% (n=2, SEM=4.16) of the cells
expressed CD105; and 54.37%.+-.13.29% (n=2, SEM=9.40) of the cells
expressed CD13.
[0351] 6.3.2.5 Chorion-Derived Cells
[0352] Chorion-derived cells were consistently positive for HLA-G,
CD117, CD10, CD44, CD200, CD90, CD38, and CD13, while the
expression of CD33, and CD105 varied (FIG. 6). The average
expression of each marker for chorion cells was the following:
53.25%.+-.32.87% (n=3, SEM=18.98) of the cells expressed HLA-G;
15.44%.+-.11.17% (n=6, SEM=4.56) of the cells expressed CD33;
70.76%.+-.11.87% (n=3, SEM=6.86) of the cells expressed CD117;
35.84%.+-.25.96% (n=3, SEM=14.99) of the cells expressed CD10;
28.76%.+-.6.09% (n=3, SEM=3.52) of the cells expressed CD44;
29.20%.+-.9.47% (n=2, SEM=6.70) of the cells expressed CD200;
54.88%.+-.0.17% (n=2, SEM=0.12) of the cells expressed CD90;
68.63%.+-.44.37% (n=2, SEM=31.37) of the cells expressed CD38;
23.81%.+-.33.67% (n=2, SEM=23.81) of the cells expressed CD105; and
53.16%.+-.62.70% (n=2, SEM=44.34) of the cells expressed CD13.
[0353] 6.3.2.6 Amnion-Chorion Plate-Derived Cells
[0354] Cells from amnion-chorion plate were consistently positive
for IILA-G, CD33, CD117, CD10, CD44, CD200, CD90, CD38, CD105, and
CD13 (FIG. 7). The average expression of each marker for
amnion-chorion plate-derived cells was the following:
78.52%.+-.13.13% (n=2, SEM=9.29) of the cells expressed HLA-G;
38.33%.+-.15.74% (n=5, SEM=7.04) of the cells expressed CD33;
69.56%.+-.26.41% (n=2, SEM=18.67) of the cells expressed CD117;
42.44%.+-.53.12% (n=2, SEM=37.56) of the cells expressed CD10;
32.47%.+-.31.78% (n=2, SEM=22.47) of the cells expressed CD44;
5.56% (n=1) of the cells expressed CD200; 83.33% (n=1) of the cells
expressed CD90; 83.52% (n=1) of the cells expressed CD38; 7.25%
(n=1) of the cells expressed CD105; and 81.16% (n=1) of the cells
expressed CD13.
[0355] 6.3.2.7 Umbilical Cord-Derived Cells
[0356] Umbilical cord-derived cells were consistently positive for
HLA-G, CD33, CD90, CD38, CD105, and CD13, while the expression of
CD117, CD10, CD44, and CD200 varied (FIG. 8). The average
expression of each marker for umbilical cord-derived cells was the
following: 62.50%.+-.53.03% (n=2, SEM=37.50) of the cells expressed
HLA-G; 25.67%.+-.11.28% (n=5, SEM=5.04) of the cells expressed
CD33; 44.45%.+-.62.85% (n=2, SEM=44.45) of the cells expressed
CD117; 8.33%.+-.11.79% (n=2, SEM=8.33) of the cells expressed CD10;
21.43%.+-.30.30% (n=2, SEM=21.43) of the cells expressed CD44; 0.0%
(n=1) of the cells expressed CD200; 81.25% (n=1) of the cells
expressed CD90; 64.29% (n=1) of the cells expressed CD38; 6.25%
(n=1) of the cells expressed CD105; and 50.0% (n=1) of the cells
expressed CD13.
[0357] A summary of all marker expression averages is shown in FIG.
9.
[0358] 6.3.2.8 BD FACS Aria Sort Report
[0359] The three distinct populations of placental cells that
expressed the greatest percentages of HLA ABC, CD45, CD34, and
CD133 (cells derived from perfusate, amnion and chorion) were
stained with 7AAD and the antibodies for these markers. The three
populations were positively sorted for live cells expressing the
phenotype of interest. The results of the BD FACS Aria sort are
listed in Table 3.
TABLE-US-00003 TABLE 3 BD FACS Aria Sort Report Events Sorted Cell
Events (Phenotype % Of Source Processed of Interest) Total
Perfusate 135540110 51215 0.037786 Amnion 7385933 4019 0.054414
Chorion 108498122 4016 0.003701
[0360] The three distinct populations of positively sorted cells
("sorted") and their corresponding non-sorted cells were plated and
the results of the culture were assessed on day 12 (Table 3).
Sorted perfusate-derived cells, plated at a cell density of
40,600/cm.sup.2, resulted in small, round, non-adherent cells. Two
out of the three sets of non-sorted perfusate-derived cells, each
plated at a cell density of 40,600/cm.sup.2, resulted in mostly
small, round, non-adherent cells with several adherent cells
located around the periphery of well. Non-sorted perfusate-derived
cells, plated at a cell density of 93,800/cm.sup.2, resulted in
mostly small, round, non-adherent cells with several adherent cells
located around the well peripheries.
[0361] Sorted amnion-derived cells, plated at a cell density of
6,300/cm.sup.2, resulted in small, round, non-adherent cells.
Non-sorted amnion-derived cells, plated at a cell density of
6,300/cm.sup.2, resulted in small, round, non-adherent cells.
Non-sorted amnion-derived cells plated at a cell density of
62,500/cm.sup.2 resulted in small, round, non-adherent cells.
[0362] Sorted chorion-derived cells, plated at a cell density of
6,300/cm.sup.2, resulted in small, round, non-adherent cells.
Non-sorted chorion-derived cells, plated at a cell density of
6,300/cm.sup.2, resulted in small, round, non-adherent cells.
Non-sorted chorion-derived cells plated at a cell density of
62,500/cm.sup.2, resulted in small, round, non-adherent cells.
[0363] A majority of the above non-adherent cells, when cultured,
adhered to the tissue culture surface and assumed a fibroblastoid
shape.
[0364] Subsequent to the performance of the experiments related
above, and further culture of the placental stem cells, it was
determined that the labeling of the antibodies for CD117 and CD133,
in which a streptavidin-conjugated antibody was labeled with
biotin-conjugated phycoerythrin (PE), produced background
significant enough to resemble a positive reading. This background
had initially resulted in the placental stem cells being deemed to
be positive for both markers. When a different label, APC or PerCP
was used, the background was reduced, and the placental stem cells
were correctly determined to be negative for both CD117 and
CD133.
6.4 Example 4: Characterization of Placental Stem Cell Marker
Expression
[0365] This example describes experiments designed to further
characterize expression of a variety of protein expression markers
by placental stem cells as follows: CD200.sup.+, CD105.sup.+,
CD90.sup.+, CD10.sup.+, cytokeratin 18.sup.+, CD34.sup.-, and
CD45.sup.-. In addition, the baseline expression of hepatocyte
markers on uninduced placental stem cells is assessed, including,
for example, expression of cytokeratin 18, secretion of hepatocyte
growth factor (HGF) and expression of asialoglycoprotein
receptor.
[0366] Placental stem cell marker expression is assessed according
to the following exemplary flow cytometry protocol. Placental stem
cells are first trypsinized, then incubated with
fluorochrome-conjugated antibodies on ice for 30 minutes in the
dark, rinsed twice with cold PBS-2% BSA and then analyzed with a
FACSCalibur (BD Biosciences). Exemplary antibodies suitable for
these assays are PerCP-conjugated anti-CD34, FITC conjugated
anti-CD105, APC-conjugated anti-CD10, and PE conjugated CD200.
Other suitable antibody reagents include, the appropriate
fluorophore combinations: APC-conjugated anti-HLA-ABC,
PE-conjugated anti-CD31, PerCP-conjugated anti-CD45,
FITC-conjugated anti-CD38, PE-conjugated anti-CD44, FITC-conjugated
anti-cytokeratin K (or cytokeratin 18), APC-conjugated anti-CD90,
APC-conjugated anti-CD86, FITC-conjugated anti-CD80,
FITC-conjugated anti-HLA DR,DQ,DP, PE-conjugated
anti-3-2-microglobulin, APC-conjugated anti-CD133 and
PerCP-conjugated anti-CD117. Except where otherwise noted, these
antibodies are available from BD-Pharmingen. Analysis and
statistics of flow cytometry data is accomplished with Flowjo (Tree
Star, Inc. Ashland, Oreg.).
[0367] Cytokines produced by placental stem cells are analyzed by
collecting supernatants of cultured cells using a LincoPlex
Adipogenic analysis kit and a Luminex instrument according to the
manufacturer's instructions.
6.5 Example 5: Induction of Placental Cell Differentiation into
Hepatocytes Using Sodium Butyrate
[0368] This example describes exemplary methods for induction of
hepatocyte differentiation of placental stem cells using sodium
butyrate. The methods are designed to characterize Na-butyrate
induced placental stem cells using a variety of protein expression
markers, expression of hepatocyte specific genes and hepatocyte
specific markers such as cytokeratin 18 (a molecule already
expressed in undifferentiated placental stem cells) and
asialoglycoprotein receptor. The ability of induced placental stem
cells to produce intracellular albumin and secretion of urea is
assessed.
[0369] To induce differentiation, placental stem cells are plated
at a density of about 10.sup.5 cells/well in 0.1% gelatin coated
six well plates in Iscove's Modified Dulbecco's medium (IMDM)
(Gibco, Cat #31980-030) containing 20% fetal bovine serum (Gibco),
4 mM L-glutamine (Gibco), 100 U/ml penicillin, 100 U/ml
streptomycin (Gibco) and 10 .mu.g/ml gentamicin (Gibco). Gelatin
solution (0.1%) is prepared by dissolving 0.5 .mu.m of porcine
gelatin (Sigma-Aldrich, Cat # G-2500) in 500 ml of Phosphate
Buffered Saline (PBS) (Gibco, Cat #20012-027) with gentle heating.
To coat plates with gelatin, 2 ml of 0.1% gelatin solution is added
to each well of a polystyrene tissue culture treated plate. The
plates are incubated for 2 hrs following which the gelatin solution
is aspirated. The plates are washed once with PBS and then 2.5 ml
of IMDM is added followed by about 10.sup.5 cells/well. Optionally,
cells are exposed to 1% DMSO (Sigma-Aldrich) for the next 4 days
followed by exposure to Na-butyrate (Sigma Aldrich, Cat # B5887) at
different concentrations (1, 2.5, 5.10 mM) for 6 days. Media is
replaced daily.
[0370] In order to measure function of the differentiated cell, a
secondary culture is initiated where cells are removed from the
primary culture dish and replated on collagen type I coated (BD
Biosciences) and polystyrene 12 well plates at a density of about
10.sup.5 cells/well. Media is changed 24 hrs after replating and
cells are tested for functional assays 48 hrs after replating.
6.6 Example 6: Aggregation of Placental Stem Cells by
Alginate-Poly-L-Lysine Microencapsulation in Preparation for
Differentiation into Hepatocytes
[0371] This example describes exemplary methods for aggregating
placental stem cells using alginate microencapsulation technology
in preparation for differentiating the cells into hepatocytes.
Alginate Poly-L-Lysine Encapsulation
[0372] An alginate solution is generated by dissolving 2.2 g of
alginate (Sigma-Aldrich, MW: 100,000-200,000 g/mol, G-Content:
65%-70%) in 100 mL of Ca.sup.2+ free DMEM (Gibco), using a heated
magnetic stir plate at a temperature of 45.degree. C. The solution
is then filtered using a 25-micron syringe filter (Fisher Brand,
Pittsburgh, Pa.). A confluent monolayer of adherent cells is
removed following trypsin incubation, centrifuged for 10 minutes at
1200 rpm, and resuspended in PBS. The cells are washed twice more
with PBS (Gibco), resuspended in 2 mL of their respective media and
both cell number and viability assessed using the method of trypan
blue (Gibco) exclusion. To create the cell-alginate mixture a 1 mL
aliquot of cell suspension with a seeding density of about
5.times.10.sup.7 cells/ml is added to 9 mL of a 2.2% (w/v) alginate
solution to yield a final cell seeding density of about
5.times.10.sup.6 cells/ml and a final alginate concentration of
2.0% (w/v). This solution is transferred to a 10 ml syringe (BD
Biosciences), which, in turn, is connected to a syringe pump (KD
Scientific, Holliston, Mass.). Alginate beads are generated using
an electrostatic bead generator (Nisco, ZUrich, Switzerland) at a
flow rate of 40 ml/hour, and an applied voltage of 6.5 kV,
resulting in beads with a diameter of 500 .mu.m. The beads are
extruded into a 200 mL bath of CaCl.sub.2 (100 mM) (Sigma-Aldrich),
containing 145 mM NaCl (Sigma-Aldrich), and 10 mM MOPS
(Sigma-Aldrich) and are left to polymerize for 10 minutes at room
temperature. Beads are transferred to a tissue culture treated T-25
flask (Falcon, BD Biosciences), following the polymerization step.
The CaCl.sub.2 solution is removed using a 5 mL pipette, and the
beads are washed with 5 mL HEPES (Gibco). The buffer is removed and
the beads are resuspended in 5 ml of poly-L-lysine (PLL)
(Sigma-Aldrich, MW: 68,600 g/mol) (0.05% w/v) for 2 minutes. The
PLL is then gently removed, replaced with HEPES to wash the beads
and the beads are ultimately resuspended into 5 ml of cell culture
media. Media is changed at, e.g., 4, 8, 11, 14 and 17 days
post-encapsulation.
Assessment of Intracapsular Viability
[0373] Viability within beads is assessed with a calcein (Molecular
Probes, Eugene, Oreg.), ethidium homodimer (Molecular Probes) stain
immediately following encapsulation. Calcein is only cleaved to
form fluorescent products in live cells while ethidium homodimer is
only incorporated into the nucleus of dead cells. Calcein and
ethidium homodimer images are acquired using a Zeiss Axiovert LSM
laser scanning confocal microscope (Germany) fitted with a 495 nm
excitation filter and emission filters of 515 nm and 635 nm,
respectively. Specifically, z-sections of 500 um diameter beads are
taken at 10 um intervals, for a total depth of 250 um. Three
experiments incorporated an analysis of 15 beads per experiment.
Digitized images are quantified using Olympus MICROSUITE.TM..
Viability is assessed for each cross-section of every bead.
Cell Recovery and Assessment Following Depolymerization
[0374] Functional analysis and aggregate size calculations are
performed on each of the analysis days following the release of
cells from the beads. A minimum of 1500 beads is analyzed per
replicate per condition. Beads are washed with PBS, and 100 mM
sodium citrate (Fisher Scientific), containing 10 mM MOPS
(Sigma-Aldrich) and 27 mM NaCl (Sigma-Aldrich) are added for 30
minutes at 37.degree. C. to induce depolymerization. To determine
recovery yield following depolymerization, a known concentration of
cells is encapsulated and immediately depolymerized. Following
centrifugation, both the cell pellet as well as the supernatant
(which contains bead particles but no intact beads) are counted
using trypan blue exclusion (which does not stain the capsule), and
after a mass balance, verify that approximately the same number of
cells are present as in the starting population. This method
demonstrates a 98% recovery of the encapsulated cell population.
The released cells are centrifuged at 1200 rpm for 10 minutes, the
sodium citrate solution aspirated, the cell pellet washed with PBS
(3.times.), and resuspended in cell specific media. The cells are
then counted using the trypan blue method described above.
Intracapsular Aggregate Size Determination
[0375] 103271 Beads are sampled from tissue culture treated T-25
flasks and transferred to 35 mm Mattek dishes (Mattek, Ashland,
Mass.) immediately following encapsulation (day 0), and on the
analysis days 8, 11, 14, 17 20. Bright field images are acquired
using a Zeiss Axiovert LSM laser scanning confocal microscope
(Germany). Specifically, z-sections of 500 um diameter beads are
taken at 50 um intervals, to avoid multiple quantification of the
same aggregate, for a total depth of 250 um. Images are quantified
using Olympus Microsuite. In short, a color threshold is first
applied in order to distinguish cellular aggregates from the image
background. The diameter of the aggregate is then determined using
the mean diameter particle measurement.
6.7 Example 7: In Situ Indirect Immunofluorescent Cytokeratin-18
and Intracellular Albumin Analysis
[0376] This example describes exemplary methods for assessing
cytokeratin-18 and albumin expression by hepatocytes obtained from
differentiated placental stem cells. Differentiated cells
(recovered following depolymerization if appropriate) are
transferred to a tissue culture treated 24 well plate (Falcon, BD
Biosciences). Specifically, the isolated cell population is diluted
to about 6.times.10.sup.4 cells in 0.75 ml of media and incubated
for one hour at 37.degree. C. to allow for cell attachment. The
cells are then washed for 10 min in cold PBS and fixed in 4%
paraformaldehyde (Sigma-Aldrich) in PBS for 15 minutes at room
temperature. The cells are washed twice for 10 min in cold PBS and
then twice for 10 min in cold saponine/PBS (SAP) membrane
permeabilization buffer containing 1% bovine serum albumin (BSA)
(Sigma-Aldrich), 0.5% saponine (Sigma-Aldrich) and 0.1% sodium
azide (Sigma-Aldrich). To detect intracellular albumin, the cells
are subsequently incubated for 30 minutes at 4.degree. C. in a SAP
solution containing rabbit anti-mouse albumin antibody (150
.mu.g/ml) (MP Biomedicals, Irvine, Calif.), or normal rabbit serum
(150 .mu.g/ml) (MP Biomedicals) as an isotype control, washed twice
for 10 min in cold SAP buffer, and then treated for 30 minutes at
4.degree. C. with the secondary antibody, FITC-conjugated donkey
anti-rabbit, diluted 1:500 (Jackson Immuno Labs, Westgrove, Pa.).
To detect cytokeratin 18, cells are incubated for 30 minutes at
4.degree. C. in a SAP solution containing rabbit anti-mouse
cytokeratin 18 antibody (IgG1) (1:50 dilution) (Santa Cruz
Biotechnology) or the IgG1 fraction of normal rabbit serum (1:100
dilution) (Santa Cruz Biotechnology) as an isotype control, and
then treated for 30 minutes at 4.degree. C. with the secondary
antibody, FITC-conjugated goat anti-rabbit, diluted 1:200 (Jackson
Immuno Labs, Westgrove, Pa.). For both stains, cells are then
washed once with cold SAP buffer and once with cold PBS.
Fluorescent images are acquired using a computer-interfaced
inverted Olympus IX70 microscope. Specimens are excited using a 515
nm filter. Fluorescent intensity values are determined for each
cell using Olympus MICROSUITE.TM.. Experimental intensity values
for each cell are calculated after subtracting the average
intensity of the isotype control.
6.8 Example 8: Glycogen Staining
[0377] This example describes exemplary methods for assessing
glycogen production by hepatocytes obtained from differentiated
placental stem cells. Following depolymerization, cells are
transferred to tissue culture treated 24 well plates (Falcon, BD
Biosciences) and fixed with 10% formalin-ethanol fixative solution
for 15 minutes at room temperature, with subsequent washes with
PBS. Fixed cells are exposed to 0.25 ml of Periodic Acid Solution
(Sigma Aldrich) per well for 5 minutes at room temperature. Glycols
are oxidized to aldehydes in this process. After washing cells with
PBS to remove the PAS, 1 ml of Schiff's reagent is added per well
and cells exposed for 15 minutes at room temperature. Schiff's
reagent, a mixture of pararosaniline and sodium metabisulfite,
reacts to release a pararosaniline product that stains the
glycol-containing cellular elements. A third PBS wash to remove the
reagent is followed by image acquisition with an Olympus IX70
microscope and Olympus digital camera.
6.9 Example 9: Glucose and Lactate Measurements
[0378] This example describes exemplary methods for assessing
glucose and lactate consumption and/or production by hepatocytes
obtained from differentiated placental stem cells. Supernatants (1
ml) were collected in triplicate for each cell type in secondary
culture and then tested using a Bioprofile Bioanalyzer 400 (Nova
Biomedical, Waltham, Mass.) for metabolite measurements of glucose
and lactate. On each day of analysis, base media glucose and
lactate measurements were measured and the mean values were
subtracted from the test values to obtain uptake or production.
Cells were counted for each condition to get the final consumption
or production rate.
6.10 Example 10: Urea Analysis
[0379] This example describes exemplary methods for assessing urea
production by hepatocytes obtained from differentiated placental
stem cells. Media samples are collected directly from cell cultures
and stored at -20.degree. C. for subsequent urea content analysis.
Urea synthesis is assayed using a commercially available kit
(StanBio, Boerne, Tex.). A standard curve is generated by creating
serial dilutions of a urea standard from 300 .mu.g/ml to 0
.mu.g/ml. Absorbance readings are obtained using a Biorad
(Hercules, Calif.) Model 680 plate reader with a 585n emission
filter. Urea values are normalized to the cell number recorded on
the day of media sample collection.
6.11 Example 11: Sandwich ELISA for Detection of Albumin
Secretion
[0380] This example describes exemplary methods for assessing
albumin secretion by hepatocytes obtained from differentiated
placental stem cells. In order to detect secreted albumin within
the media supernatants obtained on each of the analysis days, a
commercially available mouse albumin ELISA kit (Bethyl
Laboratories, # E90-134) is used. A standard curve is generated by
creating serial dilutions of an albumin standard from 7.8 to 10,000
ng/mL. Absorbance readings are obtained using a Biorad (Hercules,
Calif.) Model 680 plate reader with a 450 nm emission filter.
Albumin values are normalized to the cell number recorded on the
day of media sample collection.
6.12 Example 12: Mouse Model of Hepatitis B Infection
[0381] This Example describes a mouse model of hepatitis B virus
(HBV) infection that uses hepatocytes differentiated from the
placental stem cells described elsewhere herein. The mouse is
produced by (1) irradiating a mouse, which is then protected by
administration of SCID mouse bone marrow; and (2) administration of
HBV-infected hepatocytes that have been differentiated from
placental stem cells. The mouse is then administered a compound
that is to be tested for its ability to reduce viral replication or
viral load.
[0382] Placental Stem Cells.
[0383] Adherent placental stem cells are obtained by one or more of
the methods described in Example 1, above.
[0384] Preparation of Hepatocytes.
[0385] Placental stem cells are differentiated according to the
method described in Examples 4, above.
[0386] HBV Infection of Placental Stem Cell-Derived
Hepatocytes.
[0387] Placental stem cell-derived hepatocytes in alginate are
collected by centrifugation and resuspended in 1 mL of high-titer
HBV DNA human serum supplemented with 3 .mu.g hexidementhrine
bromide (Sigma-Aldrich, H-9268, St. Louis, Mo.) and 0.5 .mu.g human
interleukin 6 (IL-6; Preprotech, London, England).
[0388] Preparation of Mice.
[0389] CB16F or BNX (beige/nude/xid) mice, and NOD/SCID mice at age
6-10 weeks, are used. Mice are fed sterile food and acid water
containing ciprofloxacin (20 .mu.g/mL). CB16F mice (Harlan
Laboratories, Weitzmann Institute Animal Breeding Center, Rehovot,
Israel) are exposed to total body irradiation at a dose of about 4
Gy followed three days later by a does of about 11 Gy from a gamma
beam 150-A .sup.60Co source (Atomic Energy of Canada, Kanata,
Ontario, Canada) at an irradiation rate of about 0.7 Gy/min. From
about 4.times.10.sup.6 to about 6.times.10.sup.6 bone marrow cells
from NOD/SCID mice, in 0.2 mL phosphate-buffered saline, are
immediately transplanted into the irradiated mice. Bone marrow
cells are prepared by disruption of femoral and tibial bones in an
Omni-Mixer in phosphate buffered saline to obtain marrow cells,
followed by depletion of T cells from the resulting cell
suspension. See Levite et al., Transplantation 8:1-3 (1991). The
mice are injected daily with 1 mg Fortum (Glaxo) intraperitoneally
for 5 days following bone marrow transplantation. Directly after
bone marrow cell transplantation, HBV-infected placental stem
cell-derived hepatocytes (about 5.times.10.sup.7) are then
transplanted into the irradiated mice under the kidney capsule or
into the ear pinna. Engraftment of hepatocytes can be assessed by
biopsy and hematoxylin-eosin staining, and by detection of
expression of human serum albumin-encoding mRNA in the transplanted
tissue.
[0390] BNX mice are prepared as CB16F mice, except that BNX mice
are irradiated once at a dose of 11 Gy, and transplantation of the
HBV-infected hepatocytes takes place at least 10 days after bone
marrow cell transplantation.
[0391] Extraction of DNA From HBV-Injected Sera.
[0392] DNA is extracted from 100 .mu.L of serum by proteinase K
digestion in 400 .mu.L reaction mixture containing 0.25% sodium
dodecyl sulfate, 5 mmol/L EDTA, 10 mmol/L Tris HCl (pH 8.0), and
250 .mu.g/mL proteinase K (Sigma, St. Louis, Mo.). After 2.5 hours
at 65.degree. C., 1 .mu.g of a DNA carrier and 0.5 mg BSA is added.
DNA is then extracted by phenol-chloroform and precipitated in
ethanol overnight at -20.degree. C. Following centrifugation for 15
minutes at 20,000 g, the DNA pellets is washed with 70% ethanol,
dried, and resuspended in 50 .mu.L of water.
[0393] Determination of HBV DNA Level in Mouse Sera.
[0394] The HBV DNA copy number is determined by semiquantitative
PCR using HBV-specific primers. PCR products are separated on a
standard 2% agarose gel. 50 .mu.L of the products are dot blotted
and hybridized overnight at 42.degree. C. with an appropriate
[.sup.32P]-labeled DNA fragment (Rediprime DNA labeling system,
Amersham, Buckinghamshire, UK). The blot is then washed in
0.1.times.0.15 mol/L NaCl and 0.015 mol/L sodium citrate, pH 7.0
and 1% SDS at 55.degree. C., and exposed to X ray film. The
intensity of dots is read on an ELISA reader, e.g., Dynatech at 630
nm, or on a Molecular Dynamics computing densitometer Model 300A.
Viral load is determined using a standard curve composed of DNA
samples obtained from calibrated human serum diluted in normal
mouse serum comprising copy numbers from 10.sup.2 to 10.sup.7 per
100 .mu.L samples. A mouse having a viral load of less than about
5.times.10.sup.3/mL serum is considered to be uninfected. Primers
used in this procedure recognize both covalently closed circular
and relaxed forms of HBV.
[0395] HBV viral load can, alternately or additionally, be
determined by ELISA using one or more antibodies that recognize a
surface antigen of HBV.
[0396] Determination of HBV Covalently Closed Circular DNA in
Engrafted Hepatocytes.
[0397] This step can be performed if confirmation of viral
replication is needed. DNA is extracted from hepatocytes collected
by centrifugation (about 1.times.10.sup.5) and resuspended in 100
.mu.L of H.sub.2O. Fifty .mu.L is subjected to PCR using
HBV-specific primers in 100 .mu.L reaction mixture containing 13
Taq Pol buffer, 2.5 mmol/L MgCl.sub.2, 0.2 mmol/L of each dNTP, 50
pmol of each primer, 1 mg/mL BSA, and 2.5 U of Taq Pol. (Promega).
The PCR reaction is programmed for 2 minutes at 94.degree. C., and
then 30 cycles, 1 minute at
[0398] 94.degree. C., and 3 minutes at 72.degree. C., with a final
elongation reaction of 5 minutes at 72.degree. C. PCR products are
analyzed on a 2% agarose gel and by dot-blot hybridization using a
DNA fragment corresponding to a portion of the core sequence. This
procedure uses primers that recognize only the covalently closed
circular form of HBV.
[0399] Determination of Antiviral Activity of Compounds.
[0400] Once viremia is established, the mice are administered a
compound that is to be tested for anti-HBV activity. The route of
administration is determined on a compound-by-compound basis, but
is generally either intraperitoneal or intravenous. Administration
of the compound is performed at 6-17 days post-transplantation with
infected hepatocytes. On days 2 and 9 post-administration, serum is
drawn from the mice and assessed for viral load using antibody to
HBsAg (HBV surface antigen) and PCR to detect HBV covalently closed
circular DNA (cccHBV). The compound is determined to be an
antiviral compound if the viral load is detectably reduced (e.g.,
statistically significantly reduced) compared to the viral load in
mice not administered the compound. Viral load can be compared to
the amount of cccHBV present to determine whether a compound that
reduces viral load is an inhibitor of HBV replication.
6.13 Example 13: Induction of Differentiation of Placental Stem
Cells into Chondrocytes
[0401] 6.13.1 General Method
[0402] Chondrogenic differentiation of placental stem cells is
generally accomplished as follows:
[0403] 1. Placental stem cells are maintained in MSCGM (Cambrex) or
DMEM supplemented with 15% cord blood serum.
[0404] 2. Placental stem cells are aliquoted into a sterile
polypropylene tube. The cells are centrifuged (150.times.g for 5
minutes), and washed twice in Incomplete Chondrogenesis Medium
(Cambrex).
[0405] 3. After the last wash, the cells are resuspended in
Complete Chondrogenesis Medium (Cambrex) containing 0.01 .mu.g/ml
TGF-beta-3 at a concentration of 5.times.10(5) cells/ml.
[0406] 4. 0.5 ml of cells is aliquoted into a 15 ml polypropylene
culture tube. The cells are pelleted at 150.times.g for 5 minutes.
The pellet is left intact in the medium.
[0407] 5. Loosely capped tubes are incubated at 37.degree. C., 5%
CO2 for 24 hours.
[0408] 6. The cell pellets are fed every 2-3 days with freshly
prepared complete chondrogenesis medium.
[0409] 7. Pellets are maintained suspended in medium by daily
agitation using a low speed vortex.
[0410] 8. Chondrogenic cell pellets are harvested after 14-28 days
in culture.
[0411] 9. Chondrogenesis is characterized by e.g., observation of
production of esoinophilic ground substance, assessing cell
morphology, an/or RT/PCR confirmation of collagen 2 and/or collagen
9 gene expression and/or the production of cartilage matrix acid
mucopolysaccharides, as confirmed by Alcian blue cytochemical
staining.
[0412] Chondrogenesis can also be assessed by gene expression for
early stage chondrogenesis markers fibromodulin and cartilage
oligomeric matrix protein; gene expression for mid-stage
chondrogenesis markers aggrecan, versican, decorin and biglycan;
and gene expression for types II and X collagens and
chondroadherein, markers of mature chondrocytes.
[0413] Placental stem cells can also be induced to chondrogenesis
by the method above, wherein the placental stem cells are cultured
on nanofibrous scaffolds such as poly(L-lactic acid) (PLLA), type I
collagen, or a copolymer of vinylidene fluoride and
trifluoroethylene (PVDF-TrFE), beginning at step 3, without
centrifugation step 4.
[0414] 6.13.2 Differentiation of Placental and Umbilical Cord Stem
Cells into Chondrogenic Cells
[0415] This Example demonstrates the differentiation of placental
stem cells into chondrogenic cells and the development of
cartilage-like tissue from such cells.
[0416] Cartilage is an avascular, alymphatic tissue that lacks a
nerve supply. Cartilage has a low chondrocyte density (<5%),
however these cells are surprisingly efficient at maintaining the
extracellular matrix around them. Three main types of cartilage
exist in the body: (1) articular cartilage, which facilitates joint
lubrication in joints; (2) fibrocartilage, which provides shock
absorption in, e.g., meniscus and intervertebral disc; and (3)
elastic cartilage, which provides anatomical structure in, e.g.,
nose and ears. All three types of cartilage are similar in
biochemical structure.
[0417] Joint pain is a major cause of disability and provides an
unmet need of relief in the area of orthopedics. Primary
osteoarthritis (which can cause joint degeneration), and trauma are
two common causes of pain. Approximately 9% of the U.S. population
has osteoarthritis of hip or knee, and more than 2 million knee
surgeries are performed yearly. Unfortunately, current treatments
are more geared towards treatment of symptoms rather than repairing
the cartilage. Natural repair occurs when fibroblast-like cells
invade the area and fill it with fibrous tissue which is neither as
resilient or elastic as the normal tissue, hence causing more
damage. Treatment options historically included tissue grafts,
subchondral drilling, or total joint replacement. More recent
treatments however include CARTICEL.RTM., an autologous chondrocyte
injection; SYNVISC.RTM. and ORTHOVISC.RTM., which are hyaluronic
acid injections for temporary pain relief; and CHONDROGEN.TM., an
injection of adult mesenchymal stem cells for meniscus repair. In
general, the trend seems to be lying more towards cellular
therapies and/or tissue engineered products involving chondrocytes
or stem cells.
Materials and Methods.
[0418] Two placental stem cell lines from amnion/chorion,
designated AC61665, P3 (passage 3) and AC63919, P5, and two from
umbilical cord, designated UC67249, P2 and UC67477, P3 were used in
the studies outlined below. Human mesenchymal stem cells (MSC) were
used as positive controls, and an osteosarcoma cell line, MC3T3,
and human dermal fibroblasts (HDF) were used as negative
controls.
[0419] Placental and umbilical cord stem cells were isolated and
purified from full term human placenta by enzymatic digestion.
Human MSC cells and HDF cells were purchased from Cambrex, and
MC3T3 cells were purchased from American Type Culture Collection.
All cell lines used were centrifuged into pellets in polypropylene
centrifuge tubes at 800 RPM for 5 minutes and grown in both
chondrogenic induction media (Cambrex) and non-inducing basal MSC
media (Cambrex). Pellets were harvested and histologically analyzed
at 7, 14, 21 and 28 days by staining for glycosaminoglycans (GAGs)
with Alcian Blue, and/or for collagens with Sirius Red. Collagen
type was further assessed with immunostaining. RNA analysis for
cartilage-specific genes was performed at 7 and 14 days.
Results
[0420] Experiment 1: Chondrogenesis studies were designed to
achieve three main objectives: (1) to demonstrate that placental
and umbilical cord stem cells can differentiate and form cartilage
tissue; (2) to demonstrate that placental and umbilical cord stem
cells can differentiate functionally into chondrocytes; and (3) to
validate results obtained with the stem cells by evaluating control
cell lines.
[0421] For objective 1, in a preliminary study, one placental stem
cell line was cultured in chondrogenic induction medium in the form
of cell pellets, either with or without bone morphogenic protein
(BMP) at a final concentration of 500 ng/mL. Pellets were assessed
for evidence of chondrogenic induction every week for 4 weeks.
Results indicated that the pellets do increase in size over time.
However, no visual differences were noted between the BMP.sup.+ and
BMP.sup.- samples. Pellets were also histologically analyzed for
GAGs, an indicator of cartilage tissue, by staining with Alcian
Blue. BMP.sup.+ cells generally appeared more metabolically active
with pale vacuoles whereas BMP cells were smaller with
dense-stained nuclei and less cytoplasm (reflects low metabolic
activity). At 7 days, BMP+ cells had stained heavily blue, while
BMP.sup.- had stained only faintly. By 28 days of induction, both
BMP+ and BMP.sup.- cells were roughly equivalently stained with
Alcian Blue. Overall, cell density decreased over time, and matrix
overtook the pellet. In contrast, the MC3T3 negative cell line did
not demonstrate any presence of GAG when stained with Alcian
Blue.
[0422] Experiment 2: Based on the results of Experiment 1, a more
detailed study was designed to assess the chondrogenic
differentiation potential of two placental stem cell and two
umbilical cord stem cell lines. In addition to the Alcian Blue
histology, cells were also stained with Sirius Red, which is
specific for type II collagen. Multiple pellets were made for each
cell line, with and without induction media.
[0423] The pelleted, cultured cell lines were first assessed by
gross observation for macroscopic generation of cartilage. Overall,
the stein cell lines were observed to make pellets as early as day
1. These pellets grew over time and formed a tough matrix,
appearing white, shining and cartilage-like, and became
mechanically tough. By visual inspection, pellets from placental
stem cells or umbilical cord stem cells were much larger than the
MSC controls. Control pellets in non-induction media started to
fall apart by Day 11, and were much smaller at 28 days than pellets
developed by cells cultured in chondrogenic induction medium.
Visually, there were no differences between pellets formed by
placental stem cells or umbilical cord. However, the UC67249 stem
cell line, which was initiated in dexamethasone-free media, formed
larger pellets. Negative control MC3T3 cells did not form pellets;
however, HDFs did form pellets.
[0424] Representative pellets from all test groups were then
subjected to histological analysis for GAG's and collagen.
Generally, pellets formed by the stem cells under inducing
conditions were much larger and stayed intact better than pellets
formed under non-inducing conditions. Pellets formed under inducing
conditions showed production of GAGs and increasing collagen
content over time, and as early as seven days, while pellets formed
under non-inducing conditions showed little to no collagen
production, as evidenced by weak Alcian Blue staining. In general,
the placental stem cells and umbilical cord stem cells appeared, by
visual inspection, to produce tougher, larger pellets, and appeared
to be producing more collagen over time, than the mesenchymal stem
cells. Moreover, over the course of the study, the collagen
appeared to thicken, and the collagen type appeared to change, as
evidenced by changes in the fiber colors under polarized light
(colors correlate to fiber thickness which may be indicative of
collagen type). Non-induced placental stem cells produced much less
type II collagen, if any, compared to the induced stem cells. Over
the 28-day period, cell density decreased as matrix production
increased, a characteristic of cartilage tissue.
[0425] These studies confirm that placental and umbilical cord stem
cells can be differentiated along a chondrogenic pathway, and can
easily be induced to form cartilage tissue. Initial observations
indicate that such stem cells are preferable to MSCs for the
formation of cartilage tissue.
6.14 Example 14: Induction of Differentiation into Chondrocytes by
Nanofibrous Scaffolds
[0426] This example describes methods for inducing the
differentiation of stem cells, including placental stem cells or
mesenchymal stem cells from bone marrow (BM-MSC), into chondrocytes
with nanofibrous scaffolds. The objectives of this example are
threefold: 1) to characterize chondrogenic differentiation of
placental stem cells in vitro; 2) determine the optimum scaffold
for stimulating chondrogenic differentiation of placental stem
cells in vitro; and 3) evaluate in vivo repair of osteochondral
defects in a rabbit model using placental stem cells-scaffold
constructs.
[0427] To accomplish Objective 1, dynamic pellet placental stem
cells cultures are used to lengthen culture duration beyond 28
days. Temporal gene expression as well as biochemical and
histological analyses of early and late stage markers of
chondrogenesis for placental stem cells in static as well as
dynamic pellet cultures are assessed. Placental stem cells are
cultured with or without TGF-.beta..sub.3 in pellet cultures under
static or dynamic conditions for up to 56 days. By real-time PCR,
quantitative gene expression is performed for early stage markers
of fibromodulin and cartilage oligomeric matrix protein. Mid-stage
markers of aggrecan, versican, decorin and biglycan are also
assessed. Genes for types II and X collagens and chondroadherin,
which are characteristic of mature chondrocytes, are also assessed.
Biochemical assays are performed for type IT collagen,
glycosaminoglycan, and proteoglycan synthesis. Tissue morphology of
the chondrogenic pellets are also characterized by histological
staining during the time course.
[0428] To accomplish Objective 2, the ability of electrospun
nanofibrous scaffolds to promote placental stem cell
differentiation into chondrocytes is assessed. Scaffolds composed
of poly(L-lactic acid) (PLLA), type I collagen and a copolymer of
vinylidene fluoride and trifluoroethylene (PVDF-TrFE) are assessed.
Placental stem cells are loaded onto scaffolds generated from these
materials and cultured in both static and dynamic conditions.
Quantitative gene expression, biochemical and histological
analyses, and mechanical testing are performed during the study
time course.
[0429] To accomplish Objective 3, chondral repair by placental stem
cells is assessed by in vivo functional evaluation in an animal
model that will not spontaneously heal a cartilage defect such as,
for example, a rabbit osteochondral defect model. Scaffolds that
support chondrogenic differentiation are evaluated in this model in
combination with placental stem cells. Repair is evaluated by
histochemical staining, specifically accessing the extent of
cartilage union between uninjured cartilage and repaired cartilage.
Osteo-chondral repair is also measured by mechanical evaluation of
samples excised from the site of the original defect.
[0430] 6.14.1 Fibrous Scaffold Fabrication
[0431] This example describes fabrication and characterization of
exemplary nanofiber non-woven meshes. The technique of
electrospinning was employed to produce nanoscale fiber meshes. The
resulting meshes have high surface area, controllable porosity,
architecture and mechanical properties. Fibers produced with this
technique are on the same scale as the fibers of the ECM.
poly(L-lactic acid) (PLLA) and poly lactic glycolic acid (PLGA)
were used as the polymer compositions since they are one of the
most widely used biomaterials in the tissue engineering field. The
potential use of these scaffolds as tissue engineering scaffolds
was then assessed by cell proliferation and differentiation of
human MSCs.
[0432] The electrospinning process is affected by varying the
electric potential, flow rate, solution concentration,
capillary-collector distance, diameter of the needle, and ambient
parameters, e.g., temperature. To fabricate PLLA and PLGA scaffolds
of four distinct fiber diameters, namely 290 nm, 1 .mu.m, 5 .mu.m
and 9 .mu.m, the voltage [20 KV] and collector to needle distance
[30 cm] were kept constant. The needle gauge size [12 G; 22 G], the
flow rate [0.05-0.1 mL/min] and the solution concentration [10-25
w/w %] were varied.
[0433] The average diameter distribution of PLLA and PLGA
electrospun mats generated thereby is listed in Table 4. Microfiber
and the nanofiber scaffolds of both PLLA and PLGA had a porosity of
39% and 47%, respectively. The morphology of the electrospun fibers
was observed by scanning electron microscopy. By inspection, the
fibers were free of beads, appeared round and were aligned randomly
in a non-woven fashion.
TABLE-US-00004 TABLE 4 Polymer Average Diameter Polymer Average
Diameter PLLA-1 290 .+-. 84 nm PLGA-1 380 .+-. 0.80 nm PLLA-2 1
.+-. 0.4 .mu.m PLGA-2 1 .+-. 0.3 .mu.m PLLA-3 5 .+-. 1.5 .mu.m
PLGA-3 6 .+-. 1.5 .mu.m PLLA-4 9 .+-. 2.0 .mu.m PLGA-4 9 .+-. 1.6
.mu.m
[0434] 6.14.2 Seeding of Human MSCs on Nanofibrous Scaffolds
[0435] Human BM-MSCs isolated from whole bone marrow and
subcultured were seeded onto microfiber and nanofiber scaffolds.
Cell culture plastic was used as a control surface. BM-MSCs grown
on both nano and microfiber scaffolds had similar growth kinetics.
Cell morphology and uniformity of the cell layer was observed by
SEM. Cells seeded on nanofibers were uniformly distributed across
the scaffold surface whereas cells on microfiber scaffolds were
spread out along the fibrils and less uniformly distributed,
regardless of composition. This finding was also confirmed using
actin cytoskeleton staining and viewed by confocal microscopy.
[0436] Chondrogenic differentiation of BM-MSCs in static tissue
culture conditions also demonstrated an expression of Type II
collagen for cells seeded on nanofiber scaffolds without the
presence of inductive factors in the media. Thus, these findings
suggest that nanofiber architectures promote chondrogenic
differentiation, even without the presence of inductive factors in
the culture media.
[0437] 6.14.3 In Vitro Chondrogenic Differentiation of Placental
Stem Cells
[0438] To examine the chondrogenic potential of placental stem
cells isolated and expanded under typical conditions, placental
stem cells were grown in pellet culture in the presence of
chondrogenic induction media. The duration of static cell culture
experiments was limited to 28 days due to decreased cell content of
pellets. Histological sections were taken to examine the pellets
for functional differentiation and the presence of
glycosaminoglycans (GAGs) and collagen. Quantitative protein and
gene expression analyses were used to further characterize the
pellets. Immune markers on differentiated placental stem cells were
compared to undifferentiated placental stem cells, which are known
to be immunosuppressive.
[0439] From the histological sections, it was determined that
placental stem cells formed tighter pellets than BM-MSCs. Both
placental stem cells and BM-MSCs stained for GAGs and collagen,
while a control cell line (human dermal fibroblasts, HDF) formed
loosely organized pellets with no GAGs and little collagen
expression.
[0440] Protein and gene expression were used to quantify
chondrogenesis by ELISA and RT-PCR, respectively. ELISA data
confirmed the presence of GAGs and Type I collagen in pellets
produced from placental stem cells. No Type II collagen could be
found in the pellets by ELISA, but low levels of Type II collagen
gene expression were found. Further, gene expression data confirmed
the up-regulation of a number of chondrogenic markers in induced
Placental stem cell pellets compared to uninduced pellets (Table
5)
TABLE-US-00005 TABLE 5 Gene Up-regulation Aggrecan 1 + Bone
morphogeneic protein 2 ++ Cartilage oligo matrix protein ++++
Cartilage-derived ret. acid sens + Collagen, type II + Collagen,
type IX + Link protein - Matrilin 3 + Parathyroid hormone receptor
1 + Transcription factor SOX9 + (Level of up-regulation: - = no
up-regulation, + = 1-10 fold; ++ = 10-100 fold, +++ = 100-1,000
fold; and ++++ = 1,000-10,000 fold)
[0441] The potential immunogenicity of undifferentiated and
chondrogenic differentiated placental stem cells was compared using
flow cytometry to stain for the presence of the following immune
system molecules: MHC A,B,C; MHC DR,DP,DQ; .beta.-2-microglubulin,
and CD86. The lack of expression of MHC II and CD86 and the
positive expression of small amounts of MHC A,B,C and
.beta.-2-microglobulin were similar (Table 6) between
undifferentiated placental stem cells and placental stem cells
cultured in under chondrogenic conditions.
TABLE-US-00006 TABLE 6 Chondorgenic Undifferentiated Differentiated
Placental Stent Placental Stem Marker Cell Expression Cell
Expression MHC Class II None None CD 86 None None MHC A, B, C Low
Low .beta.-2 Microglobulin Low Low
[0442] 6.14.4 Characterization of Chondrogenic Differentiation of
Placental Stem Cells In Vitro.
[0443] This example provides exemplary methods for achieving
Objective 1 as set forth in Section 6.11, above. In this example,
dynamic pellet cultures are used to permit lengthened culture
duration. This extension permits assessment of temporal gene
expression as well as biochemical and histological analyses of
early and late stage markers of chondrogenesis for placental stem
cells in static as well as dynamic pellet cultures for up to 56
days.
[0444] Placental stem cells are isolated from the post-partum
placenta, for example, according to Example 1, above. Placental
stem cells are established from disrupted tissue in, for example, a
complete medium containing low concentrations of fetal calf serum
and limited growth factors, according to Example 1, above. After
reaching 80-85% confluence, placental stem cells are subcultured
and/or cryopreserved as described elsewhere herein. Flow cytometry
analysis is performed to ensure that at least 70% or more of
isolated cells, for example,
CD200.sup.+CD105.sup.+CD73.sup.+CD34.sup.- CD45.sup.-, e.g.,
according to the method disclosed in Example 3, above. Functional
characterization of placental stem cells further includes a
chondrogenic differentiation assay.
[0445] 6.14.5 Pellet Culture in Static and Dynamic Systems
[0446] Chondrogenic differentiation of placental stem cells can be
carried out as follows. Placental stem cells are cultured to near
confluence, trypsinized, washed 2.times. in incomplete
chondrogenesis media (Cambrex) and resuspended at about 500,000
cells/mL in complete chondrogenic media as described above.
Aliquots of 500 .mu.L are pipetted into 15 mL polypropylene
centrifuge tubes and centrifuged (800 rpm, 5 min) to induce pellet
formation. Serum-free chondrogenic complete medium (CCM) consisting
of 1 mM sodium pyruvate (Sigma), 0.1 mM ascorbic acid-2-phosphate
(Wako), 1.times.10.sup.-7 M dexamethasone (Sigma), 1% ITS
(insulin-transferrin-selenium) (Collaborative Biomedical Products),
and 10 ng/mL recombinant human TGF-.beta.3 (Oncogene Sciences)
dissolved in DMEM-low glucose is added to centrifuge tubes. Some
pellets are transferred to bioreactors (Synthecon, model #
RCCS-411) containing serum-free CCM for dynamic culturing. Pellets
under static or dynamic culture conditions are incubated at
37.degree. C., 5% CO.sub.2 and medium is exchanged every 2-3 days.
At days 7, 14, 28, and 56, pellets are removed from culture and
processed for gene expression, biochemical, or histological
analysis. Comparisons are made with MSCs grown under static or
dynamic culture condition.
[0447] To characterize the differentiated chondrocytes, pellets are
washed with HEPES buffered saline without calcium and magnesium and
digested with 3 mg/ml collagenase type 2, 1 mg/ml hyaluronidase,
and 0.25% trypsin citrate at 37.degree. C. After digestion the
cells are spun down, resuspended in 1 ml DPBS buffer, and washed.
The amount of cells recovered is determined by trypan blue dye cell
count. RNA is recovered by lysing the cells with a lysis buffer.
RNA is isolated using Qiagen RNEASY.RTM. kits and quantified using
a Nanodrop spectrophotometer. RT-PCR for chondrogenic gene
expression is performed using TAQMAN.RTM. gene expression probes.
Quantitative gene expression is performed for early stage markers
of fibromodulin and cartilage oligomeric matrix protein. Mid-stage
markers of aggrecan, versican, decorin and biglycan are also
examined. Genes for types II and X collagens and chondroadherin,
which are characteristic of mature chondrocytes, are also
examined.
[0448] To further characterize the differentiated chondrocytes,
Enzyme-Linked Immunosorbent Assay (ELISA) assays are used to
examine protein expression of chondro-differentiated placental stem
cells. First, the pellets are solubilized. After obtaining a dry
weight, rehydrated pellets are digested using pepsin and pancreatic
elastase. The collected supernatants are used for Collagen Type II
and proteoglycan ELISA. Glycosaminoglycan (GAGs) is measured by a
methylene blue dye binding assay.
[0449] The differentiated chondrocytes are also subjected to
histological analysis as follows. The pellets are fixed in formalin
10%, dehydrated through graded alcohols, and embedded in paraffin.
Sections are cut at a thickness of 5 m and stained with a stain for
glycosaminoglycans (e.g., Alcian blue and/or Safranin-O, and a
stain for collagen, e.g., Sirius Red. Alcian Blue is a copper
phthalocyanine basic dye that is water-soluble and colored blue
because of its copper content. When used in a 3% acetic acid
solution (pH 2.5), Alcian Blue stains both sulfated and
carboxylated acid mucopolysaccharides and sulfated and carboxylated
sialomucins. Safranin O in the orthochromatic form stains articular
cartilage, mucin and mast cell granules on formalin-fixed, paraffin
embedded tissue sections. Proteoglycans stain red, cytoplasm stains
gray green and nuclei stain black. Sirius Red dye can be used to
differentiate different collagen types in tissue sections. Under
polarized light, the fibers seemingly glow with bright colors
against a black background. The color of the fibers depends on
their thickness; as thickness increases, the color changes from
green to yellow to orange to red. Type I collagen, which tends to
form thick collagen fibers composed of closely packed thick
fibrils, appears as an intense yellow to red color. Type III
collagen forms thin fibers, composed of loosely disposed thin
fibrils and have weak green birefringence. Thus, the color
displayed is a result of the thickness of the fiber, as well as the
arrangement of the collagen molecules.
[0450] Four experimental groups are used in this study; cells are
grown in either standard growth media or in CCM and in static or
dynamic culture systems. The quantitative assays are performed on
days 7, 14, and 28, and 56. A sample size, n, of 4 is used for all
quantitative biochemical assays (glycosaminoglycan, Type II
collagen, and proteoglycan), gene expression, and histological
analyses. One way and two way ANOVAs are performed to test for
statistical differences between groups at each time point and over
time, respectively for p<0.05. The Tukey-Kramer Method,
p<0.05, is used to perform multiple comparisons between
groups.
[0451] 6.14.6 Optimization of Scaffolds for Stimulating Placental
Stem Cell Chondrogenic Differentiation In Vitro.
[0452] This example describes experiments designed to achieve
Objective 2, set forth above. In brief, the ability of nanofibrous
scaffolds to promote placental stem cell differentiation into
chondrocytes is tested. Electrospun nanofibrous scaffolds,
irrespective of composition, promote mesenchymal stem cell
differentiation into chondrocytes in vitro as shown above. The
materials to be tested as scaffold substrates include poly(L-lactic
acid) (PLLA), type I collagen and a copolymer of vinylidene
fluoride and trifluoroethylene (PVDF-TrFE). Placental stem cells
are loaded onto scaffolds generated from these materials and
cultured in both static and dynamic conditions. Quantitative gene
expression, biochemical and histological analyses as well as
mechanical testing is performed during the study time course.
[0453] 6.14.6.1 Scaffold Fabrication And Characterization
[0454] As described above, the electrospinning apparatus comprises
a syringe fitted with a needle (16-22 gauge), mounted on a Harvard
Syringe Pump Model 901. The syringe is filled with the polymer
solution. The positive output lead of a high voltage power supply
(Gamma High Voltage Power Supply ES30P) is attached to the needle.
The collector is a stainless steel plate of dimensions 25.times.30
cm, which is electrically grounded. The electrospinning process is
affected by varying the flow rate, solution concentration, and the
diameter of the needle. The voltage applied to the solution is 20
kV, and the collector to needle distance is 20 cm. Scaffolds are
collected as nonwoven mats at the collector. These scaffolds/mats
are made of fibers having diameters of approximately 200-400
nm.
[0455] 6.14.6.2 Polymer Solutions for Scaffold Preparation
[0456] Initial scaffolds are electrospun from solutions of a Poly
L-lactic acid (Alkermes Inc., Medisorb Polymer 75/25 DL High IV) in
methylene chloride. Similar scaffolds of Type I collagen (derived
from bovine tendon) are produced by dissolving collagen in
trifluoroacetic acid (TFA). The piezoelectric polymer, p(VDF-TrFE)
is spun from a 10% solution of the polymer in methyl ethyl ketone,
as previously demonstrated. See Sachlos and Czernuszka, 2003, Eur.
Cells Mater. 5:29-40.
[0457] 6.14.6.3 Scaffold Characterization
[0458] All scaffolds are examined by the following protocol.
Scaffolds are imaged by polarized light optical microscopy and
scanning electron microscopy. Image analysis techniques are
utilized to determine average fiber diameter and coefficient of
variation, as well as the area distribution between fibers. The
fiber parameters of average array pore volume and pore size are by
mercury porosimetry. Internal scaffold structure is monitored by a
variety of thermal analysis techniques including DSC, TGA, DMA, TMA
and TSC as appropriate.
[0459] For the p(VDF-TrFE) scaffold, a scan of current vs. an
applied E field with a Sawyer-Tower circuit is used to identify the
properties indicative of the polarizability of the p(VDF-TrFE)
material. These properties include remnant and saturation
polarizability, and the coercive field (the E field at which
polarity switching occurs). These properties have well known values
for p(VDF-TrFE) materials. The small currents injected or released
by a vibrating electroded electrospun p(VDF-TrFE) mat can also be
measured with an electrometer. Thermal discharge current (TSC)
instrumentation is for these studies. Electroded p(VDF-TrFE) films
undergo 1-10% strains in length, width and thickness when subjected
to high magnitude (10-100 MV/m), periodic E fields. The strains
result as the dimensions of the ferroelectric crystallite content
responds to applied field. It can be expected that electroded
electrospun p(VDF-TrFE) mats will undergo similar strains.
[0460] 6.14.6.4 Scaffold Seeding and Differentiation Protocol
[0461] Chondrogenic differentiation of placental stem cells is
carried out using media as described above, and elsewhere herein
(see, e.g., Section 5.4.5, above). All scaffolds are vacuum loaded
with about 200,000 cells using conventional techniques. See, e.g.,
Dennis et al., 1998, Biomaterials 19:1323-8. Cell-loaded scaffolds
are placed in bioreactors (Synthecon, model # RCCS-4H) containing
CCM. The cells are maintained in culture for up to 56 days. Medium
is changed every 2-3 days. Comparisons are made with positive
controls, placental stem cells grown without scaffolds using
standard pellet culture conditions with CCM as described above, and
with negative controls, and placental stem cells grown with or
without scaffolds in the absence of CCM (using standard growth
media).
[0462] 6.14.6.5 Quantitative Measurements of Cartilage-Specific
Extracellular Matrix Components and Cell Number
[0463] Chondrogenic pellets and cell-laden scaffolds are harvested
at 7, 14, 28, and 56 days and analyzed for glycosaminoglycan, Type
II collagen, and proteoglycan synthesis. These assays allow for
rapid, high throughput screening for cartilage markers using
96-well plate formats. To do so, the samples are washed with
phosphate buffered saline and digested with 200 .mu.L papain
solution (1 .mu.g/mL in 50 mM sodium phosphate pH 6.5, containing 2
mM N-acetyl cysteine and 2 mM EDTA) for 16 hours at 65.degree. C.
Glycosaminoglycan and proteoglycan synthesis are measured
quantitatively using an ELISA kit (BLYSCAN.TM. Kit, Accurate
Chemical and Scientific Corporation, Westbury, N.Y.). Type II
collagen synthesis is measured by an ELISA kit (Arthrogen-CIA,
Chondrex, Inc.). Cell number is quantified by DNA measurement,
using pico-green fluorescence assay (Molecular Probes, Inc.).
[0464] 6.14.6.6 Tissue Morphology by Histological Analysis
[0465] Histological staining and viewing at days 21 and 35 is used
to characterize cell and tissue morphology of the chondrogenic
scaffolds. Cell pellets and cell-laden scaffolds are harvested and
fixed in 10% buffered formalin for 2 hours at room temperature.
Pellet and scaffolds are dehydrated by treatment with a series of
graded alcohols, cleared by treatment with xylene and xylene
substitute, and infiltrated with paraffin. Thin sections are
slide-mounted and stained with toluidine blue and safranin O.
[0466] 6.14.6.7 Analysis of mRNA Changes From Differentiation
[0467] As described previously for pellet cultures, samples are
washed with HEPES buffered saline without calcium and magnesium and
digested with 3 mgs/ml collagenase type 2, 1 mg/ml hyaluronidase,
and 0.25% trypsin citrate at 37.degree. C. After digestion the
cells are spun down, resuspended in 1 mL DPBS (Dulbecco's
Phosphate-Buffered Saline) buffer, and washed. The amount of cells
recovered is determined by trypan blue dye cell count. RNA is
recovered by lysing the cells with a lysis buffer. RNA is isolated
using Qiagen RNEASY.RTM. kits and quantified using Nanodrop. RT-PCR
for chondrogenic gene expression is accomplished using TAQMAN.RTM.
gene expression probes from ABI. Quantitative gene expression is
performed for early stage markers of fibromodulin and cartilage
oligomeric matrix protein. Mid-stage markers of aggrecan, versican,
decorin and biglycan are also examined. Genes for types II and X
collagens and chondroadherin, which are characteristic of mature
chondrocytes, are also examined.
[0468] 6.14.6.8 Number of Groups and Statistical Analyses
[0469] Six experimental groups are used in this study: three
material compositions (PLLA, Type I Collagen, and pVDF-TrFE), and
cells are grown in either standard growth media or in CCM. The
quantitative assays are performed on days 7, 14, 21, and 28 days. A
sample size, n, of 4 is used for all quantitative assays
(glycosaminoglycan, Type II collagen, and proteoglycan). One way
and two way ANOVAs are performed to test for statistical
differences between groups at each time point and over time,
respectively for p<0.05. The Tukey-Kramer Method, p<0.05, is
used to perform multiple comparisons between groups.
[0470] 6.14.7 Evaluation of In Vivo Repair of Osteochondral Defects
in a Rabbit Model Using Placental Stem Cell-Scaffold
Constructs.
[0471] This Example describes experiments designed to achieve
Objective 3, set forth above. Briefly, scaffolds identified as
supporting chondrogenic differentiation are evaluated in
combination with placental stem cells in the repair of
osteochondral defects in rabbits.
[0472] 6.14.7.1 Animal Model
[0473] A rabbit, partial-weight bearing articular cartilage repair
model is employed to study the chondrogenic effects of
cell/scaffold constructs. Depending upon the number of scaffolds
that support placental stem cell chondrogenesis, the number of
rabbits is determined. Rabbits, New Zealand strain, are randomly
assigned to the groups. An n of 4 per group is implanted. Average
animal weight is between 3-5 kg and animals are specific pathogen
free (SPF).
[0474] A 3.5 mm cylindrical defect is created in the intercondylar
groove of the distal femur of each animal, and a cylindrical,
cell/scaffold construct is implanted into the defect in a press-fit
fashion. The contralateral knee of each rabbit serves as an
internal control. The same defect is created and a scaffold of the
same composition and architecture without cells is inserted. The
knee to act as internal control is randomized by picking an opaque
envelope, prior to the procedure, that states left or right as the
control knee.
[0475] To implant the scaffolds, intravenous pentobarbital (45
mg/kg) is administered to initiate anesthesia. Anesthesia is
maintained through inhalation of 0.5-2% isoflurane delivered in
oxygen. The rabbit is placed in the supine position and each knee
is shaved and prepped in a sterile fashion with a 70% alcohol scrub
over the incision site. The surgical site is isolated with sterile
drapes.
[0476] A lateral parapatellar incision and arthrotomy is performed.
The femoral condyles are exposed by medial dislocation of the
patella. A 3.5 mm drill bit is used to create the defect in the
intercondylar groove of the distal femur. Defect depth is 3-5 mm in
depth. The scaffold with or without cells is sutured into the
defect utilizing a small absorbable suture material (6-0/7-0
vicryl). The wound is then closed in layers with absorbable
sutures. The same procedure is performed on the contralateral knee
as outlined above to serve as an internal control.
[0477] Post-operation, all animals are allowed weight-bearing as
tolerated in their cages. Buprenorphine 0.03 mg/kg is administered
every 12 hours subcutaneously for post-operative pain control for
the first 5-7 days post-operatively. Regular diet is provided.
[0478] Rabbits are euthanized utilizing a lethal dose of
pentobarbital at 12 weeks post-operatively. The distal femora of
each rabbit are harvested. Gross evaluation of the repair site is
documented for color and appearance compared to the normal
surrounding tissue; evidence of joint arthrosis is documented as
well. Specimens (n=4 per group) are decalcified and paraffin
embedded for histological evaluation and staining. Histological
evaluation is graded utilizing a modified O'Driscoll score. N=8
specimens per group are used for mechanical testing. Compression
testing is performed based on previously reported protocols for
nanofibrous scaffolds.
6.15 Example 15: Identification of a CD34.sup.+CD45.sup.- Cell
Population in Placental Perfusate
[0479] The purpose of this study was to phenotypically characterize
and compare cells from matched placenta perfusate (HPP) and cord
blood (HUCB) units (n=10) and to identify additional useful cell
surface markers for placenta perfusate using multi-parameter flow
cytometry. To assess the quality of the cells, total nucleated
cells (TNCs) and cell viability were also measured. Since post-thaw
samples are close to the condition of cells prior to their use in
preclinical or clinical studies, characterization of the placenta
perfusate focused on these samples. For comparison purposes, a
donor matched cord blood for each placenta unit was tested.
Including matched HUCB units in this project allowed evaluation of
the differences between cell populations in the placenta perfusate
and umbilical cord blood collected from the same donor.
[0480] Materials and methods: Placental perfusates were obtained
from perfusion of placentas from normal births using 0.9% NaCl.
Matched units of umbilical cord blood were collected and
cryopreserved by standard methods, and thawed immediately prior to
use. Frozen HPP and HUCB samples were drawn from a liquid nitrogen
tank and thawed in a 37.degree. C. water bath immediately. Cells
were assessed by flow cytometry, by washing the cells in PBS with
2% fetal calf serum, staining with conjugated antibodies, and
analyzing using either a BD FacsCalibur or a BD ARIA (Becton
Dickinson, San Jose, Calif.). Antibodies used included PE-CD34 (BD
Cat #348057) and PerCP-CD45 (BD Cat #340665). Cell sorting
experiments were performed after staining with the appropriate
antibodies and cells were placed in a standard CFU assay system
using MethoCult.
[0481] Results: Flow cytometry determined that a cell having a
CD34.sup.+CD45.sup.- phenotype was 4-fold enriched in HPP as
compared to HUCB (FIG. 10). Data in Table 7 was derived from HPP
and HUCB cells that were sorted using the FACS ARIA cell sorted for
Becton Dickinson. Placental perfusate stem cells were sorted based
on the following phenotypes: CD34.sup.- CD45.sup.-,
CD34.sup.+CD45.sup.- and CD34.sup.+CD45.sup.+. The double negative
cell type was not expected to produce any CFU and served as a sort
purity control. The CD34.sup.+CD45.sup.+ cell type is the classical
CB stem cell and served as the positive control for the sort and
the CFU assay. The test phenotype of CD34.sup.+CD45.sup.- was not
present in sufficient quantities in the HUCB to allow for sorting
and CFU, but from HPP cells were obtained. As seen in Table 1,
cells from HPP gave rise to larger numbers of CFU-E and BFU-E, as
well as providing the same CFU pattern as found in HUCB. In
addition, the HPP CD34.sup.+CD45.sup.+ cells provided CFU-GEMM, a
population not detected in HUCB nor produced by
CD34.sup.+CD45.sup.+ cells in HUCB.
TABLE-US-00007 TABLE 7 CFU DATA Chart CD34-; CD45- Cells
CFU-E/BFU-E CFU-GM CFU-GEMM 1000 cells/ 3000 cells/ 9000 cells/
1000 cells/ 3000 cells/ 9000 cells/ 1000 cells/ 1000 cells/ 9000
cells/ Unit # well well well well well well well well well
327950HPP 0 0 0 0 0 0 0 0 0 327940CB 0 0 0 2 2 0 0 0 0 CD34+; CD45-
Cells CFU-E/BFU-E CFU-GM CFU-GEMM 50 cells/ 150 cells/ 450 cells/
1000 cells/ 50 cells/ 150 cells/ 450 cells/ 1000 cells/ 50 cells/
150 cells/ 450 cells/ 1000 cells/ well well well well well well
well well well well well well 327950HPP 0 2 0 1 0 0 0 1 0 0 0 0
327940CB N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A CD34+;
CD45+ Cells CFU-E/BFU-E CFU-GM CFU-GEMM 50 cells/ 150 cells/ 450
cells/ 50 cells/ 150 cells/ 450 cells/ 50 cells/ 150 cells/ 450
cells/ well well well well well well well well well 327950HPP 1 4 7
4 4 5 0 0 0 327940CB 0 0 I 5 12 0 0 0 0 Duplicate wells CD34-;
CD45- Cells CFU-E/BFU-E CFU-GM CFU-GEMM 1000 cells/ 3000 cells/
9000 cells/ 1000 cells/ 3000 cells/ 9000 cells/ 1000 cells/ 1000
cells/ 9000 cells/ well well well well well well well well well
327950HPP 0 0 0 0 0 0 0 0 0 327940CB 0 0 0 0 1 0 0 0 0 CD34+; CD45-
Cells CFU-E/BFU-E CFU-GM CFU-GEMM 50 cells/ 150 cells/ 450 cells/
1000 cells/ 50 cells/ 150 cells/ 450 cells/ 1000 cells/ 50 cells/
150 cells/ 450 cells/ 1000 cells/ well well well well well well
well well well well well well 327950HPP 0 1 2 1 0 0 1 0 0 0 0 0
327940CB N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A CD34+;
CD45+ Celts CFU-E/BFU-E CFU-GM CFU-GEMM 50 cells/ 150 cells/ 450
cells/ 50 cells/ 150 cells/ 450 cells/ 50 cells/ 150 cells/ 450
cells/ well well well well well well well well well 327950HPP 3 2 8
2 2 14 0 0 0 327940CB 0 0 0 16 14 0 0 0 0 HPP: human placental
perfusate CB: Cord blood CFU-E/BFU-E: colony forming unit,
erythrocyte/blast forming unit erythrocyte CFU-GM: colony forming
unit, granulocyte macrophage CFU-GEMM: colony forming unit,
granulocyte, erythrocyte, monocyte, megakaryocyte
[0482] In another study, CD34, CD45, CD31 and CDH5 gene expression
levels were compared in CD34.sup.+CD45.sup.+ and
CD34.sup.+CD45.sup.+ cell populations isolated from the same
placental perfusate (HPP).
[0483] Materials and Methods:
[0484] CD34 CD45.sup.- and CD34.sup.+CD45.sup.+ cell populations
were obtained as described above with cell purity greater than 90%,
and subjected to RNA preparation using RNAQUEOUS.RTM.-4PCR Kit
(Ambion, Cat # AM1914). In brief, the isolated cells
(5.times.10.sup.5 cells) were lysed in the guanidinium lysis
solution. The sample lysate was then mixed with an ethanol
solution, and applied to a silica-based filter which selectively
and quantitatively binds mRNA and the larger ribosomal RNAs. Very
small RNAs such as tRNA and 5S ribosomal RNA were not
quantitatively bound. The filter was then washed to remove residual
DNA, protein, and other contaminants, and the RNA was eluted in
nuclease-free water containing a trace amount of EDTA to chelate
heavy metals. The silica filter was housed in a small cartridge
which fits into the RNase-free microfuge tubes supplied with the
kit. The sample lysate, wash solutions, and elution solution were
moved through the filter by centrifugation or vacuum pressure.
After elution from the filter the RNA was treated with the
ultra-pure DNase 1 provided with the kit to remove trace amounts of
DNA. Finally, the DNase and divalent cations were removed by a
reagent also provided with the kit. The concentration and purity of
the recovered RNA was determined by measuring its absorbance at 260
and 280 nm. The RNA can then be used for cDNA synthesis using
TAQMAN.RTM. Reverse Transcription Reagents (Applied Biosystems, Cat
# N8080234) followed by real-time PCR analysis by 7900HT Fast
Real-Time PCR System using Taqman Gene Expression Assays of CD34
(Applied Biosystems, Cat # Hs00990732_m1), CD45 (Applied
Biosystems, Cat # Hs00236304_m1), CD31 (Applied Biosystems, Cat #
Hs01065289_m1), and CDH5 (Applied Biosystems, Cat # Hs00174344
ml).
[0485] Results:
[0486] Real-time PCR analysis determined that CD34 expression in
CD34 CD45.sup.- and CD34.sup.+CD45.sup.+ cells was comparable. As
expected, CD45 expression was not detectable in
CD34.sup.+CD45.sup.- cells, but was, however, detectable in
CD34.sup.+CD45.sup.+ cells. CD31 expression in CD34.sup.+CD45.sup.+
cells was 0.05% of that in CD34.sup.+CD45.sup.- cells. CDH5
expression in CD34.sup.+CD45.sup.+ cells was 13.66% of that in
CD34.sup.+CD45.sup.- cells.
6.16 Example 16: Enrichment of CD34.sup.+ Cells from Human Placenta
Perfusate
[0487] This example describes the enrichment of CD34.sup.+ cells
from human placenta using magnetic antibody-coated-bead separation
(MACS).
[0488] A cell suspension from human placental perfusate is obtained
and resuspended in MACS buffer (PBS pH 7.2, +0.5% BSA+2 mmol EDTA)
containing ACD (anticoagulant citrate dextrose). An aliquot is
collected for a first flow sample. 6 mL of Ficoll is added to a
separate 15 mL tube, and the cell suspension is layered over Ficoll
very slowly. The cell suspension in Ficoll is centrifuged at
300.times.g (avg.) for 35 minutes, 20.degree. C., no brake in a
Beckman coulter Allegra X12R Centrifuge with a SC4750 rotor. Upon
completion of centrifugation, the supernatant is aspirated
carefully, and the mononuclear cells settled at the interface are
collected into a separate tube. This material is resuspended to a
total volume of 10 mL with MACS buffer containing ACD. An aliquot
is collected for a second flow sample. Cells are counted and
checked for viability. The cells are then centrifuged at
400.times.g (avg.) in an SC4750 rotor for 15 minutes at 4.degree.
C. When centrifugation is completed, the supernatant is aspirated,
and the cells are resuspended to 100 .mu.L in MACS buffer
containing ACD. STEMSEP.TM. selection cocktail (StemCell
Technologies, Inc., Vancouver, BC Canada) is added at a
concentration of 100 .mu.L/1 mL of cells (at concentration of 1
.mu.L/2.times.10.sup.6 cells). The cells and cocktail are mixed
well and incubated for 10 minutes at 4-8.degree. C. Magnetic
colloid is added at 60 .mu.L/1 mL of cells (at concentration of 1
.mu.L/3.33.times.10.sup.6 cells). The cells and colloid are mixed
well and incubated for 10 minutes at 4-8.degree. C. A 10.times.
volume of refrigerated MACS buffer containing ACD is then added to
the cells, and the resulting solution is centrifuged at 400.times.g
(avg.) in an SC4750 rotor for 8 minutes at room temperature. The
supernatant is aspirated, and the cells are resuspended in 2 mL of
MACS buffer containing ACD. The suspension is optionally filtered
at this point. A third aliquot is collected for a flow sample.
Cells are then analyzed on an AUTOMACS.TM. automated magnetic cell
sorter (Miltenyi Biotec) using program POSSELD2, placing collection
tubes at positions "POS 2" and "NEG1". About 2 mL of positively
selected cells are collected. The cell suspension is then washed
and centrifuged at 400.times.g (SC4750 rotor) for 10 minutes at
room temperature and resuspended to 1 mL in PBS containing 1%
FBS.
6.17 Example 17: In Vitro Colony Forming Unit Assay
[0489] Total nucleated cells are isolated from a unit of cord blood
by Hetastarch separation. Total nucleated placental cells are
obtained from 750 milliliters of placental perfusate by Ficoll
separation. The total nucleated cells from placenta and cord blood
are combined in triplicate in 35 mm culture dishes in Methocult
GF+H4435 medium (Stem Cell Technologies, Vancouver, Canada), or
RPMI 1640 medium supplemented with 2% fetal calf serum and 1%
Stemspan CC 100 cytokine cocktail (Stem Cell Technologies,
Vancouver, Canada). Cells are combined in at least two ratios
(e.g., 2.times.10.sup.5:2.times.10.sup.5;
1.times.10.sup.5:3.times.10.sup.5;
3.times.10.sup.5:1.times.10.sup.5), and are cultured for 14 days.
The morphology of the cells is then examined under phase contrast
microscope, and the total number of colony-forming units (e.g.,
CFU-GM, CFU-L, CFU-M, CFU-G, CFU-DC, CFU-GEMM, CFU-E) are recorded.
A determination is then made as to which ratio produces the highest
number of colony-forming units.
EQUIVALENTS
[0490] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described 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.
[0491] Various publications, patents and patent applications are
cited herein, the disclosures of which are incorporated by
reference in their entireties.
* * * * *