U.S. patent application number 15/013590 was filed with the patent office on 2016-06-02 for methods and compositions for treatment of bone defects with placental cell populations.
This patent application is currently assigned to ANTHROGENESIS CORPORATION. The applicant listed for this patent is ANTHROGENESIS CORPORATION. Invention is credited to Sascha D. Abramson, Marian Guelakis, Mohammad A. Heidaran, Kristen Labazzo, Shmuel Yaccoby.
Application Number | 20160151425 15/013590 |
Document ID | / |
Family ID | 41395029 |
Filed Date | 2016-06-02 |
United States Patent
Application |
20160151425 |
Kind Code |
A1 |
Abramson; Sascha D. ; et
al. |
June 2, 2016 |
METHODS AND COMPOSITIONS FOR TREATMENT OF BONE DEFECTS WITH
PLACENTAL CELL POPULATIONS
Abstract
Provided herein are osteogenic placental adherent cells (OPACs),
methods of using OPACs and OPAC populations, and methods of
culturing, proliferating, expanding, or differentiating the OPACs.
Further provided herein are methods of using the OPACs to formulate
implantable or injectable compositions suitable for administration
to a subject. Still further provided herein are provides methods
for treating bone defects with OPACs and compositions comprising
OPACs. Also provided herein are methods of using OPACs in the
treatment and management of multiple myeloma, e.g., reducing the
progression of, halting the progression of, or improving, one or
more symptoms of multiple myeloma in an individual having multiple
myeloma, comprising administering a plurality of OPACs to the
individual.
Inventors: |
Abramson; Sascha D.;
(Holland Township, NJ) ; Guelakis; Marian;
(Shelton, CT) ; Heidaran; Mohammad A.; (Potomac,
MD) ; Labazzo; Kristen; (Springfield, NJ) ;
Yaccoby; Shmuel; (Little Rock, AR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ANTHROGENESIS CORPORATION |
Warren |
NJ |
US |
|
|
Assignee: |
ANTHROGENESIS CORPORATION
Warren
NJ
|
Family ID: |
41395029 |
Appl. No.: |
15/013590 |
Filed: |
February 2, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14218465 |
Mar 18, 2014 |
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15013590 |
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12546556 |
Aug 24, 2009 |
8728805 |
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14218465 |
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61090897 |
Aug 22, 2008 |
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61090898 |
Aug 22, 2008 |
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Current U.S.
Class: |
424/93.7 ;
435/325 |
Current CPC
Class: |
A61P 7/06 20180101; A61P
13/12 20180101; A61P 35/00 20180101; C12N 5/0605 20130101; A61K
35/50 20130101; A61K 35/12 20130101 |
International
Class: |
A61K 35/50 20060101
A61K035/50; C12N 5/073 20060101 C12N005/073 |
Claims
1. A method of treating an individual having multiple myeloma,
comprising administering to said individual isolated osteogenic
placental adherent cells (OPACs), wherein said OPACs are obtained
from chorion, and are adherent to tissue culture plastic, and
wherein said OPACs are negative for CD200 or are CD200.sup.dim, and
positive for CD105, and wherein said administering detectably
reduces the progression of, halts the progression of, or improves,
one or more symptoms of said multiple myeloma.
2. The method of claim 1, wherein said OPACs are SSEA3.sup.+ or
SSEA4.sup.+.
3. The method of claim 1, wherein said OPACs are SSEA3.sup.+ and
SSEA4.sup.+.
4. The method of claim 1, wherein said OPACs: express one or more
genes at a detectably higher level than an equivalent number of
CD200.sup.+ adherent placental stem cells, wherein said one or more
genes comprise one or more of BMP3 (bone morphogenetic protein 3),
CDH11, COL10A1, COL14A1, COL15A1, DMP1 (dentin matrix acidic
phosphoprotein 1), DSPP (dentin sialophosphoprotein), ENAM
(enamelin), FGFR2 (fibroblast growth factor receptor 2), MMP10
(matrix metalloprotease 10 (stromelysin 2)), TGFB3, and/or TGFBR1,
when said OPACs and said CD200.sup.+ placental stem cells are
cultured in growth medium, as assessed by Ct values from
quantitative real-time PCR; express one or more genes at a
detectably higher level than an equivalent number of CD200.sup.+
adherent placental stem cells, wherein said one or more genes
comprise one or more of AMBN (ameloblastin (enamel matrix
protein)), BMP2 (bone morphogenetic protein 2), CALCR (calcitonin
receptor), CDH11, COL11A1, COL14A1, COL15A1, COL2A1, CSF2, CSF3,
DMP1, DSPP, ENAM, FGF3, GDF10 (growth differentiation factor 10),
IGF1 (insulin-like growth factor 1), ITGA1 (integrin, alpha 1
(CD49)), ITGA2 (integrin, alpha 2 (CD49B)), MMP10, MMP8 (matrix
metalloprotease 8 (neutrophil collagenase)), MMP9, PDGFA
(platelet-derived growth factor A), SMAD1, TGFB3, TGFBR1 and/or
TGFBR2 (transforming growth factor beta, receptor 2) when said
OPACs and said placental stem cells are cultured in osteogenic
medium, as assessed by Ct values from quantitative real-time PCR;
express one or more genes at a detectably higher level than an
equivalent number of adherent CD200.sup.+ placental stem cells that
are not trophoblasts or cytotrophoblasts, wherein said one or more
genes comprise one or more, or all, of CDH11, COL14A1, COL15A1,
DMP1, DSPP, ENAM, MMP10, TGFB3 and/or TGFBR1 regardless of whether
said OPACs and said placental stem cells are cultured in growth
medium or osteogenic medium, as assessed by Ct values from
quantitative real-time PCR; express one or more genes at a
detectably lower level than an equivalent number of adherent
CD200.sup.+ placental stem cells that are not trophoblasts or
cytotrophoblasts, wherein said one or more genes comprise one or
more, or all, of AHSG (alpha-2-HS-glycoprotein), ALPL (alkaline
phosphatase liver/bone/kidney), EGF (epidermal growth factor), FLT1
(fins-related tyrosine kinase 1 (vascular endothelial growth
factor/vascular permeability factor receptor)), IGF2, ITGA2, ITGAM
(integrin, alpha M (complement component 3 receptor 3 subunit)),
SCARB1 (scavenger receptor class B, member 1), SOX9 (SRY (sex
determining region Y)-box 9), TNF, TWIST1 (Twist homolog 1;
formerly blepharophimosis, epicanthus inversus and ptosis 3,
acrocephalosyndactyly 3), VCAM1 (vascular cell adhesion molecule 1)
and/or VDR (vitamin D (1,25-dihydroxyvitamin D3) receptor) when
said OPACs and said placental stem cells are cultured in growth
medium, as assessed by Ct values from quantitative real-time PCR;
express one or more genes at a detectably lower level than an
equivalent number of adherent CD200.sup.+ placental stem cells that
are not trophoblasts or cytotrophoblasts, wherein said one or more
genes comprise one or more, or all, of BGN (biglycan), COL11A1,
COMP (cartilage oligomeric matrix protein), FGF1 and/or VCAM1 when
said OPACs and said placental stem cells are cultured in osteogenic
medium, as assessed by Ct values from quantitative real-time PCR;
express VCAM1 at a detectably lower level than an equivalent number
of adherent CD200.sup.+ placental stem cells that are not
trophoblasts or cytotrophoblasts, regardless of whether said OPACs
and said placental stem cells are cultured in growth medium or
osteogenic medium, as assessed by Ct values from quantitative
real-time PCR; express one or more genes at a detectably higher
level than an equivalent number of bone marrow-derived mesenchymal
stem cells, wherein said one or more genes comprise one or more, or
all, of BMP4, CALCR, CD36, CDH11, COL12A1, COL14A1, COL15A1,
COL3A1, COL5A1, DMP1, DSPP, FLT1, MSX1, PDGFA, TGFB3, TGFBR1 and/or
TUFT1 (Tuftelin 1), when the OPACs and stem cells are cultured in
growth medium, as assessed by Ct values from quantitative real-time
PCR; express one or more genes at a detectably higher level than an
equivalent number of bone marrow-derived mesenchymal stem cells,
wherein said one or more genes comprise one or more, or all, of
AMBN, CALCR, COL14A1, COL15A1, CSF3, DMP1, DSPP, ITGA1, ITGA2,
MMP10, MMP9, MSX1, PDGFA, TGFB1, TGFB3, TGFBR1 and/or TGFBR2, when
the OPACs and stem cells are cultured in osteogenic medium, as
assessed by Ct values from quantitative real-time PCR; express one
or more genes at a detectably higher level than an equivalent
number of bone marrow-derived mesenchymal stem cells, wherein said
one or more genes comprise one or more, or all, of CALCR, COL14A1,
COL15A1, DMP1, DSPP, MSX1, PDGFA, TGFB3 and/or TGFBR1 regardless of
whether said OPACs and said placental stem cells are cultured in
growth medium or osteogenic medium, as assessed by Ct values from
quantitative real-time PCR; express one or more genes at a
detectably lower level than an equivalent number of bone
marrow-derived mesenchymal stem cells, wherein said one or more
genes comprise one or more, or all, of ALPL, BGLAP (bone
gamma-carboxyglutamate (gla) protein), IGF2, ITGA2, ITGAM, SCARB1
and/or SOX1, when the OPACs and mesenchymal stem cells are cultured
in growth medium, as assessed by Ct values from quantitative
real-time PCR; express one or more genes at a detectably lower
level than an equivalent number of bone marrow-derived mesenchymal
stem cells, wherein said one or more genes comprise one or more, or
all, of AHSG, ALPL, BGLAP, BGN, BMP3, BMP5, CD36, COL10A1, COL11A1,
COL12A1, COL2A1, COL4A3, COMP, EGF, FGF1, FGFR2, IGF2, MMP8, PHEX
(phosphate regulating endopeptidase homolog, X-linked), RUNX2
(runt-related transcription factor 2), SCARB1, SOX1, VCAM1 and/or
VEGFB, when the OPACs and stem cells are cultured in osteogenic
medium, as assessed by Ct values from quantitative real-time PCR;
or express one or more genes at a detectably lower level than an
equivalent number of bone marrow-derived mesenchymal stem cells,
wherein said one or more genes comprise one or more, or all, of
ALPL, BGLAP, IGF2, SCARB1 and/or SOX9, regardless of whether said
OPACs and said placental stem cells are cultured in growth medium
or osteogenic medium, as assessed by Ct values from quantitative
real-time PCR.
5. The method of claim 1, wherein said OPACs: express one or more
genes at a detectably higher level than an equivalent number of
bone marrow derived mesenchymal stem cells, wherein said one or
more genes comprise one or more of BMP4, BMP6, CD36, CDH11,
COL14A1, COL15A1, COL1A1, COL3A1, COL5A1, CSF2, CTSK, FGF2, FGFR1,
FLT1, ITGA1, MINPP1, MMP9, MSX1, PDGFA, SERPINH1, TGFB3 and TGFBR1,
wherein said OPACs and said mesenchymal stem cells have undergone
an equivalent number of passages; or express one or more genes at a
detectably higher level than an equivalent number of fibroblast
cells, wherein said one or more genes comprise one or more of BMP4,
BMP6, CDH11, COL14A1, COL15A1, COL1A1, COL3A1, COL5A1, FLT1, IGF1R,
ITGA1, MINPP1, PDGFA, SERPINH1, SMAD3, TGFB1, TGFB2, TGFB3, TGFBR1,
TNF, TUFT1, VCAM1 and VEGFA, and wherein said fibroblast cells have
undergone an equivalent number of passages.
6. The method of claim 1, wherein said OPACs secrete one or more of
the proteins decorin, epiregulin, IGFBP-3, IGFBP-6, IL-2 R alpha,
IL-17RC, IL-27, Latent TGF-beta binding protein 1 (LTBP), NCAM-1,
Smad4, TFPI, TGF-beta R1/ALK5 or TIMP-2.
7. The method of claim 1, wherein said OPACs secrete the proteins
decorin, epiregulin, IGFBP-3, IGFBP-6, IL-2 R alpha, IL-17RC,
IL-27, Latent TGF-beta binding protein 1 (LTBP), NCAM-1, Smad4,
TFPI, TGF-beta R1/ALK5 and TIMP-2.
8. The method of claim 1, wherein said one or more symptoms are
bone pain, osteocytic lesions, anemia, or renal failure.
9. The method of claim 1, comprising administering at least
1.times.10.sup.8 OPACs/kg to said individual.
10. An isolated osteogenic placental adherent cell (OPAC), wherein
said cell is obtained from chorion, and is adherent to tissue
culture plastic, and wherein said cell is negative for CD200 or is
CD200.sup.dim, and positive for CD105.
11. The isolated cell of claim 9 that is SSEA3.sup.+ or
SSEA4.sup.+.
12. The isolated cell of claim 9 that is SSEA3.sup.+ and
SSEA4.sup.+.
13. The isolated cell of claim 9, wherein said cell produces
osteoprotegerin.
14. The isolated cell of claim 9, wherein said cell is additionally
negative for expression of .alpha.-smooth muscle actin, negative
for expression of RANKL, positive for expression of NG2, positive
for expression of osteoprotegerin, or exhibits inducible alkaline
phosphatase activity.
15. The isolated cell of claim 13, wherein said cell is
additionally negative for expression of .alpha.-smooth muscle
actin, negative for expression of RANKL, positive for expression of
NG2, positive for expression of osteoprotegerin, and exhibits
inducible alkaline phosphatase activity.
16. The isolated cell of claim 9, wherein said cell: expresses one
or more genes at a detectably higher level than a CD200.sup.+
adherent placental stem cell, wherein said one or more genes
comprise one or more of BMP6, CDH11, COL10A1, COL14A1, COL15A1,
COL1A1, COL1A2, COL3A1, COL4A3, COL5A1, CSF3, CTSK, IGF1R, MINPP1,
MMP2, MMP9, MSX1, SMAD1, SMAD3, TGFB3, TGFBR1 and VEGFB, wherein
said OPAC and said CD200.sup.+ adherent placental stem cell have
undergone an equivalent number of passages; expresses one or more
genes at a detectably higher level than a bone marrow derived
mesenchymal stem cell, wherein said one or more genes comprise one
or more of BMP4, BMP6, CD36, CDH11, COL14A1, COL15A1, COL1A1,
COL3A1, COL5A1, CSF2, CTSK, FGF2, FGFR1, FLT1, ITGA1, MINPP1, MMP9,
MSX1, PDGFA, SERPINH1, TGFB3 and TGFBR1, wherein said OPAC and said
mesenchymal stem cell have undergone an equivalent number of
passages; and/or expresses one or more genes at a detectably higher
level than a fibroblast cell, wherein said one or more genes
comprise one or more of BMP4, BMP6, CDH11, COL14A1, COL15A1,
COL1A1, COL3A1, COL5A1, FLT1, IGF1R, ITGA1, MINPP1, PDGFA,
SERPINH1, SMAD3, TGFB1, TGFB2, TGFB3, TGFBR1, TNF, TUFT1, VCAM1 and
VEGFA, wherein said fibroblast cell has undergone a number of
passages in culture that is equivalent to the number of passages
said fibroblast cell has undergone.
17. The isolated cell of claim 9, wherein said cell secretes one or
more of the proteins decorin, epiregulin, IGFBP-6, IL-2 R alpha,
IL-17RC, IL-27, Latent TGF-beta binding protein 1 (LTBP), NCAM-1,
Smad4, TFPI, TGF-beta R1/ALK5 or TIMP-2.
18. The isolated cell of claim 16, wherein said cell secretes the
proteins decorin, epiregulin, IGFBP-3, IGFBP-6, IL-2 R alpha,
IL-17RC, IL-27, Latent TGF-beta binding protein 1 (LTBP), NCAM-1,
Smad4, TFPI, TGF-beta R1/ALK5 and TIMP-2.
19. An isolated population of cells comprising the cells of claim
9.
20. The isolated population of cells of claim 9, wherein said
cells: express one or more genes at a detectably higher level than
an equivalent number of CD200.sup.+ adherent placental stem cells,
wherein said one or more genes comprise one or more of BMP3 (bone
morphogenetic protein 3), CDH11, COL10A1, COL14A1, COL15A1, DMP1
(dentin matrix acidic phosphoprotein 1), DSPP (dentin
sialophosphoprotein), ENAM (enamelin), FGFR2 (fibroblast growth
factor receptor 2), MMP10 (matrix metalloprotease 10 (stromelysin
2)), TGFB3, and/or TGFBR1, when said OPACs and said CD200.sup.+
placental stem cells are cultured in growth medium, as assessed by
Ct values from quantitative real-time PCR; express one or more
genes at a detectably higher level than an equivalent number of
CD200.sup.+ adherent placental stem cells, wherein said one or more
genes comprise one or more of AMBN (ameloblastin (enamel matrix
protein)), BMP2 (bone morphogenetic protein 2), CALCR (calcitonin
receptor), CDH11, COL11A1, COL14A1, COL15A1, COL2A1, CSF2, CSF3,
DMP1, DSPP, ENAM, FGF3, GDF10 (growth differentiation factor 10),
IGF1 (insulin-like growth factor 1), ITGA1 (integrin, alpha 1
(CD49)), ITGA2 (integrin, alpha 2 (CD49B)), MMP10, MMP8 (matrix
metalloprotease 8 (neutrophil collagenase)), MMP9, PDGFA
(platelet-derived growth factor A), SMAD1, TGFB3, TGFBR1 and/or
TGFBR2 (transforming growth factor beta, receptor 2) when said
OPACs and said placental stem cells are cultured in osteogenic
medium, as assessed by Ct values from quantitative real-time PCR;
express one or more genes at a detectably higher level than an
equivalent number of adherent CD200.sup.+ placental stem cells that
are not trophoblasts or cytotrophoblasts, wherein said one or more
genes comprise one or more, or all, of CDH11, COL14A1, COL15A1,
DMP1, DSPP, ENAM, MMP10, TGFB3 and/or TGFBR1 regardless of whether
said OPACs and said placental stem cells are cultured in growth
medium or osteogenic medium, as assessed by Ct values from
quantitative real-time PCR; express one or more genes at a
detectably lower level than an equivalent number of adherent
CD200.sup.+ placental stem cells that are not trophoblasts or
cytotrophoblasts, wherein said one or more genes comprise one or
more, or all, of AHSG (alpha-2-HS-glycoprotein), ALPL (alkaline
phosphatase liver/bone/kidney), EGF (epidermal growth factor), FLT1
(fms-related tyrosine kinase 1 (vascular endothelial growth
factor/vascular permeability factor receptor)), IGF2, ITGA2, ITGAM
(integrin, alpha M (complement component 3 receptor 3 subunit)),
SCARB1 (scavenger receptor class B, member 1), SOX9 (SRY (sex
determining region Y)-box 9), TNF, TWIST1 (Twist homolog 1;
formerly blepharophimosis, epicanthus inversus and ptosis 3,
acrocephalosyndactyly 3), VCAM1 (vascular cell adhesion molecule 1)
and/or VDR (vitamin D (1,25-dihydroxyvitamin D3) receptor) when
said OPACs and said placental stem cells are cultured in growth
medium, as assessed by Ct values from quantitative real-time PCR;
express one or more genes at a detectably lower level than an
equivalent number of adherent CD200.sup.+ placental stem cells that
are not trophoblasts or cytotrophoblasts, wherein said one or more
genes comprise one or more, or all, of BGN (biglycan), COL11A1,
COMP (cartilage oligomeric matrix protein), FGF1 and/or VCAM1 when
said OPACs and said placental stem cells are cultured in osteogenic
medium, as assessed by Ct values from quantitative real-time PCR;
express VCAM1 at a detectably lower level than an equivalent number
of adherent CD200.sup.+ placental stem cells that are not
trophoblasts or cytotrophoblasts, regardless of whether said OPACs
and said placental stem cells are cultured in growth medium or
osteogenic medium, as assessed by Ct values from quantitative
real-time PCR; express one or more genes at a detectably higher
level than an equivalent number of bone marrow-derived mesenchymal
stem cells, wherein said one or more genes comprise one or more, or
all, of BMP4, CALCR, CD36, CDH11, COL12A1, COL14A1, COL15A1,
COL3A1, COL5A1, DMP1, DSPP, FLT1, MSX1, PDGFA, TGFB3, TGFBR1 and/or
TUFT1 (Tuftelin 1), when the OPACs and stem cells are cultured in
growth medium, as assessed by Ct values from quantitative real-time
PCR; express one or more genes at a detectably higher level than an
equivalent number of bone marrow-derived mesenchymal stem cells,
wherein said one or more genes comprise one or more, or all, of
AMBN, CALCR, COL14A1, COL15A1, CSF3, DMP1, DSPP, ITGA1, ITGA2,
MMP10, MMP9, MSX1, PDGFA, TGFB1, TGFB3, TGFBR1 and/or TGFBR2, when
the OPACs and stem cells are cultured in osteogenic medium, as
assessed by Ct values from quantitative real-time PCR; express one
or more genes at a detectably higher level than an equivalent
number of bone marrow-derived mesenchymal stem cells, wherein said
one or more genes comprise one or more, or all, of CALCR, COL14A1,
COL15A1, DMP1, DSPP, MSX1, PDGFA, TGFB3 and/or TGFBR1 regardless of
whether said OPACs and said placental stem cells are cultured in
growth medium or osteogenic medium, as assessed by Ct values from
quantitative real-time PCR; express one or more genes at a
detectably lower level than an equivalent number of bone
marrow-derived mesenchymal stem cells, wherein said one or more
genes comprise one or more, or all, of ALPL, BGLAP (bone
gamma-carboxyglutamate (gla) protein), IGF2, ITGA2, ITGAM, SCARB1
and/or SOX1, when the OPACs and mesenchymal stem cells are cultured
in growth medium, as assessed by Ct values from quantitative
real-time PCR; express one or more genes at a detectably lower
level than an equivalent number of bone marrow-derived mesenchymal
stem cells, wherein said one or more genes comprise one or more, or
all, of AHSG, ALPL, BGLAP, BGN, BMP3, BMP5, CD36, COL10A1, COL11A1,
COL12A1, COL2A1, COL4A3, COMP, EGF, FGF1, FGFR2, IGF2, MMP8, PHEX
(phosphate regulating endopeptidase homolog, X-linked), RUNX2
(runt-related transcription factor 2), SCARB1, SOX1, VCAM1 and/or
VEGFB, when the OPACs and stem cells are cultured in osteogenic
medium, as assessed by Ct values from quantitative real-time PCR;
or express one or more genes at a detectably lower level than an
equivalent number of bone marrow-derived mesenchymal stem cells,
wherein said one or more genes comprise one or more, or all, of
ALPL, BGLAP, IGF2, SCARB1 and/or SOX9, regardless of whether said
OPACs and said placental stem cells are cultured in growth medium
or osteogenic medium, as assessed by Ct values from quantitative
real-time PCR.
21. The isolated population of cells of claim 18, wherein cells in
said population of cells express one or more of the proteins
decorin, epiregulin, IGFBP-3, IGFBP-6, IL-2 R alpha, IL-17RC,
IL-27, Latent TGF-beta binding protein 1 (LTBP), NCAM-1, Smad4,
TFPI, TGF-beta R1/ALK5 or TIMP-2.
22. The isolated population of cells of claim 18, wherein cells in
said population of cells express the proteins decorin, epiregulin,
IGFBP-3, IGFBP-6, IL-2 R alpha, IL-17RC, IL-27, Latent TGF-beta
binding protein 1 (LTBP), NCAM-1, Smad4, TFPI, TGF-beta R1/ALK5 and
TIMP-2.
23. The isolated population of cells of claim 18, wherein at least
50% of the cells of said population are the cells of claim 9.
24. The isolated population of cells of claim 18, wherein at least
80% of the cells of said population are the cells of claim 9.
25. The isolated population of cells of claim 18, wherein at least
95% of the cells of said population are the cells of claim 9.
26. The isolated population of cells of claim 18 consisting
essentially of the cells of claim 9.
Description
[0001] This application claims benefit of U.S. Provisional Patent
Application No. 61/090,898, filed Aug. 22, 2008, and U.S.
Provisional Patent Application No. 61/090,897, filed Aug. 22, 2008,
the disclosures of which are hereby incorporated by reference in
their entireties.
1. FIELD
[0002] Provided herein are methods of using isolated populations of
osteogenic adherent placental cells (OPACs), and methods of using
OPACs, e.g., in the treatment of multiple myeloma, and of reducing,
stopping, or reversing bone loss associated with or caused by
multiple myeloma.
2. BACKGROUND
[0003] Multiple myeloma (also known as MM, myeloma, plasma cell
myeloma, or Kahler's disease) is a type of cancer of plasma cells,
which are antibody-producing immune system cells. Symptoms of
multiple myeloma include bone pain, infection, renal failure,
anemia, and bone lesions. The disease is considered incurable, and
only a few treatments, such as lenalidomide (REVLIMID.RTM.) are
available and show promise. As such, a need exists for new
treatments for multiple myeloma.
3. SUMMARY
[0004] Provided herein are isolated osteogenic placental adherent
cells (OPACs), populations of OPACs, and cell populations
comprising OPACs, wherein the OPACs are present in, and isolated
from chorion. In certain embodiments, the OPACs are not isolated
from chorionic skirt (laeve). The OPACs exhibit one or more
characteristics of stem cells or multipotent cells (e.g., exhibit
markers associated with stem cells or multipotent cells, replicate
at least 10-20 times in culture in an undifferentiated state,
differentiate into adult cells representative at least one of the
three germ layers, etc.), and can adhere to a tissue culture
substrate (e.g., tissue culture plastic such as the surface of a
tissue culture dish or multiwell plate). Further provided herein
are methods of using OPACs in the treatment of bone defects, and in
the treatment of bone-related cancers, e.g., multiple myeloma, and
methods of using the OPACs to reduce, stop, or reverse bone loss
associated with or caused by multiple myeloma.
[0005] In one aspect, provided herein is an isolated OPAC, i.e., an
isolated osteogenic placental adherent cell that is adherent to
tissue culture plastic, osteogenic, and isolated from, chorion,
excluding the chorionic skirt (laeve). OPACs are not trophoblasts,
cytotrophoblasts, embryonic stem cells, or embryonic germ cells as
those cells are known an understood in the art.
[0006] In one embodiment, an OPAC is an isolated CD200.sup.- or
CD200.sup.dim cell, e.g., one that is isolated from chorion, but
not from chorionic skirt (laeve). In a specific embodiment, an OPAC
is osteogenic. In a specific embodiment, an OPAC is positive for
secretion of osteoprotegerin (OPG), e.g., as detected by flow
cytometry. Osteoprotegerin is an osteoblast-secreted decoy receptor
that specifically binds to osteoclast differentiation factor (ODF)
and inhibits osteoclast maturation. Thus, OPACs promote bone
formation and reduce osteoclast-mediated bone loss. In another
specific embodiment, an OPAC does not express RANKL (Receptor
Activator of Nuclear Factor .kappa. B Ligand; see, e.g., GenBank
Accession No. AAB86811.1), e.g., as detected by quantitative
RT-PCR. RANKL is a protein that activates osteoclasts, which are
involved in bone resorption. Thus, OPACs do not to promote bone
resorption. In another specific embodiment, an OPAC is CD200.sup.-
or CD200.sup.dim, and CD105.sup.+. In another specific embodiment,
an OPAC is negative for expression of .alpha.-smooth muscle actin
(see, e.g., GenBank Accession No. NP_001604), negative for RANKL,
or positive for expression of NG2 (neural/glial cell 2 chondroitin
sulfate proteoglycan), e.g., as determined by antibody staining. In
certain embodiments, staining for said NG2 in OPACs is diffuse, as
compared to CD200.sup.+ (non-dim) tissue culture plastic-adherent
placental stem cells, in which NG2 staining is focused. In another
specific embodiment, an OPAC is negative for expression of
.alpha.-smooth muscle actin, negative for RANKL, positive for
expression of NG2 and positive for secretion of osteoprotegerin. In
another specific embodiment, an OPAC exhibits inducible alkaline
phosphatase activity, e.g., as determined by a colorimetric assay.
In a more specific embodiment, an OPAC is CD200.sup.- or
CD200.sup.dim, CD105.sup.+, as detected by flow cytometry, and is
negative for expression of .alpha.-smooth muscle actin, negative
for RANKL, positive for expression of NG2, positive for expression
of osteoprotegerin or exhibits inducible alkaline phosphatase
activity. In another more specific embodiment, an OPAC is
CD200.sup.- or CD200.sup.dim, CD105.sup.+, and is negative for
expression of .alpha.-smooth muscle actin, positive for expression
of NG2, positive for expression of osteoprotegerin, and exhibits
inducible alkaline phosphatase activity.
[0007] In another specific embodiment, an OPAC is SSEA3.sup.+ or
SSEA4.sup.+. In a more specific embodiment, an OPAC is SSEA3.sup.+
and SSEA4.sup.+. In another more specific embodiment, an OPAC is
CD200.sup.-, CD105.sup.+, SSEA3.sup.+, and is also negative for
expression of .alpha.-smooth muscle actin, negative for expression
of RANKL, positive for expression of NG2, positive for expression
of osteoprotegerin or exhibits inducible alkaline phosphatase
activity. In another more specific embodiment, an OPAC is
CD200.sup.-, CD105.sup.+, SSEA4.sup.+, and is also negative for
expression of .alpha.-smooth muscle actin, negative for RANKL,
positive for expression of NG2, positive for expression of
osteoprotegerin or exhibits inducible alkaline phosphatase
activity. In a more specific embodiment, an OPAC is CD200.sup.-,
CD105.sup.+, SSEA3.sup.+, SSEA4.sup.+, and is also negative for
expression of .alpha.-smooth muscle actin, negative for RANKL,
positive for expression of NG2, positive for expression of
osteoprotegerin or exhibits inducible alkaline phosphatase
activity.
[0008] In certain embodiments, the OPAC, population of OPACs, or
population of cells comprising the OPACs facilitates formation of a
mineralized matrix in a population of placental cells when said
population is cultured under conditions that allow the formation of
a mineralized matrix.
[0009] Also provided herein are populations of cells comprising
OPACs, wherein the population of cells is CD200.sup.- or
CD200.sup.dim. Thus, in one embodiment, provided herein is an
isolated population of cells comprising OPACs, wherein said
population of cells is not isolated from chorionic skirt (laeve),
and wherein said population of cells is CD200.sup.- or
CD200.sup.dim. In a specific embodiment, the population of cells
consists essentially of OPACs. In a specific embodiment, said
population of cells is osteogenic. In another specific embodiment,
said population of cells is CD200.sup.- and CD105.sup.+ as detected
by flow cytometry. In another specific embodiment, said population
of cells is CD200.sup.dim and CD105.sup.+ as detected by flow
cytometry. In another specific embodiment, said population of cells
is negative for expression of .alpha.-smooth muscle actin, positive
for expression of NG2, or positive for secretion of
osteoprotegerin. In certain embodiments, staining for said NG2 in
OPACs is diffuse, as compared to CD200.sup.+ (non-dim) tissue
culture plastic-adherent placental stem cells, in which NG2
staining is focused. In another embodiment, said population of
cells is negative for expression of RANKL, e.g., as detected by
quantitative RT-PCR. In another embodiment, said population of
cells is negative for expression of .alpha.-smooth muscle actin,
positive for expression of NG2 and positive for secretion of
osteoprotegerin. In another specific embodiment, said population of
cells exhibits inducible alkaline phosphatase activity. In a more
specific embodiment, said population is CD200.sup.-, CD105.sup.+ or
CD200.sup.dim, CD105.sup.+ and is also one or more of negative for
expression of .alpha.-smooth muscle actin, negative for expression
of RANKL, positive for expression of NG2, positive for expression
of osteoprotegerin or exhibits inducible alkaline phosphatase
activity. In a more specific embodiment, said population of cells
is CD200.sup.-, CD105.sup.+ or CD200.sup.dim, CD105.sup.+; is
negative for expression of .alpha.-smooth muscle actin, negative
for RANKL, positive for expression of NG2, positive for expression
of osteoprotegerin; and exhibits inducible alkaline phosphatase
activity.
[0010] In another specific embodiment, said population of cells is
SSEA3.sup.+ or SSEA4.sup.+. In yet another embodiment, said
population cells is SSEA3.sup.+ and SSEA4.sup.+. In yet another
embodiment, said population of cells is CD200.sup.-, CD105.sup.+ or
CD200.sup.dim, CD105.sup.+, SSEA3.sup.+, and also negative for
expression of .alpha.-smooth muscle actin, negative for expression
of RANKL, positive for expression of NG2, positive for expression
of osteoprotegerin or exhibits inducible alkaline phosphatase
activity. In another more specific embodiment, said population of
cells is CD200.sup.- or CD200.sup.dim, is CD105.sup.+ and
SSEA4.sup.+, and is also negative for expression of .alpha.-smooth
muscle actin, negative for expression of RANKL, positive for
expression of NG2, positive for expression of osteoprotegerin or
exhibits inducible alkaline phosphatase activity. In another more
specific embodiment, said population of cells is CD200.sup.- or
CD200.sup.dim; is CD105.sup.+, SSEA3.sup.+, SSEA4.sup.+, negative
for expression of .alpha.-smooth muscle actin, negative for
expression of RANKL, positive for expression of NG2, positive for
expression of osteoprotegerin, and/or exhibits inducible alkaline
phosphatase activity.
[0011] In another specific embodiment, the population of cells
comprising OPACs expresses matrix metallopeptidase 9 (MMP9) at a
detectably higher level in osteogenic medium than an equivalent
number of CD200.sup.+, non-dim, adherent placental stem cells, as
assessed by Ct values from quantitative real-time PCR.
[0012] In other specific embodiments, the population of cells
comprising OPACs expresses one or more genes at a detectably higher
level than an equivalent number of CD200.sup.+, non-dim, adherent
placental stem cells, wherein said one or more genes comprise one
or more, or all, of BMP3 (bone morphogenetic protein 3), CDH11
(cadherin type 11), COL10A1 (collagen type X, alpha 1), COL14A1
(collagen, type XIV, alpha 1), COL15A1 (collagen, type XV, alpha
1), DMP1 (dentin matrix acidic phosphoprotein 1), DSPP (dentin
sialophosphoprotein), ENAM (enamelin), FGFR2 (fibroblast growth
factor receptor 2), MMP10 (matrix metalloprotease 10 (stromelysin
2)), TGFB3 (transforming growth factor, .beta.3), and/or TGFBR1
(transforming growth factor .beta., receptor 1) when an OPAC and
said CD200.sup.+ placental stem cell are cultured in growth medium,
as assessed by Ct values from quantitative real-time PCR.
[0013] In another specific embodiment, the population of cells
comprising OPACs expresses one or more genes at a detectably higher
level than an equivalent number of CD200.sup.+, non-dim, adherent
placental stem cells, wherein said one or more genes comprise one
or more, or all, of AMBN (ameloblastin (enamel matrix protein)),
BMP2 (bone morphogenetic protein 2), CALCR (calcitonin receptor),
CDH11, COL11A1 (collagen, type XI, alpha 1), COL14A1, COL15A1,
COL2A1 (collagen, type II, alpha 1), CSF2 (colony stimulating
factor 2 (granulocyte-macrophage)), CSF3 (colony stimulating factor
3 (granulocyte)), DMP1, DSPP, ENAM, FGF3 (fibroblast growth factor
3), GDF10 (growth differentiation factor 10), IGF1 (insulin-like
growth factor 1), ITGA1 (integrin, alpha 1 (CD49)), ITGA2
(integrin, alpha 2 (CD49B)), MMP8 (matrix metalloprotease 8
(neutrophil collagenase)), MMP9 (matrix metallopeptidase 9
(gelatinase B, 92 kDa gelatinase, 92 kDa type IV collagenase)),
MMP10, PDGFA (platelet-derived growth factor A), SMAD1 (SMAD family
member 1), TGFB3, TGFBR1 and/or TGFBR2 (transforming growth factor
beta, receptor 2) when the OPACs and said placental stem cells are
cultured in osteogenic medium, as assessed by Ct values from
quantitative real-time PCR.
[0014] In another specific embodiment, the population of cells
comprising OPACs expresses one or more genes at a detectably higher
level than an equivalent number of adherent CD200.sup.+, non-dim,
placental stem cells, wherein said one or more genes comprise one
or more, or all, of CDH11, COL14A1, COL15A1, DMP1, DSPP, ENAM,
MMP10, TGFB3 and/or TGFBR1 regardless of whether the OPACs and said
placental stem cells are cultured in growth medium or osteogenic
medium, as assessed by Ct values from quantitative real-time
PCR.
[0015] In another specific embodiment, the population of cells
comprising OPACs expresses one or more genes at a detectably lower
level than an equivalent number of adherent CD200.sup.+, non-dim,
placental stem cells, wherein said one or more genes comprise one
or more, or all, of AHSG (alpha-2-HS-glycoprotein), ALPL (alkaline
phosphatase liver/bone/kidney), EGF (epidermal growth factor), FLT1
(fms-related tyrosine kinase 1 (vascular endothelial growth
factor/vascular permeability factor receptor)), IGF2, ITGA2, ITGAM
(integrin, alpha M (complement component 3 receptor 3 subunit)),
SCARB1 (scavenger receptor class B, member 1), SOX9 (SRY (sex
determining region Y)-box 9), TNF (tumor necrosis factor), TWIST1
(Twist homolog 1; formerly blepharophimosis, epicanthus inversus
and ptosis 3, acrocephalosyndactyly 3), VCAM1 (vascular cell
adhesion molecule 1) and/or VDR (vitamin D (1,25-dihydroxyvitamin
D3) receptor) when said OPACs and said placental stem cells are
cultured in growth medium, as assessed by Ct values from
quantitative real-time PCR
[0016] In another specific embodiment, the population of cells
comprising OPACs expresses one or more genes at a detectably lower
level than an equivalent number of adherent CD200.sup.+, non-dim,
placental stem cells, wherein said one or more genes comprise one
or more, or all, of BGN (biglycan), COL11A1, COMP (cartilage
oligomeric matrix protein), FGF1 and/or VCAM1 when said OPACs and
said placental stem cells are cultured in osteogenic medium, as
assessed by Ct values from quantitative real-time PCR.
[0017] In another specific embodiment, the population of cells
comprising OPACs expresses VCAM1 at a detectably lower level than
an equivalent number of adherent CD200.sup.+, non-dim, placental
stem cells, regardless of whether said OPACs and said placental
stem cells are cultured in growth medium or osteogenic medium, as
assessed by Ct values from quantitative real-time PCR
[0018] In another specific embodiment, the population of cells
comprising OPACs expresses one or more genes at a detectably higher
level than an equivalent number of bone marrow-derived mesenchymal
stem cells, wherein said one or more genes comprise one or more, or
all, of BMP4, CALCR, CD36, CDH11, COL12A1, COL14A1, COL15A1,
COL3A1, COL5A1, DMP1, DSPP, FLT1, MSX1, PDGFA, TGFB3, TGFBR1 and/or
TUFT1 (Tuftelin 1), when the OPACs and mesenchymal stem cells are
cultured in growth medium, as assessed by Ct values from
quantitative real-time PCR;
[0019] In another specific embodiment, the population of cells
comprising OPACs expresses one or more genes at a detectably higher
level than an equivalent number of bone marrow-derived mesenchymal
stem cells, wherein said one or more genes comprise one or more, or
all, of AMBN, CALCR, COL14A1, COL15A1, CSF3, DMP1, DSPP, ITGA1,
ITGA2, MMP10, MMP9, MSX1, PDGFA, TGFB1, TGFB3, TGFBR1 and/or
TGFBR2, when the OPACs and mesenchymal stem cells are cultured in
osteogenic medium, as assessed by Ct values from quantitative
real-time PCR.
[0020] In another specific embodiment, the population of cells
comprising OPACs expresses one or more genes at a detectably higher
level than an equivalent number of bone marrow-derived mesenchymal
stem cells, wherein said one or more genes comprise one or more, or
all, of CALCR, COL14A1, COL15A1, DMP1, DSPP, MSX1, PDGFA, TGFB3
and/or TGFBR1 regardless of whether said OPACs and said mesenchymal
stem cells are cultured in growth medium or osteogenic medium, as
assessed by Ct values from quantitative real-time PCR;
[0021] In another specific embodiment, the population of cells
comprising OPACs expresses one or more genes at a detectably lower
level than an equivalent number of bone marrow-derived mesenchymal
stem cells, wherein said one or more genes comprise one or more, or
all, of ALPL, BGLAP (bone gamma-carboxyglutamate (gla) protein),
IGF2, ITGA2, ITGAM, SCARB1 and/or SOX1, when the OPACs and
mesenchymal stem cells are cultured in growth medium, as assessed
by Ct values from quantitative real-time PCR.
[0022] In another specific embodiment, the population of cells
comprising OPACs expresses one or more genes at a detectably lower
level than an equivalent number of bone marrow-derived mesenchymal
stem cells, wherein said one or more genes comprise one or more, or
all, of AHSG, ALPL, BGLAP, BGN, BMP3, BMP5, CD36, COL10A1, COL11A1,
COL12A1, COL2A1, COL4A3, COMP, EGF, FGF1, FGFR2, IGF2, MMP8, PHEX
(phosphate regulating endopeptidase homolog, X-linked), RUNX2
(runt-related transcription factor 2), SCARB1, SOX1, VCAM1 and/or
VEGFB, when the OPACs and mesenchymal stem cells are cultured in
osteogenic medium, as assessed by Ct values from quantitative
real-time PCR.
[0023] In another specific embodiment, the population of cells
comprising OPACs expresses one or more genes at a detectably lower
level than an equivalent number of bone marrow-derived mesenchymal
stem cells, wherein said one or more genes comprise one or more, or
all, of ALPL, BGLAP, IGF2, SCARB1 and/or SOX9, regardless of
whether said OPACs and said mesenchymal stem cells are cultured in
growth medium or osteogenic medium, as assessed by Ct values from
quantitative real-time PCR. In a more specific embodiment of each
of the above embodiments, OPACs and said CD200.sup.+ placental stem
cells or mesenchymal stem cells have undergone an equivalent number
of passages or cell doublings.
[0024] In another embodiment, provided herein is a population of
cells comprising OPACs, e.g., a population of OPACs, wherein a gene
encoding matrix metallopeptidase 9 (MMP9) is induced in said OPACs
in osteogenic medium, as compared to expression of MMP9 in growth
medium, at least 2, 3, 4 or 5 orders of magnitude greater than said
MMP9 is induced in placental stem cells, e.g., CD34.sup.-,
CD10.sup.+, CD105.sup.+ tissue culture-adherent multipotent
placental cells, in said osteogenic medium, as compared to
expression of MMP9 in said growth medium, e.g., as assessed by Ct
values from quantitative real-time PCR. In another embodiment,
provided herein is a population of cells comprising OPACs, e.g., a
population of OPACs, wherein a gene encoding matrix
metallopeptidase 9 (MMP9) is induced in said OPACs in osteogenic
medium, as compared to expression of MMP9 in growth medium, at
least 2, 3, 4 or 5 orders of magnitude greater than said MMP9 is
induced in bone marrow-derived mesenchymal stem cells (MSCs) in
said osteogenic medium, as compared to expression of MMP9 in said
growth medium, e.g., as assessed by Ct values from quantitative
real-time PCR. In another embodiment, provided herein is a
population of cells comprising OPACs, e.g., a population of OPACs,
wherein a gene encoding matrix metallopeptidase 9 (MMP9) is induced
in said OPACs in osteogenic medium, as compared to expression of
MMP9 in growth medium, at least 2, 3, 4 or 5 orders of magnitude
greater than said MMP9 is induced in fibroblasts in said osteogenic
medium, as compared to expression of MMP9 in said growth medium,
e.g., as assessed by Ct values from quantitative real-time PCR.
[0025] In specific embodiments, at least 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90% or 95% of the cells in the population of cells
comprising OPACs are CD200.sup.- and/or CD200.sup.dim. In other
specific embodiments, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90% or 95% of the OPACs in the population of cells comprising OPACs
are CD200.sup.- and/or CD200.sup.dim.
[0026] Further provided herein is an isolated population of OPACs,
wherein said population is produced by isolating chorionic tissue
from a placenta, wherein said chorionic tissue is not chorionic
skirt (laeve) tissue; digesting the isolated chorionic tissue with
a tissue-disrupting enzyme to obtain a population of chorion cells
comprising OPACs; and isolating said OPACs from said chorion cells.
In a specific embodiment, the tissue-disrupting enzyme is trypsin,
dispase or collagenase. In various embodiments, the OPACs,
contained within a population of cells obtained from digesting
chorionic tissue, are at least about 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, 95%, 99% or at least 99.5% of said population of
chorionic cells.
[0027] The OPACs, and cell populations comprising OPACs provided
herein, include OPACs and OPAC-containing cell populations that
have been cultured, e.g., for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20 or more passages, or for 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20
population doublings. OPAC populations also includes populations
of, e.g., two or more, OPACs in culture, and a population in a
container, e.g., a bag.
[0028] In another aspect, provided herein is a method of producing
osteogenic cells with the ability to mineralize matrix, comprising
culturing a plurality of OPACs provided herein or a population of
isolated OPACs provided herein, under conditions in which said
OPACs differentiate into osteogenic cells, said culturing being for
a time sufficient for said osteogenic cells to produce, or
facilitate the production of, detectable amounts of mineralized
matrix comprising calcium and/or phosphate. In certain embodiments,
the OPACs produce, or facilitate the production of, bone.
[0029] In another aspect, provided herein is a composition, e.g.,
an implantable composition, comprising OPACs. In a specific
embodiment, the implantable composition comprises a matrix. 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, 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. In certain embodiments, the
matrix is a synthetic degradable polymer such as, for example,
polylactic acid or polyglycolic acid. In certain embodiments, the
implantable scaffolding substrate is a .beta.-tricalcium phosphate
substrate, a .beta.-tricalcium phosphate-collagen substrate, a
collagen substrate, a calcium phosphate substrate, a mineralized
human placental collagen substrate, a hyaluronic acid substrate, or
a ceramic substrate. In certain embodiments, the implantable
scaffolding substrate is .beta.-tricalcium phosphate substrate. In
certain embodiments, the implantable scaffolding substrate is a
.beta.-tricalcium phosphate-collagen substrate. In certain
embodiments, the implantable scaffolding substrate is a collagen
substrate. In certain embodiments, the implantable scaffolding
substrate is a calcium phosphate substrate. In certain embodiments,
the implantable scaffolding substrate is a mineralized human
placental collagen substrate.
[0030] In another aspect, provided herein is a method for treating
bone defects in a subject, comprising administering to a subject in
need thereof an implantable or injectable composition comprising a
population of OPACs provided herein, thereby treating the bone
defect in the subject. In certain embodiments, the bone defect is
an osteolytic lesion associated with a cancer, a bone fracture, or
a spine, e.g., in need of fusion. In certain embodiments, the
osteolytic lesion is associated with multiple myeloma, bone cancer,
or metastatic cancer. In certain embodiments, the bone fracture is
a non-union fracture. In certain embodiments, an implantable
composition is surgically implanted, e.g., at the site of the bone
defect In certain embodiments, an injectable composition is
surgically administered to the region of the bone defect. In
certain embodiments, the injectable composition is systemically
administered.
[0031] In another aspect, provided herein is a method of producing
osteogenic cells comprising culturing a plurality of OPACs or a
population of isolated OPACs under conditions in which said OPACs
differentiate into osteogenic cells, said culturing being for a
time sufficient for said OPACs to produce, or facilitate the
production of, detectable amounts of mineralized calcium, bone
tissue, or bone.
[0032] In certain embodiments, provided herein is a method for
formulating an injectable composition, comprising combining a
population of OPACs with injectable hyaluronic acid or collagen, In
certain embodiments, the composition comprises injectable
hyaluronic acid. In certain embodiments, the composition comprises
injectable collagen. Also provided herein are compositions
comprising a population of OPACs and injectable hyaluronic acid or
collagen.
[0033] In another aspect, provided herein are methods of treating
individuals having a bone-related cancer, e.g., multiple myeloma,
bone cancer, breast cancer, lung cancer, neuroblastoma,
osteosarcoma, Ewing's sarcoma, chondrosarcoma, chordoma, malignant
fibrous histiocytoma of bone, fibrosarcoma of bone, metastatic
cancer, multiple myeloma, and any form of metastatic cancer
characterized by bone metastases. In one embodiment, provided
herein is a method of treating an individual having a bone-related
cancer, comprising administering to said individual isolated OPACs,
e.g., an isolated population of cells comprising OPACs, wherein
said OPACs are obtained from chorion, and are adherent to tissue
culture plastic, and wherein said OPACs are negative for CD200 or
are CD200.sup.dim, and positive for CD105, and wherein said
administering detectably reduces the progression of halts the
progression of, or improves, one or more symptoms of said multiple
myeloma. In a specific embodiment, said OPACs are SSEA3.sup.+ or
SSEA4.sup.+. In another specific embodiment, said OPACs are
SSEA3.sup.+ and SSEA4.sup.+. In another specific embodiment, the
OPACs are CD200.sup.-, CD105.sup.+, SSEA3.sup.+, and are also
negative for expression of .alpha.-smooth muscle actin, negative
for expression of RANKL, positive for expression of NG2, positive
for expression of osteoprotegerin or exhibits inducible alkaline
phosphatase activity. In another more specific embodiment, the
OPACs are CD200.sup.- and/or CD200.sup.dim, CD105.sup.+,
SSEA4.sup.+, and are also negative for expression of .alpha.-smooth
muscle actin, negative for RANKL, positive for expression of NG2,
positive for expression of osteoprotegerin or exhibits inducible
alkaline phosphatase activity. In a more specific embodiment, the
OPACs are CD200.sup.- and/or CD200.sup.dim, CD105.sup.+,
SSEA3.sup.+, SSEA4.sup.+, and are also negative for expression of
.alpha.-smooth muscle actin, negative for RANKL, positive for
expression of NG2, positive for expression of osteoprotegerin or
exhibits inducible alkaline phosphatase activity. In other specific
embodiments, the OPACs comprise any of the characteristics, or
combinations of characteristics, recited in Section 5.1, below.
[0034] In another specific embodiment of the method of treatment,
said OPACs express one or more genes at a detectably higher level
than an equivalent number of CD200.sup.+, non-dim, adherent
placental stem cells, wherein said one or more genes comprise one
or more, or all, of BMP3, CDH11, COL10A1, COL14A1, COL15A1, DMP1,
DSPP, ENAM, FGFR2, MMP10, TGFB3, and/or TGFBR1, when said OPACs and
said CD200.sup.+ placental stem cells are cultured in growth
medium, as assessed by Ct values from quantitative real-time
PCR.
[0035] In another specific embodiment of the method of treatment,
said OPACs express one or more genes at a detectably higher level
than an equivalent number of adherent CD200.sup.+, non-dim,
placental stem cells, wherein said one or more genes comprise one
or more, or all, of AMBN, BMP2, CALCR, CDH11, COL11A1, COL14A1,
COL15A1, COL2A1, CSF2, CSF3, DMP1, DSPP, ENAM, FGF3, GDF10, IGF1,
ITGA1, ITGA2, MMP10, MMP8, MMP9, PDGFA, SMAD1, TGFB3, TGFBR1 and/or
TGFBR2 when said OPACs and said placental stem cells are cultured
in osteogenic medium, as assessed by Ct values from quantitative
real-time PCR.
[0036] In another specific embodiment of the method of treatment,
said OPACs express one or more genes at a detectably higher level
than an equivalent number of adherent CD200.sup.+, non-dim,
placental stem cells, wherein said one or more genes comprise one
or more, or all, of CDH11, COL14A1, COL15A1, DMP1, DSPP, ENAM,
MMP10, TGFB3 and/or TGFBR1 regardless of whether said OPACs and
said placental stem cells are cultured in growth medium or
osteogenic medium, as assessed by Ct values from quantitative
real-time PCR.
[0037] In another specific embodiment of the method of treatment,
said OPACs express one or more genes at a detectably lower level
than an equivalent number of adherent CD200.sup.+, non-dim,
placental stem cells, wherein said one or more genes comprise one
or more, or all, of AHSG, ALPL, EGF, FLT1, IGF2, ITGA2, ITGAM,
SCARB1, SOX9, TNF, TWIST1, VCAM1 or VDR when said OPACs and said
placental stem cells are cultured in growth medium, as assessed by
Ct values from quantitative real-time PCR.
[0038] In another specific embodiment of the method of treatment,
said OPACs express one or more genes at a detectably lower level
than an equivalent number of adherent CD200.sup.|, non-dim,
placental stem cells, wherein said one or more genes comprise one
or more, or all, of BGN, COL11A1, COMP, FGF1 and/or VCAM1 when said
OPACs and said placental stem cells are cultured in osteogenic
medium, as assessed by Ct values from quantitative real-time
PCR.
[0039] In another specific embodiment of the method of treatment,
said OPACs express VCAM1 at a detectably lower level than an
equivalent number of adherent CD200.sup.+, non-dim, placental stem
cells, regardless of whether said OPACs and said placental stem
cells are cultured in growth medium or osteogenic medium, as
assessed by Ct values from quantitative real-time PCR.
[0040] In another specific embodiment of the method of treatment,
said OPACs express one or more genes at a detectably higher level
than an equivalent number of bone marrow-derived mesenchymal stem
cells, wherein said one or more genes comprise one or more, or all,
of BMP4, CALCR, CD36, CDH11, COL12A1, COL14A1, COL15A1, COL3A1,
COL5A1, DMP1, DSPP, FLT1, MSX1, PDGFA, TGFB3, TGFBR1 and/or TUFT1,
when the OPACs and mesenchymal stem cells are cultured in growth
medium, as assessed by Ct values from quantitative real-time
PCR.
[0041] In another specific embodiment of the method of treatment,
said OPACs express one or more genes at a detectably higher level
than an equivalent number of bone marrow-derived mesenchymal stem
cells, wherein said one or more genes comprise one or more, or all,
of AMBN, CALCR, COL14A1, COL15A1, CSF3, DMP1, DSPP, ITGA1, ITGA2,
MMP10, MMP9, MSX1, PDGFA, TGFB1, TGFB3, TGFBR1 and/or TGFBR2, when
the OPACs and mesenchymal stem cells are cultured in osteogenic
medium, as assessed by Ct values from quantitative real-time
PCR.
[0042] In another specific embodiment of the method of treatment,
said OPACs express one or more genes at a detectably higher level
than an equivalent number of bone marrow-derived mesenchymal stem
cells, wherein said one or more genes comprise one or more, or all,
of CALCR, COL14A1, COL15A1, DMP1, DSPP, MSX1, PDGFA, TGFB3 and/or
TGFBR1 regardless of whether said OPACs and said mesenchymal stem
cells are cultured in growth medium or osteogenic medium, as
assessed by Ct values from quantitative real-time PCR.
[0043] In another specific embodiment of the method of treatment,
said OPACs express one or more genes at a detectably lower level
than an equivalent number of bone marrow-derived mesenchymal stem
cells, wherein said one or more genes comprise one or more, or all,
of ALPL, BGLAP (bone gamma-carboxyglutamate (gla) protein), IGF2,
ITGA2, ITGAM, SCARB1 and/or SOX1, when the OPACs and mesenchymal
stem cells are cultured in growth medium, as assessed by Ct values
from quantitative real-time PCR.
[0044] In another specific embodiment of the method of treatment,
said OPACs express one or more genes at a detectably lower level
than an equivalent number of bone marrow-derived mesenchymal stem
cells, wherein said one or more genes comprise one or more, or all,
of AHSG, ALPL, BGLAP, BGN, BMP3, BMP5, CD36, COL10A1, COL11A1,
COL12A1, COL2A1, COL4A3, COMP, EGF, FGF1, FGFR2, IGF2, MMP8, PHEX,
RUNX2, SCARB1, SOX1, VCAM1 and/or VEGFB, when the OPACs and
mesenchymal stem cells are cultured in osteogenic medium, as
assessed by Ct values from quantitative real-time PCR.
[0045] In another specific embodiment of the method of treatment,
said OPACs or express one or more genes at a detectably lower level
than an equivalent number of bone marrow-derived mesenchymal stem
cells, wherein said one or more genes comprise one or more, or all,
of ALPL, BGLAP, IGF2, SCARB1 and/or SOX9, regardless of whether
said OPACs and said mesenchymal stem cells are cultured in growth
medium or osteogenic medium, as assessed by Ct values from
quantitative real-time PCR.
[0046] In another specific embodiment of the method of treating
multiple myeloma, said OPACs: express one or more genes at a
detectably higher level than an equivalent number of bone marrow
derived mesenchymal stem cells, wherein said one or more genes
comprise one or more of BMP4, BMP6, CD36, CDH11, COL14A1, COL15A1,
COL1A1, COL3A1, COL5A1, CSF2, CTSK, FGF2, FGFR1, FLT1, ITGA1,
M1NPP1, MMP9, MSX1, PDGFA, SERPINH1, TGFB3 and TGFBR1, wherein said
OPACs and said mesenchymal stem cells have undergone an equivalent
number of passages or cell doublings; or express one or more genes
at a detectably higher level than an equivalent number of
fibroblast cells, wherein said one or more genes comprise one or
more of BMP4, BMP6, CDH11, COL14A1, COL15A1, COL1A1, COL3A1,
COL5A1, FLT1, IGF1R, ITGA1, MINPP1, PDGFA, SERPINH1, SMAD3, TGFB1,
TGFB2, TGFB3, TGFBR1, TNF, TUFT1, VCAM1 and VEGFA, and wherein said
fibroblast cells have undergone an equivalent number of passages or
cell doublings.
[0047] In another specific embodiment of the method of treating an
individual having multiple myeloma, said OPACs secrete one or more
of the proteins decorin, epiregulin, IGFBP-3, IGFBP-6, IL-2 R
alpha, IL-17RC, IL-27, Latent TGF-beta binding protein 1 (LTBP),
NCAM-1, Smad4, TFPI, TGF-beta R1/ALK5 or TIMP-2. In a more specific
embodiment, said OPACs secrete the proteins decorin, epiregulin,
IGFBP-3, IGFBP-6, IL-2 R alpha, IL-17RC, IL-27, Latent TGF-beta
binding protein 1 (LTBP), NCAM-1, Smad4, TFPI, TGF-beta R1/ALK5 and
TIMP-2.
[0048] In another specific embodiment of the method of treatment,
said one or more symptoms of multiple myeloma are bone pain,
osteocytic lesions (e.g., visible by X-ray or magnetic resonance
imaging (MRI)), osteoporosis, anemia, hypercalcemia or a symptom
due to hypercalcemia, or renal failure. In another specific
embodiment, said administering causes a detectable increase in, or
lessening of the reduction of, bone mineral density or bone mineral
content in said individual. In another specific embodiment, said
administering comprises administering at least 1.times.10.sup.8
OPACs/kg to said individual. In other specific embodiments, said
individual has never been treated for multiple myeloma; said
individual has been treated for multiple myeloma and responds to
non-OPAC therapy; said individual has been treated for multiple
myeloma and has not responded to non-OPAC therapy, but the course
of multiple myeloma in said individual has not progressed; or said
individual has progressive multiple myeloma.
[0049] In yet another aspect, provided herein is a method for
treating bone defects in a subject, comprising administering to a
subject in need thereof an implantable or injectable composition
comprising a population of OPACs, thereby treating the bone defect
in the subject. In certain embodiments, the bone defect is (a) an
osteolytic lesion associated with a cancer, (b) a bone fracture,
(c) a spine in need of fusion, (d) a nonunion fracture, or (e)
osteoporosis. In certain embodiments, the osteolytic lesion is
associated with multiple myeloma, bone cancer, or metastatic
cancer. In certain embodiments, the bone fracture is a non-union
fracture. In certain embodiments, an implantable composition
comprising a population of OPACs is administered to the subject. In
certain embodiments, the implantable composition is surgically
implanted. In certain embodiments, an injectable composition
comprising a population of OPACs is administered to the subject. In
certain embodiments, the injectable composition is surgically
administered to the region of the bone defect. In certain
embodiments, the injectable composition is systemically
administered.
[0050] In yet another aspect, provided herein is a method for
treating bone defects in a subject, comprising administering to a
subject in need thereof an implantable or injectable composition
comprising a population of OPACs, wherein said OPACs cause or
facilitate the formation of bone or bone tissue in the subject, and
thereby treating the bone defect in the subject. In certain
embodiments, the bone defect is (a) an osteolytic lesion associated
with a cancer, (b) a bone fracture, (c) a spine in need of fusion,
(d) a nonunion fracture, or (e) osteoporosis. In certain
embodiments, the osteolytic lesion is associated with multiple
myeloma, bone cancer, or metastatic cancer. In certain embodiments,
the bone fracture is a non-union fracture. In certain embodiments,
an implantable composition comprising a population of OPACs is
administered to the subject. In certain embodiments, the
implantable composition is surgically implanted. In certain
embodiments, an injectable composition comprising a population of
OPACs is administered to the subject. In certain embodiments, the
injectable composition is surgically administered to the region of
the bone defect. In certain embodiments, the injectable composition
is systemically administered.
3.1 Definitions
[0051] As used herein, "consisting essentially of," in the context
of a population of cells comprising OPACs, means that the
population of cells is, e.g., at least 95%, at least 96%, at least
97%, at least 98%, at least 95% or at least 95.5% OPACs.
[0052] As used herein, the terms "OPAC" and OPACs" refer to the
cells described in Section 5.1, below.
[0053] As used herein, "CD200.sup.dim", when referring to a cell,
means that the cell displays fluorescence intensity for CD200 in
flow cytometry that is no more than 20-30% above isotype control.
As used herein, a population of cells is CD200.sup.dim if at least
60% of the cells in the population either do not express CD200 or
are CD200.sup.dim, and the cell population, as a whole, in a flow
cytometric assay, displays a CD200 fluorescence intensity that is
no more than 40-60% above isotype control. It is noted that a
CD200.sup.dim population can comprise CD200.sup.+ (non-dim)
cells.
[0054] 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.+.
[0055] 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.+.
[0056] As used herein, the term "isolated OPAC" means an OPAC that
is substantially separated from other, non-OPAC of the tissue,
e.g., chorion, from which the OPACs is derived. An OPAC is
"isolated" if at least about 50%, 60%, 70%, 80%, 90%, 95%, or at
least 99% of the non-OPACs with which the OPACs are naturally
associated are removed from the OPACs, e.g., during collection
and/or culture of the OPACs.
[0057] 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 population of OPACs is "isolated"
if at least about 50%, 60%, 70%, 80%, 90%, 95%, or at least 99% of
the cells with which the population of OPACs, or cells from which
the population of OPACs is derived, is naturally associated are
removed from the population of OPACs, e.g., during collection
and/or culture.
[0058] As used herein, the term "placental stem cell" refers to a
stem cell or progenitor 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. 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, the
ability to differentiate into cells of all 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.
[0059] As used herein, a cell is "positive" for a particular marker
when that marker is detectable. For example, an OPACs is positive
for, e.g., CD105 because CD105 is detectable on placental stem
cells 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. similarly,
a population of cells is positive for a marker when, e.g., at least
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of cells in the
population express the marker.
[0060] As used herein, an "osteogenic cell" is a cell that is
capable of either depositing hydroxyapatite, the main component of
bone, or differentiating into a cell that is capable of depositing
hydroxyapatite. An "osteogenic cell" is specifically contemplated
as encompassing a cell ordinarily referred to as an osteoblast or
an osteocyte. See Section 5.1.6, below, for exemplary conditions
under which an osteogenic placental adherent cell can differentiate
into a cell that can deposit hydroxyapatite.
[0061] As used herein, a "matrix" refers to a three-dimensional
substance that is characterized by pores dispersed throughout the
substance. The pores are suitable, for example, for growth of
cells, e.g., stem cells, OPACs, and/or osteogenic cells, within the
matrix. Exemplary matrices include, but are not limited to, a
.beta.-tricalcium phosphate substrate, a .beta.-tricalcium
phosphate-collagen substrate, a collagen substrate, a calcium
phosphate substrate, a mineralized human placental collagen
substrate, a hyaluronic acid substrate, and a ceramic substrate.
Preferably, the matrix can be mineralized by an osteogenic cell
present in the pores of the matrix.
4. BRIEF DESCRIPTION OF THE FIGURES
[0062] FIG. 1: Gene expression of OPACs obtained by selective
adhesion. X axis: log.sub.10 relative quantification alkaline
phosphatase gene expression. X axis: Experimental conditions.
[0063] FIG. 2: Immunophenotype of OPACs obtained by selective
adhesion with respect to CD34, CD105 and CD200. LN: laminin. VN:
vitronectin. FN: fibronectin. COL: collagen. 10: 10% fetal bovine
serum. 20: 20% fetal bovine serum. Ctrl: culture of OPACs on the
indicated surface coating for 6 days.
[0064] FIG. 3: Immunophenotype of osteogenic markers of OPACs
obtained by selective adhesion. LN: laminin. VN: vitronectin. FN:
fibronectin. COL: collagen. 10: 10% fetal bovine serum. 20: 20%
fetal bovine serum. Ctrl: culture of OPACs on the indicated surface
coating for 6 days. Asterisks: significant difference compared to
CD10.sup.+, CD34.sup.-, CD105.sup.+ placental stem cell control
(horizontal line).
[0065] FIG. 4: Immunophenotype of embryonic stem cells markers of
OPACs obtained by selective adhesion. LN: laminin. VN: vitronectin.
FN: fibronectin. COL: collagen. 10: 10% fetal bovine serum. 20: 20%
fetal bovine serum. Ctrl: culture of OPACs on the indicated surface
coating for 6 days. Upper horizontal line: CD10.sup.+, CD34.sup.-,
CD105.sup.+ placental stem cell expression of SSEA-3; lower
horizontal line: CD10.sup.+, CD34.sup.-, CD105.sup.+ placental stem
cell expression of SSEA-4.
[0066] FIG. 5: Alkaline Phosphatase (AP) activity of OPACs obtained
by selective adhesion.
[0067] FIG. 6: Immunophenotype of culture expanded OPACs.
[0068] FIG. 7: Alkaline phosphatase activity of culture expanded
OPACs. Basal: growth medium. OS: osteogenic medium. m1: medium
1--20% FBS (Hyclone)/.alpha.-MEM comprising 100 units/mL
penicillin, 100 .mu.g/mL streptomycin, 2 mM L-glutamine. m2: medium
2--Mesenchymal Stem Cell Growth Medium (MSCGM; Lonza). m3: medium
3--10% FBS (Mesenchymal Stem Cell Qualified FBS, Stem Cell
Technologies)/.alpha.-MEM comprising 100 Units/mL, 100 .mu.g/mL
streptomycin and 2 mM L-glutamine.
[0069] FIG. 8: Immunophenotype (CD200, CD105) of OPACs after
magnetic activated cell sorting. LN: laminin. VN: vitronectin. FN:
fibronectin. m1: medium 1--20% FBS (Hyclone)/.alpha.-MEM comprising
100 units/mL penicillin, 100 .mu.g/mL streptomycin, 2 mM
L-glutamine. m3: medium 3--10% FBS (Mesenchymal Stem Cell Qualified
FBS, Stem Cell Technologies)/.alpha.-MEM comprising 100 Units/mL,
100 .mu.g/mL streptomycin and 2 mM L-glutamine.
[0070] FIG. 9: Alkaline phosphatase activity of a CD200.sup.+
population and a flow-through fraction (comprising CD200.sup.-
cells) after magnetic activated cell sorting. LN: laminin. VN:
vitronectin. FN: fibronectin. m1: medium 1--20% FBS
(Hyclone)/.alpha.-MEM comprising 100 units/mL penicillin, 100
.mu.g/mL streptomycin, 2 mM L-glutamine. m3: medium 3--10% FBS
(Mesenchymal Stem Cell Qualified FBS, Stem Cell
Technologies)/.alpha.-MEM comprising 100 Units/mL, 100 .mu.g/mL
streptomycin and 2 mM L-glutamine. Basal: AP expression in growth
medium. Induced: AP expression in osteogenic medium.
[0071] FIG. 10: Colony forming unit--alkaline phosphatase (CFU-AP)
activity of populations of chorion derived stem cells after
magnetic activated cell sorting. LN: laminin. VN: vitronectin. FN:
fibronectin. m1: medium 1--20% FBS (Hyclone)/.alpha.-MEM comprising
100 units/mL penicillin, 100 .mu.g/mL streptomycin, 2 mM
L-glutamine. m3: medium 3--10% FBS (Mesenchymal Stem Cell Qualified
FBS, Stem Cell Technologies)/.alpha.-MEM comprising 100 Units/mL,
100 .mu.g/mL streptomycin and 2 mM L-glutamine. CD200.sup.+
condition is substantially zero for AP expression.
[0072] FIG. 11: Total colony formation of fractions of OPACs after
magnetic activated cell sorting. FN: fibronectin. m1: medium 1--20%
FBS (Hyclone)/.alpha.-MEM comprising 100 units/mL penicillin, 100
.mu.g/mL streptomycin, 2 mM L-glutamine. m3: medium 3--10% FBS
(Mesenchymal Stein Cell Qualified FBS, Stem Cell
Technologies)/.alpha.-MEM comprising 100 Units/mL, 100 .mu.g/mL
streptomycin and 2 mM L-glutamine. PDAC: CD10.sup.+, CD34.sup.-,
CD105.sup.+, CD200.sup.+ tissue culture plastic-adherent placental
multipotent cells.
[0073] FIG. 12: Inducible osteoprotegerin secretion by OPACs after
osteogenic stimulation.
[0074] FIG. 13: List of secreted proteins identified in OPACs,
PDACs.TM., MSCs, and fibroblasts using RayBiotech 507 protein
RayBio.RTM. Biotin Label-based Antibody Array. Protein expression
is classified as + (low), ++ (medium), and +++ (high) compared to
an internal positive control. The implication of the protein in
bone formation ( ) or resorption ( ) is indicated.
[0075] FIG. 14: Mean amount of osseous tissue formation by
treatment group; scored as 0-4, with 4 as the largest amount.
[0076] FIG. 15: Bone mineral density of cranial defect site by
treatment group.
[0077] FIG. 16: Distribution of animals with >50% defect closure
as determined by measurement of residual defect from X-ray scans at
the time of sacrifice.
[0078] FIG. 17: Effects of OPACs and MSCs on osteoclastic
differentiation. OC: osteoclast. control: osteoclasts without OPACs
or MSCs.
[0079] FIG. 18: Effect of OPACs and MSCs on the proliferation of
multiple myeloma cells. OB: osteoblasts or osteoblast-like cells
obtained from MSCs or PDACs under osteogenic conditions. MTT OD:
optical density in MTT assay; higher values indicate a higher
degree of multiple myeloma cell survival. Con MM: Multiple myeloma
cells from 27 human multiple myeloma patients.
[0080] FIG. 19: Effect of OPACs on bone mineral density in primary
myelomatous SCID-rab mice. Control: PBS only. Pre-Rx: bone mineral
density (BMD) prior to administration of OPACs. Final: Bone mineral
density after 8-16 weeks post-injection. Changes in the bone
mineral density (BMD) of the implanted bones were determined using
a PIXImus DEXA (GE Medical Systems LUNAR, Madison, Wis.)
[0081] FIG. 20: Effect of OPACs on human immunoglobulin (Mg) levels
in primary myelomatous mice. Ig: Human immunoglobulin in mouse
sera.
[0082] FIG. 21: Effect of OPACs on bone mass in primary myelomatous
SCID-rab mice. BMD: bone mineral density. BMC: bone mineral
content.
5. DETAILED DESCRIPTION
5.1 Osteogenic Placental Adherent Cells (OPACS)
[0083] 5.1.1 Characteristics
[0084] 5.1.1.1 Physical and Morphological Characteristics
[0085] The osteogenic placental adherent cells (OPACs) provided
herein, when cultured in primary cultures or in cell culture,
adhere to the tissue culture substrate, e.g., tissue culture
container surface (e.g., tissue culture plastic). The OPACs in
culture assume a generally fibroblastoid, stellate appearance, with
a number of cytoplasmic processes extending from the central cell
body. The OPACs are, however, morphologically distinguishable from
fibroblasts cultured under the same conditions, as the OPACs
generally exhibit a greater number of such processes than do
fibroblasts. Morphologically, OPACs are also distinguishable from
hematopoietic stem cells, which generally assume a more rounded, or
cobblestone, morphology in culture.
[0086] 5.1.1.2 Cell Surface, Molecular and Genetic Markers
[0087] In one embodiment, provided herein is an isolated OPAC
(osteogenic placental adherent cell), i.e., an isolated cell that
is adherent, osteogenic, and isolated from chorion. In one
embodiment, the OPACs are not isolated from the chorionic skirt
(laeve). OPACs provided herein, and populations of OPACs, express a
plurality of cellular and genetic markers that can be used to
identify and/or isolate the OPACs, or populations of cells that
comprise the OPACs. The OPACs, and cell populations comprising
OPACs provided herein, include OPACs and OPACs-containing cell
populations obtained directly from the chorion, e.g., primary
cultures. OPAC populations also include populations of, e.g., two
or more, OPACs in culture, OPACs in single-cell suspension, and a
population in a container, e.g., a bag. In a specific embodiment, a
population of OPACs is a plurality of OPACs in cell culture. OPACs
are not trophoblasts, cytotrophoblasts, embryonic stem cells, or
embryonic germ cells as those cells are known an understood in the
art.
[0088] Provided herein is an isolated OPAC, where the OPAC is
CD200.sup.- or CD200.sup.dim. In a specific embodiment, an OPAC is
osteogenic. In a specific embodiment, an OPAC is positive for
secretion of osteoprotegerin (OPG; see, e.g., GenBank Accession No.
AAB53709). Osteoprotegerin is an osteoblast-secreted decoy receptor
that specifically binds to osteoclast differentiation factor and
inhibits osteoclast maturation. Thus, OPACs promote bone formation
and reduce osteoclast-mediated bone loss. In another specific
embodiment, an OPAC is negative for expression of RANKL (Receptor
Activator of Nuclear Factor .kappa.B). RANKL is a protein that
activates osteoclasts, which are involved in bone resorption. Thus,
OPACs do not to promote bone resorption. In another specific
embodiment, an OPAC is CD200.sup.- or CD200.sup.dim, and
CD105.sup.+. In another specific embodiment, an OPAC is negative
for expression of .alpha.-smooth muscle actin, e.g., as determined
by immunofluorescence staining. It is noted that other populations
of cells from placenta, e.g., the cells described in U.S. Patent
Application Publication No. 2005/0058631, are positive for
.alpha.-smooth muscle actin. In another specific embodiment, an
OPAC is one or more of negative for expression of .alpha.-smooth
muscle actin, negative for expression of RANKL, or positive for
expression of NG2 (neural/glial cell 2 chondroitin sulfate
proteoglycan). In another specific embodiment, an OPAC is negative
for expression of .alpha.-smooth muscle actin, negative for
expression of RANKL, positive for expression of NG2, and positive
for secretion of osteoprotegerin. In another specific embodiment,
an OPAC exhibits inducible alkaline phosphatase activity. In a more
specific embodiment, an OPAC is CD200.sup.- or CD200.sup.dim, and
CD105.sup.+, and is one or more of negative for expression of
.alpha.-smooth muscle actin, negative for expression of RANKL,
positive for expression of NG2, positive for expression of
osteoprotegerin, or exhibits inducible alkaline phosphatase
activity. In another more specific embodiment, an OPAC is
CD200.sup.- or CD200.sup.dim, and CD105.sup.+, and also negative
for expression of .alpha.-smooth muscle actin, negative for
expression of RANKL, positive for expression of NG2, positive for
expression of osteoprotegerin, and exhibits inducible alkaline
phosphatase activity. The lack of expression, or the low
expression, of CD200 by OPACs distinguishes OPACs from other tissue
culture plastic-adherent placenta-derived multipotent cells, e.g.,
PDACs.TM., e.g., the placental multipotent cells described in
Edinger et al., U.S. Patent Application Publication No.
2007/0275362.
[0089] In another specific embodiment, an OPAC is SSEA3.sup.+ or
SSEA4.sup.+. In a more specific embodiment, an OPAC is SSEA3.sup.+
and SSEA4.sup.+. In another more specific embodiment, an OPAC is
CD200.sup.- or CD200.sup.dim, and CD105.sup.+, SSEA3.sup.+, and
also negative for expression of .alpha.-smooth muscle actin,
negative for expression of RANKL, positive for expression of NG2,
positive for expression of osteoprotegerin, and/or exhibits
inducible alkaline phosphatase activity. In another more specific
embodiment, an OPAC is CD200 or CD200.sup.dim, and CD105.sup.+,
SSEA4.sup.+, and is also negative for expression of .alpha.-smooth
muscle actin, negative for expression of RANKL, positive for
expression of NG2, positive for expression of osteoprotegerin or
exhibits inducible alkaline phosphatase activity. In a more
specific embodiment, an OPAC is CD200.sup.- or CD200.sup.dim,
CD105.sup.+, SSEA3.sup.+, SSEA4.sup.+, and is also negative for
expression of .alpha.-smooth muscle actin, negative for expression
of RANKL, positive for expression of NG2, positive for expression
of osteoprotegerin or exhibits inducible alkaline phosphatase
activity. The expression of SSEA3 and SSEA4 by OPACs serves to
distinguish OPACs from other placenta-derived cells, e.g., tissue
culture plastic adherent placental stem cells described in Hariri,
U.S. Pat. No. 7,468,276.
[0090] In certain embodiments, an OPAC facilitates formation of a
mineralized matrix in a population of placental cells when said
population is cultured under conditions that allow the formation of
a mineralized matrix.
[0091] Also provided herein are populations of cells comprising
OPACs, wherein the population of cells is CD200.sup.- or
CD200.sup.dim. Thus, in one embodiment, provided herein is an
isolated population of cells comprising OPACs, wherein said
population of cells is not isolated from chorionic skirt (laeve),
and wherein said population of cells is CD200.sup.- or
CD200.sup.dim. In a specific embodiment, the population of cells
consists essentially of OPACs. In a specific embodiment, said
population of cells is osteogenic. In another specific embodiment,
said population of cells is CD200.sup.- and CD105.sup.+ as detected
by flow cytometry. In another specific embodiment, said population
of cells is CD200.sup.dim and CD105.sup.+ as detected by flow
cytometry. In another specific embodiment, said population of cells
is negative for expression of .alpha.-smooth muscle actin, negative
for expression of RANKL, positive for expression of NG2, and/or
positive for secretion of osteoprotegerin. In another embodiment,
said population of cells is negative for expression of
.alpha.-smooth muscle actin, negative for expression of RANKL,
positive for expression of NG2, and positive for secretion of
osteoprotegerin. In another specific embodiment, said population of
cells exhibits inducible alkaline phosphatase activity. In a more
specific embodiment, said population is CD200.sup.-, CD105.sup.+ or
CD200.sup.dim, CD105.sup.+ and is also one or more of negative for
expression of .alpha.-smooth muscle actin, negative for expression
of RANKL, positive for expression of NG2, positive for expression
of osteoprotegerin or exhibits inducible alkaline phosphatase
activity. In a more specific embodiment, said population of cells
is CD200.sup.-, CD105.sup.+ or CD200.sup.dim, CD105.sup.+; is
negative for expression of .alpha.-smooth muscle actin, negative
for expression of RANKL, positive for expression of NG2, positive
for expression of osteoprotegerin; and exhibits inducible alkaline
phosphatase activity.
[0092] In another specific embodiment, said population of cells is
SSEA3.sup.+ or SSEA4.sup.+. In yet another embodiment, said
population cells is SSEA3.sup.+ and SSEA4.sup.+. In yet another
embodiment, said population of cells is CD200.sup.- and/or
CD200.sup.dim, CD105.sup.+, CD105.sup.+, SSEA3.sup.+, and also
negative for expression of .alpha.-smooth muscle actin, negative
for expression of RANKL, positive for expression of NG2, positive
for expression of osteoprotegerin or exhibits inducible alkaline
phosphatase activity. In another more specific embodiment, said
population of cells is CD200.sup.- or CD200.sup.dim, is CD105.sup.+
and SSEA4.sup.+, and is also negative for expression of
.alpha.-smooth muscle actin, negative for expression of RANKL,
positive for expression of NG2, positive for expression of
osteoprotegerin or exhibits inducible alkaline phosphatase
activity. In another more specific embodiment, said population of
cells is CD200.sup.- or CD200.sup.dim; is CD105.sup.+, SSEA3.sup.+,
SSEA4.sup.+, negative for expression of .alpha.-smooth muscle
actin, negative for expression of RANKL, positive for expression of
NG2, positive for expression of osteoprotegerin, and/or exhibits
inducible alkaline phosphatase activity.
[0093] In specific embodiments, at least 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90% or 95% of the cells in the population are
CD200.sup.-.
[0094] Further provided herein is an isolated population of OPACs,
wherein said population is produced by isolating chorionic tissue
from a placenta, wherein said chorionic tissue is not chorionic
skirt (laeve) tissue; digesting the isolated chorionic tissue with
a tissue-disrupting enzyme to obtain a population of chorion cells
comprising OPACs; and isolating said OPACs from said chorion cells.
In a specific embodiment, the tissue-disrupting enzyme is trypsin,
dispase or collagenase. In various embodiments, the chorionic stem
cells, contained within a population of cells obtained from
digesting chorionic tissue, are at least about 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, 95%, 99% or at least 99.5% of said
population of chorionic cells.
[0095] Isolated populations of cells comprising OPACs, e.g.,
populations of OPACs, are distinguishable from other cells, e.g.,
CD200.sup.+, non-dim, placenta-derived adherent cells (referred to
herein as PDAC.TM.s), as described, for example, in U.S. Pat. Nos.
7,468,276 and 7,255,879, and in U.S. Patent Publication No.
2007/02753621, the disclosures of which are hereby incorporated by
reference in their entireties, e.g., as shown by gene profiling.
PDACs.TM. are identified, e.g., as CD10.sup.+, CD34.sup.-,
CD105.sup.+, CD200.sup.+, non-dim, cells from placenta or umbilical
cord, which are adherent to tissue culture surfaces, e.g., tissue
culture plastic. PDACs.TM. can be further characterized as being
CD10.sup.+, CD34.sup.-, CD45.sup.-, CD90.sup.+, CD105.sup.+, and
CD200.sup.+ cells from placenta or umbilical cord, which are
adherent to tissue culture surfaces, e.g., tissue culture plastic.
In a specific embodiment, the population of cells consists
essentially of OPACs.
[0096] The OPACs described herein can be distinguished from
PDACs.TM. on the basis of the expression of one or more genes, the
expression of which, or the degree of expression of which, is
specific to OPACs as compared to PDACs.TM.. In one embodiment, for
example, provided herein is a population of cells comprising OPACs,
wherein said population of cells express one or more genes at a
detectably higher level (e.g., at least a twofold higher level)
than an equivalent number of adherent CD200.sup.+, non-dim,
placental stem cells that are not trophoblasts or cytotrophoblasts
(PDACs.TM.), wherein said one or more genes comprise one or more,
or all, of BMP6 (bone morphogenetic protein 6; see, e.g., GenBank
Accession No. NM_001718), CDH11 (cadherin 11, type 2, osteoblast
cadherin; see, e.g., GenBank Accession No. NM_001797), COL10A1
(collagen, type X, alpha 1; see, e.g., GenBank Accession No.
NM_000493), COL14A1 (collagen, type XIV, alpha 1; see, e.g.,
GenBank Accession No. NM_021110), COL15A1 (collagen, type XV, alpha
1; see, e.g., GenBank Accession No. NM_001855), COL1A1 (collagen,
type I, alpha 1; see, e.g., GenBank Accession No. NM_000088),
COL1A2 (collagen, type I, alpha 2; see, e.g., GenBank Accession No.
NM_000089), COL3A1 (collagen, type III, alpha 1; see, e.g., GenBank
Accession No. NM_000090), COL4A3 (collagen, type IV, alpha 3; see,
e.g., GenBank Accession No. NM_000091), COL5A1 (collagen, type V,
alpha 1; see, e.g., GenBank Accession No. NM_000093), CSF3
(colony-stimulating factor 3 (granulocyte); see, e.g., GenBank
Accession No. NM_000759), CTSK (cathepsin K; see, e.g., GenBank
Accession No. NM_000396), IGF1R (insulin-like growth factor 1
receptor; see, e.g., GenBank Accession No. NM_000875), MINPP1
(multiple inositol polyphosphate histidine phosphatase 1; see,
e.g., GenBank Accession No. NM_004897), MMP2 (matrix
metalloprotease 2 (also known as gelatinase A, 72 kDa gelatinase,
72 kDa type IV collagenase); see, e.g., GenBank Accession No.
NM_004530), MMP9 (matrix metallopeptidase 9 (also known as
gelatinase B, 92 kDa gelatinase, 92 kDa type IV collagenase); see,
e.g., GenBank Accession No. NM_004994), MSX1 (msh homeobox 1; see,
e.g., GenBank Accession No. NM_002448), SMAD1 (SMAD family member
1; see, e.g., GenBank Accession No. NM_001003688), SMAD3 (SMAD
family member 3; see, e.g., GenBank Accession No. NM_005902), TGFB3
(transforming growth factor, beta 3; see, e.g., GenBank Accession
No. NM_003239), TGFBR1 (transforming growth factor, beta receptor
1; see, e.g., GenBank Accession No. NM_004612) and VEGFB (vascular
endothelial growth factor B; see, e.g., GenBank Accession No.
XM_001128909), when the OPACs and placental stem cells are grown
under equivalent conditions, e.g., as assessed by Ct values from
quantitative real-time PCR. In a specific embodiment, population of
cells consists essentially of OPACs. In a specific embodiment, the
population of cells consists essentially of OPACs.
[0097] In another embodiment, provided herein is a population of
cells comprising OPACs, wherein said population of cells express
one or more genes at a detectably higher level (e.g., at least a
twofold higher level) than an equivalent number of adherent
CD200.sup.+, non-dim, placental stem cells that are not
trophoblasts or cytotrophoblasts, wherein said one or more genes
comprise one or more, or all, of BMP3 (bone morphogenetic protein
3; see, e.g., GenBank Accession No. NM_001201), CDH11, COL10A1,
COL14A1, COL15A1, DMP1 (dentin matrix acidic phosphoprotein 1; see,
e.g., GenBank Accession No. NG_008988), DSPP (dentin
sialophosphoprotein; see, e.g., GenBank Accession No. NM_014208),
ENAM (enamelin; see, e.g., GenBank Accession No. NM_031889), FGFR2
(fibroblast growth factor receptor 2; see, e.g., GenBank Accession
No. NM_000141), MMP10 (matrix metalloprotease 10 (stromelysin 2);
see, e.g., GenBank Accession No. NM_002425), TGFB3, and/or TGFBR1
when said OPACs and said placental stem cells are cultured in
growth medium, e.g., as assessed by Ct values from quantitative
real-time PCR. In a specific embodiment, said growth medium is
.alpha.MEM/20% Fetal Bovine Serum containing 100 units/mL
penicillin, 100 .mu.g/mL streptomycin and 2 mM L-glutamine. In a
specific embodiment, the population of cells consists essentially
of OPACs.
[0098] In another embodiment, provided herein is a population of
cells comprising OPACs, wherein said population of cells, e.g., the
OPACs, express one or more genes at a detectably higher level
(e.g., at least a twofold higher level) than an equivalent number
of adherent CD200.sup.+, non-dim, placental stem cells that are not
trophoblasts or cytotrophoblasts, wherein said one or more genes
comprise one or more, or all, of AMBN (ameloblastin (enamel matrix
protein); see, e.g., GenBank Accession No. NM_031889), BMP2 (bone
morphogenetic protein 2; see, e.g., GenBank Accession No.
NM_001200), CALCR (calcitonin receptor; see, e.g., GenBank
Accession No. NM_001742), CDH11, COL11A1 (collagen type XI, alpha
1; NM_001854), COL14A1, COL15A1, COL2A1 (collagen type II, alpha 1;
see, e.g., GenBank Accession No. NM_001844), CSF2
(colony-stimulating factor 2; NM_000758), CSF3, DMP1, DSPP, ENAM,
FGF3, GDF10 (growth differentiation factor 10; see, e.g., GenBank
Accession No. NM_004962), IGF1 (insulin-like growth factor 1; see,
e.g., GenBank Accession No. NM_000618), ITGA1 (integrin, alpha 1
(CD49); see, e.g., GenBank Accession No. NM_181501), ITGA2
(integrin, alpha 2 (CD49B); see, e.g., GenBank Accession No.
NM_002203), MMP10, MMP8 (matrix metalloprotease 8 (neutrophil
collagenase); see, e.g., GenBank Accession No. NM_002424), MMP9,
PDGFA (platelet-derived growth factor A; see, e.g., GenBank
Accession No. XM_001126441), SMAD1, TGFB3, TGFBR1 and/or TGFBR2
(transforming growth factor beta, receptor 2; see, e.g., GenBank
Accession No. NM_001024847) when said OPACs and said placental stem
cells are cultured in osteogenic medium, e.g., as assessed by Ct
values from quantitative real-time PCR. In a specific embodiment,
said osteogenic medium is .alpha.MEM/20% Fetal Bovine Serum
containing 100 units/mL penicillin, 100 .mu.g/mL streptomycin, 2 mM
L-glutamine, 50 .mu.g/mL ascorbic acid, and 100 nM dexamethasone.
In a specific embodiment, the population of cells consists
essentially of OPACs.
[0099] In another embodiment, provided herein is a population of
cells comprising OPACs, wherein said population of cells, e.g., the
OPACs, express one or more genes at a detectably higher level
(e.g., at least a twofold higher level) than an equivalent number
of adherent CD200.sup.+, non-dim, placental stem cells that are not
trophoblasts or cytotrophoblasts, wherein said one or more genes
comprise one or more, or all, of CDH11, COL14A1, COL15A1, DMP1,
DSPP, ENAM, MMP10, TGFB3 and/or TGFBR1 regardless of whether said
OPACs and said placental stem cells are cultured in growth medium
or osteogenic medium, e.g., as assessed by Ct values from
quantitative real-time PCR. In a specific embodiment, the
population of cells consists essentially of OPACs.
[0100] In another embodiment, provided herein is a population of
cells comprising OPACs, wherein said population of cells, e.g., the
OPACs, express one or more genes at a detectably lower level (e.g.,
at least a twofold lower level) than an equivalent number of
adherent CD200.sup.+, non-dim, placental stem cells that are not
trophoblasts or cytotrophoblasts, wherein said one or more genes
comprise one or more, or all, of AHSG (alpha-2-HS-glycoprotein;
see, e.g., GenBank Accession No. NM_001622), ALPL (alkaline
phosphatase liver/bone/kidney; see, e.g., GenBank Accession No.
NM_000478), EGF (epidermal growth factor; see, e.g., GenBank
Accession No. NM_001963), FLT1 (fms-related tyrosine kinase 1
(vascular endothelial growth factor/vascular permeability factor
receptor); see, e.g., GenBank Accession No. NM_002019), IGF2,
ITGA2, ITGAM (integrin, alpha M (complement component 3 receptor 3
subunit); see, e.g., GenBank Accession No. NM_000632), SCARB1
(scavenger receptor class B, member 1; see, e.g., GenBank Accession
No. NM_005505), SOX9 (SRY (sex determining region Y)-box 9; see,
e.g., GenBank Accession No. NM_000346), TNF, TWIST1 (Twist homolog
1; formerly blepharophimosis, epicanthus inversus and ptosis 3,
acrocephalosyndactyly 3; see, e.g., GenBank Accession No.
NM_000474), VCAM1 (vascular cell adhesion molecule 1; see, e.g.,
GenBank Accession No. NM_001078) and/or VDR when said OPACs and
said placental stem cells are cultured in growth medium, e.g., as
assessed by Ct values from quantitative real-time PCR. In a
specific embodiment, said growth medium is osteogenic medium is
.alpha.MEM/20% Fetal Bovine Serum comprising 100 units/mL
penicillin, 100 .mu.g/mL streptomycin and 2 mM L-glutamine. In a
specific embodiment, the population of cells consists essentially
of OPACs.
[0101] In another embodiment, provided herein is a population of
cells comprising OPACs, wherein said population of cells, e.g., the
OPACs, express one or more genes at a detectably lower level (e.g.,
at least a twofold lower level) than an equivalent number of
adherent CD200.sup.+, non-dim, placental stem cells that are not
trophoblasts or cytotrophoblasts, wherein said one or more genes
comprise one or more, or all, of BGN, COL11A1, COMP (cartilage
oligomeric matrix protein), FGF1 and/or VCAM1 when said OPACs and
said placental stem cells are cultured in osteogenic medium, e.g.,
as assessed by Ct values from quantitative real-time PCR. In a
specific embodiment, said osteogenic medium is osteogenic medium is
.alpha.MEM/20% Fetal Bovine Serum comprising 100 units/mL
penicillin, 100 .mu.g/mL streptomycin, 2 mM L-glutamine, 50
.mu.g/mL ascorbic acid, and 100 nM dexamethasone. In a specific
embodiment, the population of cells consists essentially of
OPACs.
[0102] In another embodiment, provided herein is a population of
cells comprising OPACs, wherein said population of cells, e.g., the
OPACs, express VCAM1 at a detectably lower level (e.g., at least a
twofold lower level) than an equivalent number of adherent
CD200.sup.+, non-dim, placental stem cells that are not
trophoblasts or cytotrophoblasts, regardless of whether said OPACs
and said placental stem cells are cultured in growth medium or
osteogenic medium, e.g., as assessed by Ct values from quantitative
real-time PCR. In a specific embodiment, the population of cells
consists essentially of OPACs.
[0103] Gene profiling also shows that isolated populations of cells
comprising OPACs, e.g., populations of OPACs, are distinguishable
from mesenchymal stem cells, e.g., bone-marrow derived mesenchymal
stem cells. In one embodiment, for example, provided herein is a
population of cells comprising OPACs, wherein said population of
cells, e.g., the OPACs, express one or more genes at a detectably
higher level (e.g., at least a twofold higher level) than an
equivalent number of bone marrow-derived mesenchymal stem cells,
wherein said one or more genes comprise one or more, or all, of
BMP4, BMP6, CD36, CDH11, COL14A1, COL15A1, COL1A1, COL3A1, COL5A1,
CSF2, CTSK, FGF2, FGFR1, FLT1, ITGA1, MINPP1, MMP9, MSX1, PDGFA,
SERPINH1, TGFB3 and TGFBR1, when the OPACs and stem cells are grown
under equivalent conditions, e.g., as assessed by Ct values from
quantitative real-time PCR. In a specific embodiment, the
population of cells consists essentially of OPACs.
[0104] In another embodiment, provided herein is a population of
cells comprising OPACs, wherein said population of cells, e.g., the
OPACs, express one or more genes at a detectably higher level
(e.g., at least a twofold higher level) than an equivalent number
of bone marrow-derived mesenchymal stem cells, wherein said one or
more genes comprise one or more, or all, of BMP4, CALCR, CD36,
CDH11, COL12A1, COL14A1, COL15A1, COL3A1, COL5A1, DMP1, DSPP, FLT1,
MSX1, PDGFA, TGFB3, TGFBR1 and/or TUFT1, when the OPACs and
mesenchymal stem cells are cultured in growth medium, e.g., as
assessed by Ct values from quantitative real-time PCR. In a
specific embodiment, said growth medium is osteogenic medium is
.alpha.MEM/20% Fetal Bovine Serum comprising 100 units/mL
penicillin, 100 .mu.g/mL streptomycin and 2 mM L-glutamine. In a
specific embodiment, the population of cells consists essentially
of OPACs.
[0105] In another embodiment, provided herein is a population of
cells comprising OPACs, wherein said population of cells, e.g., the
OPACs, express one or more genes at a detectably higher level
(e.g., at least a twofold higher level) than an equivalent number
of bone marrow-derived mesenchymal stem cells, wherein said one or
more genes comprise one or more, or all, of AMBN, CALCR, COL14A1,
COL15A1, CSF3, DMP1, DSPP, ITGA1, ITGA2, MMP10, MMP9, MSX1, PDGFA,
TGFB1, TGFB3, TGFBR1 and/or TGFBR2, when the OPACs and mesenchymal
stem cells are cultured in osteogenic medium, e.g., as assessed by
Ct values from quantitative real-time PCR. In a specific
embodiment, said osteogenic medium is osteogenic medium is
.alpha.MEM/20% Fetal Bovine Serum comprising 100 units/mL
penicillin, 100 .mu.g/mL streptomycin, 2 mM L-glutamine, 50
.mu.g/mL ascorbic acid, and 100 nM dexamethasone. In a specific
embodiment, the population of cells consists essentially of OPACs.
In a specific embodiment, the population of cells consists
essentially of OPACs.
[0106] In another embodiment, provided herein is a population of
cells comprising OPACs, wherein said population of cells, e.g., the
OPACs, express one or more genes at a detectably higher level
(e.g., at least a twofold higher level) than an equivalent number
of bone marrow-derived mesenchymal stem cells, wherein said one or
more genes comprise one or more, or all, of CALCR, COL14A1,
COL15A1, DMP1, DSPP, MSX1, PDGFA, TGFB3 and/or TGFBR1 regardless of
whether said OPACs and said mesenchymal stem cells are cultured in
growth medium or osteogenic medium, e.g., as assessed by Ct values
from quantitative real-time PCR. In another specific embodiment,
the population of cells consists essentially of OPACs.
[0107] In another embodiment, provided herein is a population of
cells comprising OPACs, wherein said population of cells, e.g., the
OPACs, express one or more genes at a detectably lower level (e.g.,
at least a twofold lower level) than an equivalent number of bone
marrow-derived mesenchymal stem cells, wherein said one or more
genes comprise one or more, or all, of ALPL, BGLAP, IGF2, ITGA2,
ITGAM, SCARB1 and/or SOX1, when the OPACs and mesenchymal stem
cells are cultured in growth medium, e.g., as assessed by Ct values
from quantitative real-time PCR. In a specific embodiment, said
growth medium is osteogenic medium is .alpha.MEM/20% Fetal Bovine
Serum comprising 100 units/mL penicillin, 100 .mu.g/mL streptomycin
and 2 mM L-glutamine. In another embodiment, provided herein is a
population of cells comprising OPACs, wherein said population of
cells express one or more genes at a detectably lower level (e.g.,
at least a twofold lower level) than an equivalent number of bone
marrow-derived mesenchymal stem cells, wherein said one or more
genes comprise one or more, or all, of AHSG, ALPL, BGLAP, BGN,
BMP3, BMP5, CD36, COL10A1, COL11A1, COL12A1, COL2A1, COL4A3, COMP,
EGF, FGF1, FGFR2, IGF2, MMP8, PHEX, RUNX2 (runt-related
transcription factor 2; see, e.g., GenBank Accession No.
NM_001015051), SCARB1, SOX1, VCAM1 and/or VEGFB, when the OPACs and
mesenchymal stem cells are cultured in osteogenic medium, e.g., as
assessed by Ct values from quantitative real-time PCR. In a
specific embodiment, said osteogenic medium is osteogenic medium is
.alpha.MEM/20% Fetal Bovine Serum comprising 100 units/mL
penicillin, 100 .mu.g/mL streptomycin, 2 mM L-glutamine, 50
.mu.g/mL ascorbic acid, and 100 nM dexamethasone. In a specific
embodiment, the population of cells consists essentially of
OPACs.
[0108] In another embodiment, provided herein is a population of
cells comprising OPACs, wherein said population of cells, e.g., the
OPACs, express one or more genes at a detectably lower level (e.g.,
at least a twofold lower level) than an equivalent number of bone
marrow-derived mesenchymal stem cells, wherein said one or more
genes comprise one or more, or all, of ALPL, BGLAP, IGF2, SCARB 1
and/or SOX9, regardless of whether said OPACs and said mesenchymal
stem cells are cultured in growth medium or osteogenic medium,
e.g., as assessed by Ct values from quantitative real-time PCR. In
another specific embodiment, the population of cells consists
essentially of OPACs.
[0109] In another embodiment, provided herein is a population of
cells comprising OPACs, wherein said population of cells, e.g., the
OPACs, express one or more genes at a detectably higher level
(e.g., at least a twofold higher level) than an equivalent number
of adherent CD200.sup.+, non-dim, placental stem cells, and an
equivalent number of bone marrow-derived mesenchymal stem cells,
wherein said one or more genes comprise one or more, or all, of
COL14A1, COL14A2, DMP, DSPP, TGFB3 and/or TGFBR1, regardless of
whether said OPACs, placental stem cells, and bone marrow-derived
mesenchymal stem cells are cultured in growth medium or osteogenic
medium, e.g., as assessed by Ct values from quantitative real-time
PCR. In another specific embodiment, the population of cells
consists essentially of OPACs.
[0110] Gene profiling also confirms that isolated populations of
cells comprising OPACs, e.g., populations of OPACs, are
distinguishable from human dermal fibroblast cells. In one
embodiment, for example, provided herein is a population of cells
comprising OPACs, wherein said population of cells express one or
more genes at a detectably higher level (e.g., at least a twofold
higher level) than an equivalent number of dermal fibroblast cells,
wherein said one or more genes comprise one or more, or all, of
BMP4, BMP6, CDH11, COL14A1, COL15A1, COL1A1, COL3A1, COL5A1, FLT1,
IGF1R, ITGA1, MINPP1, PDGFA, SERPINH1, SMAD3, TGFB1, TGFB2, TGFB3,
TGFBR1, TNF, TUFT1, VCAM1 and VEGFA, wherein the expression of
these genes is higher in OPACs than in fibroblast cells, when the
OPACs and fibroblasts are grown under equivalent conditions, e.g.,
as assessed by Ct values from quantitative real-time PCR. In a
specific embodiment, the population of cells consists essentially
of OPACs.
[0111] In another embodiment, provided herein is a population of
cells comprising OPACs, wherein an increase in expression in said
population of cells, e.g., in the OPACs, of one or more genes in
osteogenic medium, as compared to growth medium, is at least
tenfold higher than an increase in said one or more of said genes
in osteogenic medium, as compared to growth medium, in an
equivalent number of adherent CD200.sup.+, non-dim, placental stem
cells, wherein said adherent CD200.sup.+ placental stem cells are
not trophoblasts or cytotrophoblasts, and wherein said one or more
genes comprise one or more, or all, of BMP2, CSF3, ITGA2, MMP9, MMP
10, and/or TGFB2. In a specific embodiment, the population of cells
consists essentially of OPACs.
[0112] In another embodiment, provided herein is a population of
cells comprising OPACs, wherein an increase in expression in said
population of cells, e.g., in the OPACs, of one or more genes in
osteogenic medium, as compared to growth medium, is at least
tenfold lower than an increase in said one or more of said genes in
osteogenic medium, as compared to growth medium, in an equivalent
number of adherent CD200.sup.+, non-dim, placental stem cells,
wherein said adherent CD200.sup.+ non-dim, placental stem cells are
not trophoblasts or cytotrophoblasts, and wherein said one or more
genes comprise one or more, or all, of COL1A1, COL11A1, COL4A3,
COL5A1, COMP (cartilage oligomeric matrix protein; see, e.g.,
GenBank Accession No. NM_000095), CTSK, FGF1 (fibroblast growth
factor 1; see, e.g., GenBank Accession No. NM_000800), and/or
FGFR2. In a specific embodiment, the population of cells consists
essentially of OPACs.
[0113] In another embodiment, provided herein is a population of
cells comprising OPACs, wherein an increase in expression in said
population of cells, e.g., in the OPACs, of one or more genes in
osteogenic medium, as compared to growth medium, is at least
tenfold higher than an increase in said one or more of said genes
in osteogenic medium, as compared to growth medium, in an
equivalent number of bone marrow-derived mesenchymal stem cells,
and wherein said one or more genes comprise one or more, or all, of
CSF3, IGF2, ITGA2, ITGA3, MMP9, MMP10, and/or TGFB2. In a specific
embodiment, the population of cells consists essentially of
OPACs.
[0114] In another embodiment, provided herein is a population of
cells comprising OPACs, wherein an increase in expression in said
population of cells, e.g., in the OPACs, of one or more genes in
osteogenic medium, as compared to growth medium, is at least
tenfold lower than an increase in said one or more of said genes in
osteogenic medium, as compared to growth medium, in an equivalent
number of bone marrow-derived mesenchymal stem cells, and wherein
said one or more genes comprise one or more, or all, of ALPL
(alkaline phosphatase, liver/bone/kidney), CD36, COL10A1, COL11A1,
COL12A1, COL1A1, COL4A3, COMP, CTSK, FGF1, and/or FGFR2. In a
specific embodiment, the population of cells consists essentially
of OPACs.
[0115] In another embodiment, provided herein is a population of
cells comprising OPACs, wherein an increase in expression in said
population of cells, e.g., in the OPACs, of one or more genes in
osteogenic medium, as compared to growth medium, is at least
tenfold higher than an increase in said one or more of said genes
in osteogenic medium, as compared to growth medium, in an
equivalent number of fibroblast cells, and wherein said one or more
genes comprise one or more, or all, of BMP2, CSF2, CSF3, IGF1,
ITGA2, MMP9 and/or MMP10. In a specific embodiment, the population
of cells consists essentially of OPACs.
[0116] In another embodiment, provided herein is a population of
cells comprising OPACs, wherein an increase in expression in said
population of cells, e.g., in the OPACs, of one or more genes in
osteogenic medium, as compared to growth medium, is at least
tenfold lower than an increase in said one or more of said genes in
osteogenic medium, as compared to growth medium, in an equivalent
number of fibroblast cells, and wherein said one or more genes
comprise one or more, or all, of BMP4, COL12A1, COMP, FGF1, and/or
MMP8. In a specific embodiment, the population of cells consists
essentially of OPACs.
[0117] In another embodiment, provided herein is a population of
cells comprising OPACs, e.g., a population of OPACs, wherein a gene
encoding matrix metallopeptidase 9 (MMP9) is induced in said OPACs
in osteogenic medium, as compared to expression of MMP9 in growth
medium, at least 2, 3, 4 or 5 orders of magnitude greater than said
MMP9 is induced in said osteogenic medium, as compared to
expression of MMP9 in said growth medium, e.g., as assessed by Ct
values from quantitative real-time PCR.
[0118] In another embodiment, provided herein is a population of
cells comprising OPACs, e.g., a population of OPACs, wherein said
population expresses one or more genes at least tenfold higher than
an equivalent number of adherent CD200.sup.+, non-dim, placental
stem cells, wherein said one or more genes are CHRD (chordin; see,
e.g., GenBank Accession No. NM_003741), GDF7 (growth
differentiation factor 7; see, e.g., GenBank Accession No.
NM_182828), IGFBP3 (Insulin-like growth factor binding protein 3;
see, e.g., GenBank Accession No. NM_000598), and/or INHA (inhibin
alpha; see, e.g., GenBank Accession No. NM_002191). In another
embodiment, provided herein is a population of cells comprising
OPACs, e.g., a population of OPACs, wherein said population
expresses TGFB2 at at least a tenfold lower level than an
equivalent number of adherent CD200.sup.+ (non-dim) placental stem
cells.
[0119] In other embodiments, provided herein is a population of
cells comprising OPACs, e.g., a population of OPACs, wherein the
OPACs express .alpha.-smooth muscle actin, as detectable by
immunofluorescent staining, and wherein said cells express a
fibronectin-1 gene (FN1) and/or TGF-.beta.2 gene (TGFB2) at
approximately the same level as, or at an increased level compared
to, an equivalent number of bone marrow-derived mesenchymal stem
cells.
[0120] The level of expression of these genes can be used to
confirm the identity of a population of OPACs, to identify a
population of cells as comprising at least a plurality of OPACs, or
the like. The population of OPACs can be clonal, e.g., a population
of OPACs expanded form a single OPACs, or a mixed population of
OPACs, e.g., a population of cells comprising solely OPACs that are
expanded from multiple OPACs, or a population of cells comprising
OPACs and at least one other type of cell.
[0121] The level of expression of these genes can be used to select
populations of OPACs. For example, a population of cells, e.g.,
clonally-expanded cells, can be selected if the expression of one
or more of these genes is significantly higher in a sample from the
population of cells than in an equivalent population of mesenchymal
stem cells. Such selecting can be of a population from a plurality
of placental stem cell or chorionic stem cell populations, from a
plurality of cell populations, the identity of which is not known,
etc.
[0122] OPACs, and populations of cells comprising OPACs, can be
selected on the basis of the level of expression of one or more
such genes as compared to the level of expression in said one or
more genes in a mesenchymal stem cell control. In one embodiment,
the level of expression of said one or more genes in a sample
comprising an equivalent number of mesenchymal stem cells is used
as a control. In another embodiment, the control, for OPACs tested
under certain conditions, is a numeric value representing the level
of expression of said one or more genes in mesenchymal stem cells
under said conditions.
[0123] OPACs, and populations of cells comprising OPACs, can also
be selected on the basis of the expression of one or more secreted
proteins as compared to the level of expression in a control, for
example a placental stem cell, a mesenchymal stem cell or a
fibroblast cell. In one embodiment, OPACs can be distinguished from
placental stem cells, mesenchymal stem cells or fibroblast cells on
the basis of secretion of one or more of decorin, epiregulin,
IGFBP-3, IGFBP-6, IL-2 R alpha, IL-17RC, IL-27, Latent TGF-beta
binding protein 1 (LTBP), NCAM-1, Smad4, TFPI, TGF-beta R1/ALK5 and
TIMP-2, which are unique to OPACs as compared to adherent
CD200.sup.+ (non-dim) placental stem cells, mesenchymal stem cells
or fibroblast cells. In another embodiment, proteins secreted by
OPACs but not by PDACs.TM. include one or more of Tissue Factor,
Follistatin-like 1, IGF-IIR, sFRP-4 and TSG-6.
[0124] The isolated populations of OPACs described above, and
populations of OPACs generally, 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 OPACs.
[0125] 5.1.1.3 Growth in Culture
[0126] The growth of the OPACs, as for any mammalian cell, depends
in part upon the particular medium selected for growth. Under
optimum conditions, OPACs typically double in number in 1-3 days.
During culture, the OPACs 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.
[0127] 5.1.2 Methods of Obtaining OPACs
[0128] 5.1.2.1 Cell Collection Composition
[0129] Further provided herein are methods of collecting and
isolating OPACs. Generally, OPACs are obtained from chorion using a
physiologically-acceptable solution, e.g., a cell collection
composition. A cell collection composition is described in detail
in U.S. Application Publication No. 2007-0190042, the disclosure of
which is incorporated by reference herein in its entirety.
[0130] The cell collection composition can comprise any
physiologically-acceptable solution suitable for the collection
and/or culture of cells, e.g., OPACs, 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.
[0131] The cell collection composition can comprise one or more
components that tend to preserve OPACs, that is, prevent the OPACs
from dying, or delay the death of the OPACs, reduce the number of
OPACs 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.).
[0132] The cell collection composition can comprise one or more
tissue-degrading enzymes, e.g., a metalloprotease, a serine
protease, a neutral protease, an RNase, or a DNase, or the like.
Such enzymes include, but are not limited to, collagenases
collagenase I, II, III or IV, a collagenase from Clostridium
histolyticum, etc.); dispase, thermolysin, elastase, trypsin,
LIBERASE, hyaluronidase, and the like.
[0133] The cell collection composition can comprise a
bacteriocidally or bacteriostatically effective amount of an
antibiotic. In certain non-limiting embodiments, the antibiotic is
a macrolide (e.g., tobramycin), a cephalosporin (e.g., cephalexin,
cephradine, cefuroxime, cefprozil, cefaclor, cefixime or
cefadroxil), a clarithromycin, an erythromycin, a penicillin (e.g.,
penicillin V) or a quinolone (e.g., ofloxacin, ciprofloxacin or
norfloxacin), a tetracycline, a streptomycin, etc. In a particular
embodiment, the antibiotic is active against Gram(+) and/or Gram(-)
bacteria, e.g., Pseudomonas aeruginosa, Staphylococcus aureus, and
the like.
[0134] The cell collection composition can also comprise one or
more of the following compounds: adenosine (about 1 mM to about 50
mM); D-glucose (about 20 mM to about 100 mM); magnesium ions (about
1 mM to about 50 mM); a macromolecule of molecular weight greater
than 20,000 daltons, in one embodiment, present in an amount
sufficient to maintain endothelial integrity and cellular viability
(e.g., a synthetic or naturally occurring colloid, a polysaccharide
such as dextran or a polyethylene glycol present at about 25 g/l to
about 100 g/l, or about 40 g/l to about 60 g/l); an antioxidant
(e.g., butylated hydroxyanisole, butylated hydroxytoluene,
glutathione, vitamin C or vitamin E present at about 25 .mu.M to
about 100 .mu.M); a reducing agent (e.g., N-acetylcysteine present
at about 0.1 mM to about 5 mM); an agent that prevents calcium
entry into cells (e.g., verapamil present at about 2 .mu.M to about
25 .mu.M); nitroglycerin (e.g., about 0.05 g/L to about 0.2 g/L);
an anticoagulant, in one embodiment, present in an amount
sufficient to help prevent clotting of residual blood (e.g.,
heparin or hirudin present at a concentration of about 1000 units/l
to about 100,000 units/l); or an amiloride containing compound
(e.g., amiloride, ethyl isopropyl amiloride, hexamethylene
amiloride, dimethyl amiloride or isobutyl amiloride present at
about 1.0 .mu.M to about 5 .mu.M).
[0135] 5.1.2.2 Collection and Handling of Placenta
[0136] 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 chorion or OPACs isolated therefrom. For
example, OPACs can be used, in light of the medical history, for
personalized medicine for the infant associated with the placenta
from which the chorion is obtained, or for parents, siblings or
other relatives of the infant.
[0137] In certain embodiments, prior to recovery of OPACs, the
umbilical cord blood and placental blood are removed from the
placenta from which the chorion is to be removed. In certain
embodiments, after delivery, the cord blood in the placenta is
recovered. The placenta can be subjected to a conventional cord
blood recovery process. Typically a needle or cannula is used, with
the aid of gravity, to exsanguinate the placenta (see, e.g.,
Anderson, U.S. Pat. No. 5,372,581; Hessel et al., U.S. Pat. No.
5,415,665). The needle or cannula is usually placed in the
umbilical vein and the placenta can be gently massaged to aid in
draining cord blood from the placenta. Such cord blood recovery may
be performed commercially, e.g., LifeBank USA, Cedar Knolls, N.J.,
ViaCord, Cord Blood Registry and Cryocell. Preferably, the placenta
is gravity drained without further manipulation so as to minimize
tissue disruption during cord blood recovery.
[0138] 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 cells. The placenta is preferably
transported in a sterile, thermally insulated transport device
(maintaining the temperature of the placenta between 20-28.degree.
C.), for example, by placing the placenta, with clamped proximal
umbilical cord, in a sterile zip-lock plastic bag, which is then
placed in an insulated container. In another embodiment, the
placenta is transported in a cord blood collection kit
substantially as described in pending U.S. patent application Ser.
No. 11/230,760, filed Sep. 19, 2005. Preferably, the placenta is
delivered to the laboratory four to twenty-four hours following
delivery. In certain embodiments, the proximal umbilical cord is
clamped, preferably within 4-5 cm (centimeter) of the insertion
into the placental disc prior to cord blood recovery. In other
embodiments, the proximal umbilical cord is clamped after cord
blood recovery but prior to further processing of the placenta.
[0139] The placenta, prior to collection or OPACs, can be stored
under sterile conditions and at either room temperature or at a
temperature of 5 to 25.degree. C. (centigrade). The placenta may be
stored for a period of longer than forty eight hours, and
preferably for a period of four to twenty-four hours prior to
perfusing the placenta to remove any residual cord blood. The
placenta is preferably stored in an anticoagulant solution at a
temperature of 5 to 25.degree. C. (centigrade). Suitable
anticoagulant solutions are well known in the art. For example, a
solution of heparin or warfarin sodium can be used. In a preferred
embodiment, the anticoagulant solution comprises a solution of
heparin (e.g., 1% w/w in 1:1000 solution). The exsanguinated
placenta is preferably stored for no more than 36 hours before
OPACs are collected.
[0140] 5.1.2.3 Physical Disruption and Enzymatic Digestion of
Chorion Tissue
[0141] In one embodiment, OPACs are collected from a mammalian
placenta by physical disruption, e.g., enzymatic digestion, of the
organ. For example, the chorion, or a portion thereof, may be,
e.g., crushed, sheared, minced, diced, chopped, macerated or the
like, while in contact with the cell collection composition
provided herein, and the resulting tissue subsequently digested
with one or more enzymes. The chorion, 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
cell collection composition. 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 about 60%,
70%, 80%, 90%, 95%, 98%, or 99% of the cells in said organ viable,
as determined by, e.g., trypan blue exclusion. Typically, OPACs can
be obtained by disruption of a small block of chorion, 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.
[0142] A preferred cell collection composition comprises one or
more tissue-disruptive enzyme(s). Enzymatic digestion of chorion
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 other embodiments,
enzymatic digestion of chorionic 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 chorionic
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 (tissue and cells
resulting from enzymatic digestion) is preferably diluted so as to
avoid trapping OPACs within the viscous digest.
[0143] 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 OPACs.
For example, in one embodiment, chorionic tissue is digested first
with an appropriate amount of collagenase I at 2 mg/ml for 30
minutes, followed by digestion with trypsin, 0.25%, for 10 minutes,
at 37.degree. C. Serine proteases are preferably used consecutively
following use of other enzymes.
[0144] In a specific embodiment, OPACs are obtained by separation
of amnion from chorionic tissue; mincing the chorionic tissue,
e.g., into pieces approximately 1 mm.sup.3; digesting the tissue in
dispase II, e.g., at about 1, 2, 3, 4 or 5 U/mL, e.g., 2.4 U/mL for
a sufficient time, e g., about 1 hour; digesting with collagenase
II at about 100, 200, 300, 400 or 500 U/mL, e.g., about 270 U/ml
for a sufficient time, e.g., about 1 hour, followed by enzyme
neutralization, collection of single cells, and culture of the
chorionic cells in a suitable medium, e.g., 20% FBS/.alpha.MEM.
[0145] 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 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 cell collection composition.
[0146] It will be appreciated that where an entire chorion, or
portion of a chorion comprising both fetal and maternal cells, the
OPACs collected can comprise a mix of OPACs derived from both fetal
and maternal sources. Where a portion of the chorion that comprises
no, or a negligible number of, maternal cells (for example,
amnion), the OPACs collected will comprise almost exclusively fetal
placental cells.
[0147] In one embodiment, OPACs are isolated from chorionic tissue
as follows. Chorionic tissue is obtained and minced, e.g., into
pieces approximately 1-2 mm.sup.3. The minced tissue is digested
with dispase II at a concentration of, e.g., about 1 U/mL to about
10 U/mL, e.g., 2.4 U/mL until digestion is complete, for example,
for 1 hour at 37.degree. C. The digested tissue is then digested
with collagenase II, e.g., about 100 U/mL to about 1000 U/mL, e.g.,
about 27 U/mL, until digestion is complete, e.g, for about 1 hour
at 37.degree. C. Cells are collected by centrifugation, washed, and
cultured on vitronectin-coated or laminin-coated tissue culture
vessels and cultured in 10% FBS/DMEM or 20% FBS/.alpha.MEM for
approximately 6 days. At 6 days' culture, non-adherent cells are
removed, and adherent cells are allowed to proliferate. When the
cells achieve about 80% to 90% confluence, the cells are removed,
e.g., using trypsin, and transferred to vitronectin-coated or
laminin-coated tissue culture vessels. Where cells are initially
cultured on laminin-coated vessels, transfer to laminin-coated
culture vessels is preferred; similarly, where cells are initially
cultured on vitronectin-coated vessels, transfer to
vitronectin-coated culture vessels is preferred Cells are
optionally cryopreserved before transfer to second plates. Cells
are analyzed, e.g., by flow cytometry for and are determined to be,
e.g. CD34.sup.- , CD105.sup.+, CD200.sup.- and/or CD200.sup.dim,
positive for alkaline phosphatase (AP), .alpha.-smooth muscle actin
(.alpha.-SMA) and osteoprotegerin (OP).
[0148] Thus, in one aspect, provided herein is a method of
isolating osteogenic placental adherent cells (OPACs), comprising
digesting chorion tissue serially with dispase II, then with
collagenase II to produce a cell population; culturing said cell
population on a first vitronectin-coated surface or laminin-coated
surface for about six days, wherein said cell population comprises
adherent cells and non-adherent cells; and removing non-adherent
cells; and transferring the adherent cells to a second
vitronectin-coated surface or laminin-coated surface, wherein said
adherent cells are OPACs. In specific embodiments, said OPACs are
one or more of CD34.sup.-, CD105.sup.+, CD200.sup.dim, AP.sup.+,
.alpha.-SMA.sup.+ and OP.sup.+. In other embodiments, the OPACs
have any of the cellular or genetic characteristics described
elsewhere herein.
[0149] 5.1.3 Culture of OPACs
[0150] 5.1.3.1 Culture Media
[0151] Isolated OPACs, or populations of OPACs, can be used to
initiate, or seed, cell cultures. Cells are generally transferred
to sterile tissue culture vessels. The vessels are preferably
coated with vitronectin, fibronectin, or both. In certain other
embodiments, the tissue culture vessels are either uncoated or
coated with extracellular matrix or ligands such as laminin,
collagen (e.g., native or denatured), gelatin, ornithine, and/or
extracellular membrane protein (e.g., MATRIGEL (BD Discovery
Labware, Bedford, Mass.)).
[0152] Preferably, OPACs are obtained as follows. Chorionic cells
comprising OPACs, obtained by digesting chorionic tissue, e.g.,
with dispase II and collagenase II as described above, are
initially cultured on a tissue culture surface coated with
collagen, vitronectin, or laminin, e.g., for about 1-6 days in 20%
FBS/.alpha.MEM, followed by, or accompanied by, removal of
non-adherent cells; non-adherent cells may be removed several
times, e.g., after 3 hours, 1 day and 6 days of culture, or once,
e.g., after 6 days of culture. Following selective adhesion, OPACs
are selected by removal of CD200.sup.+ (non-dim) cells, e.g., using
an antibody to CD200, leaving a population of
CD200.sup.dim/CD200.sup.- cells.
[0153] OPACs 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. OPACs
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.
[0154] Other media that can be used to culture OPACs 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/F 12, RPMI 1640, advanced DMEM (Gibco),
DMEM/MCDB201 (Sigma), and CELL-GRO FREE.
[0155] The culture medium can be supplemented with one or more
components including, for example, serum (e.g., fetal bovine serum
(FBS), preferably about 2-15% (v/v); equine (horse) serum (ES);
human serum (HS)); beta-mercaptoethanol (BME), preferably about
0.001% (v/v); one or more growth factors, for example,
platelet-derived growth factor (PDGF), epidermal growth factor
(EGF), basic fibroblast growth factor (bFGF), insulin-like growth
factor-1 (IGF-1), leukemia inhibitory factor (LIF), vascular
endothelial growth factor (VEGF), and erythropoietin (EPO); amino
acids, including L-valine; and one or more antibiotic and/or
antimycotic agents to control microbial contamination, such as, for
example, penicillin G, streptomycin sulfate, amphotericin B,
gentamicin, and nystatin, either alone or in combination.
[0156] OPACs can be cultured in standard tissue culture conditions,
e.g., in tissue culture dishes or multiwell plates. OPACs can also
be cultured using a hanging drop method. In this method, OPACs 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 cells are
cultured.
[0157] 5.1.3.2 Expansion and Proliferation of OPACs
[0158] Once an isolated OPAC, or isolated population of OPACs
(e.g., an OPAC or population of OPACs separated from at least about
50% of the chorionic cells with which an OPAC or population of
OPACs is normally associated in vivo) is obtained, the OPAC or
population of OPACs can be proliferated and expanded in vitro. For
example, a population of OPACs can be cultured in tissue culture
containers, e.g., dishes, flasks, multiwell plates, or the like,
for a sufficient time for the cells to proliferate to 70-90%
confluence, that is, until the cells and their progeny occupy
70-90% of the culturing surface area of the tissue culture
container.
[0159] OPACs 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. OPACs preferably are grown under low
oxidative stress (e.g., with addition of glutathione, ascorbic
acid, catalase, tocopherol, N-acetylcysteine, or the like).
[0160] 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
cells, preferably about 50,000 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 cells were
removed. Provided herein are populations of placental 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.
[0161] 5.1.3.3 OPAC Populations
[0162] Further provided herein are populations of OPACs. OPACs can
be isolated directly from chorions from one or more placentas.
Isolated OPACs provided herein can also be cultured and expanded to
produce populations of OPACs. Populations of chorionic cells
comprising OPACs can also be cultured and expanded to produce
populations of OPACs.
[0163] OPACs populations provided herein comprise OPACs, for
example, the OPACs as described herein. In various embodiments, at
least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or
99% of the cells in an isolated cell population are OPACs. That is,
a population of OPACs can comprise, e.g., as much as 1%, 5%, 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% non-OPAC cells.
[0164] Provided herein are methods of producing isolated
populations of OPACs by, e.g., selecting cells from chorion that
express particular markers and/or particular culture or
morphological characteristics. In one embodiment, for example,
provided herein is a method of producing a cell population
comprising selecting chorionic cells that adhere to a substrate,
and express CD105 but do not express CD200; and isolating said
cells from other chorionic cells to form a cell population, e.g., a
population of OPACs. In a specific embodiment CD105.sup.+,
CD200.sup.- chorionic cells that are also NG2.sup.+,
osteoprotegerin.sup.+, alpha smooth muscle actin negative, and/or
exhibit inducible alkaline phosphatase activity, are isolated from
other chorionic cells.
[0165] In the above embodiments, the substrate can be any surface
on which culture and/or selection of cells, e.g., OPACs, 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, vitronectin, collagen
or fibronectin.
[0166] OPACs, and populations of OPACs, can be selected by any
means known in the art of cell selection. For example, cells can be
selected using an antibody or antibodies to one or more cell
surface markers, for example, in flow cytometry or FACS. Selection
can be accomplished using antibodies in conjunction with magnetic
beads. Antibodies that are specific for certain stem cell-related
markers are known in the art. For example, CD200 (Abcam), or CD105
(Abcam; BioDesign International, Saco, Me.), etc. can be used to
select OPACs or populations of OPACs.
[0167] Populations of OPACs can comprise chorionic cells that are
not OPACs, or cells that are not chorionic cells or OPACs.
[0168] Isolated OPAC populations can be combined with one or more
populations of non-OPAC cells or non-chorionic cells. For example,
an isolated population of OPACs 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 OPAC
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 OPAC
population can be combined with a plurality of cells of a plurality
of cell types, as well.
[0169] In one embodiment, an isolated population of OPACs is
combined with a plurality of hematopoietic stem cells. Such
hematopoietic stem cells can be, for example, contained within
unprocessed placental blood, 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.
[0170] 5.1.4 Combinations of OPACs and Placental Perfusate or
Placental Perfusate Cells
[0171] Provided herein are combinations of placental perfusate with
isolated placental perfusate cells and/or the OPACs provided
herein. In one embodiment, for example, provided herein is a volume
of placental perfusate supplemented with a plurality of placental
perfusate cells and/or a plurality of OPACs. In specific
embodiments, for example, each milliliter of placental perfusate is
supplemented with about 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 more placental perfusate cells or OPACs. In
another embodiment, a plurality of placental perfusate cells is
supplemented with placental perfusate and/or OPACs. In another
embodiment, a plurality of OPACs is supplemented with placental
perfusate and/or a plurality of placental perfusate cells. In
certain embodiments, when perfusate is used for supplementation,
the volume of perfusate is about, greater than about, or less than
about, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 8%, 6%, 4%, 2%
or 1% of the total volume of cells (in solution) plus perfusate.
When placental perfusate cells are used to supplement a plurality
of OPACs, the placental perfusate cells generally comprise about,
greater than about, or fewer than about, 50%, 45%, 40%, 35%, 30%,
25%, 20%, 15%, 10%, 8%, 6%, 4%, 2% or 1% of the total number of
placental perfusate cells plus OPACs. Similarly, when OPACs are
used to supplement a plurality of placental perfusate cells, the
OPACs generally comprise about, greater than about, or fewer than
about, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 8%, 6%, 4%, 2%
or 1% of the total number of placental perfusate cells plus OPACs.
When OPACs or placental perfusate cells are used to supplement
placental perfusate, the volume of solution (e.g., saline solution,
culture medium or the like) in which the cells are suspended
comprises about, greater than about, or less than about, 50%, 45%,
40%, 35%, 30%, 25%, 20%, 15%, 10%, 8%, 6%, 4%, 2% or 1% of the
total volume of perfusate plus cells, where the OPACs are suspended
to about 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 or more cells per milliliter prior to
supplementation.
[0172] Further provided herein is pooled placental perfusate that
is obtained from two or more sources, e.g., two or more placentas,
and combined, e.g., pooled. Such pooled perfusate can comprise
approximately equal volumes of perfusate from each source, or can
comprise different volumes from each source. The relative volumes
from each source can be randomly selected, or can be based upon,
e.g., a concentration or amount of one or more cellular factors,
e.g., cytokines, growth factors, hormones, or the like; the number
of placental cells in perfusate from each source; or other
characteristics of the perfusate from each source. Perfusate from
multiple perfusions of the same placenta can similarly be
pooled.
[0173] Similarly, provided herein are placental perfusate cells,
and OPACs, that are obtained from two or more sources, e.g., two or
more placentas and/or chorions, and pooled. Such pooled cells can
comprise approximately equal numbers of cells from the two or more
sources, or different numbers of cells from one or more of the
pooled sources. The relative numbers of cells from each source can
be selected based on, e.g., the number of one or more specific cell
types in the cells to be pooled, e.g., the number of CD34.sup.-
stem cells, etc.
[0174] Pools can comprise, e.g., placental perfusate supplemented
with placental perfusate cells; placental perfusate supplemented
with OPACs; placental perfusate supplemented with both placental
perfusate cells and OPACs; placental perfusate cells supplemented
with placental perfusate; placental perfusate cells supplemented
with OPACs; placental perfusate cells supplemented with both
placental perfusate and OPACs; OPACs supplemented with placental
perfusate; OPACs supplemented with placental perfusate cells; or
OPACs supplemented with both placental perfusate cells and
placental perfusate.
[0175] In certain embodiments, placental perfusate, placental
perfusate cells, and OPACs are provided as pharmaceutical grade
administrable units. Such units can be provided in discrete
volumes, e.g., 100 mL, 150 mL, 200 mL, 250 mL, 300 mL, 350 mL, 400
mL, 450 mL, 500 mL, or the like. Such units can be provided so as
to contain a specified number of, e.g., placental perfusate cells,
placental perfusate-derived intermediate natural killer cells, or
both, e.g., 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 or more cells per milliliter, or 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, 1.times.10.sup.11 or more cells per unit. Such
units can be provided to contain specified numbers of any two, or
all three, of placental perfusate, placental perfusate cells,
and/or OPACs.
[0176] In the above combinations of placental perfusate, placental
perfusate cells and/or OPACs, any one, any two, or all three of the
placental perfusate, placental perfusate cells and/or OPACs can be
autologous to a recipient (that is, obtained from the recipient),
or homologous to a recipient (that is, obtained from at last one
other individual from said recipient).
[0177] Also provided herein are compositions comprising OPACs in
combination with placental perfusate cells and/or placental
perfusate. Thus, in another aspect, provided herein is a
composition comprising isolated OPACs, wherein said placental stem
are isolated from placental perfusate, and wherein said OPACs
comprise at least 50% of cells in the composition. In a specific
embodiment, said OPACs comprise at least 80% of cells in the
composition. In another specific embodiment, the composition
comprises isolated placental perfusate. In a more specific
embodiment, said placental perfusate is from the same individual as
said OPACs. In another more specific embodiment, said placental
perfusate comprises placental perfusate from a different individual
than said OPACs. In another specific embodiment, the composition
comprises placental perfusate cells. In a more specific embodiment,
said placental perfusate cells are from the same individual as said
OPACs. In another more specific embodiment, said placental
perfusate cells are from a different individual than said OPACs. In
another specific embodiment, the composition additionally comprises
isolated placental perfusate and isolated placental perfusate
cells, wherein said isolated perfusate and said isolated placental
perfusate cells are from different individuals. In another more
specific embodiment of any of the above embodiments comprising
placental perfusate, said placental perfusate comprises placental
perfusate from at least two individuals. In another more specific
embodiment of any of the above embodiments comprising placental
perfusate cells, said isolated placental perfusate cells are from
at least two individuals.
[0178] 5.1.5 Production of an OPAC Cell Bank
[0179] OPACs from postpartum chorion can be cultured in a number of
different ways to produce a set of lots, e.g., a set of
individually-administrable doses. Sets of lots of OPACs, obtained
from a plurality of chorions, can be arranged in a bank of OPACs
for, e.g., long-term storage. Generally, OPACs are obtained from an
initial culture of chorionic 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 chorionic tissue of a single placenta,
but can be derived from the tissue of a plurality of placentas.
[0180] In one embodiment, OPACs lots are obtained as follows.
Chorionic tissue is first disrupted, e.g., by mincing, digested
with a suitable enzyme, e.g., dispase or dispase and collagenase
(see Section 5.2.3, above). The chorionic tissue preferably
comprises, e.g., the entire chorion from a single placenta, but can
comprise only a part of the 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.
[0181] 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 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 chorion(s) from which the OPACs are obtained.
[0182] 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.
[0183] 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.
[0184] 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
an OPAC-containing composition.
[0185] 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 OPACs 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.
[0186] 5.1.6 Differentiation of OPACs
[0187] OPACs can be induced to differentiate, e.g., down an
osteogenic pathway. Osteogenic differentiation of OPACs can be
induced, for example, by placing OPACs in cell culture conditions
that induce differentiation into osteogenic cells. A preferred
osteocytic medium comprises MSCGM (Cambrex) or DMEM supplemented
with 15% cord blood serum, followed by Osteogenic Induction Medium
(Cambrex) containing 0.1 .mu.M dexamethasone, 0.05 mM ascorbic
acid-2-phosphate, 10 mM beta glycerophosphate. In another
embodiment, OPACs are cultured in medium (e.g., DMEM-low glucose)
containing about 10.sup.-7 to about 10.sup.-9 M dexamethasone,
about 10-50 .mu.M ascorbate phosphate salt (e.g.,
ascorbate-2-phosphate) and about 10 nM to about 10 mM
.beta.-glycerophosphate. Osteogenic medium can also include serum,
one or more antibiotic/antimycotic agents, transforming growth
factor-beta (e.g., TGF-.beta.1) and/or bone morphogenic protein
(e.g., BMP-2, BMP-4, or a combination thereof).
[0188] Differentiation can be assayed using a calcium-specific
stain, e.g., von Kossa staining, and RT/PCR detection of, e.g.,
alkaline phosphatase, osteocalcin, bone sialoprotein and/or
osteopontin gene expression.
[0189] 5.1.7 Preservation of OPACs
[0190] OPACs 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.
[0191] OPACs can be preserved using, e.g., a composition comprising
an apoptosis inhibitor, necrosis inhibitor and/or an
oxygen-carrying perfluorocarbon, as described in related U.S.
Provisional Application No. 60/754,969, entitled "Improved Medium
for Collecting Placental Stem Cells and Preserving Organs," filed
on Dec. 25, 2005. In one embodiment, provided herein is a method of
preserving a population of OPACs comprising contacting said
population of OPACs with a cell collection composition comprising
an inhibitor of apoptosis and an oxygen-carrying perfluorocarbon,
wherein said inhibitor of apoptosis is present in an amount and for
a time sufficient to reduce or prevent apoptosis in the population
of OPACs, as compared to a population of OPACs 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 OPACs. In another
embodiment, said cell collection composition comprises said
inhibitor of apoptosis and said oxygen-carrying perfluorocarbon in
separate phases. In another embodiment, said cell collection
composition comprises said inhibitor of apoptosis and said
oxygen-carrying perfluorocarbon in an emulsion. In another
embodiment, the cell collection composition additionally comprises
an emulsifier, e.g., lecithin. In another embodiment, said
apoptosis inhibitor and said perfluorocarbon are between about
0.degree. C. and about 25.degree. C. at the time of contacting the
OPACs. 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 OPACs. In another more
specific embodiment, said contacting is performed during transport
of said population of OPACs. In another more specific embodiment,
said contacting is performed during freezing and thawing of said
population of OPACs.
[0192] In another embodiment, provided herein is a method of
preserving a population of OPACs comprising contacting said
population of OPACs 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 OPACs, as compared to a population
of OPACs not contacted with the inhibitor of apoptosis. In a
specific embodiment, the organ-preserving compound is UW solution
(described in U.S. Pat. No. 4,798,824; also known as ViaSpan; see
also Southard et al., Transplantation 49(2):251-257 (1990)) or a
solution described in 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 cell collection composition additionally
comprises an oxygen-carrying perfluorocarbon, either in two phases
or as an emulsion.
[0193] In another embodiment of the method, OPACs are contacted
with a cell collection composition comprising an apoptosis
inhibitor and oxygen-carrying perfluorocarbon, organ-preserving
compound, or combination thereof, during perfusion. In another
embodiment, said OPACs are contacted during a process of tissue
disruption, e.g., enzymatic digestion. In another embodiment, OPACs
are contacted with said cell collection compound after collection
by perfusion, or after collection by tissue disruption, e.g.,
enzymatic digestion.
[0194] Typically, during 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, OPACs are 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 OPACs are exposed to said hypoxic
condition for less than two hours during said preservation. In
another more specific embodiment, OPACs are exposed to said hypoxic
condition for less than one hour, or less than thirty minutes, or
are not exposed to a hypoxic condition, during collection,
enrichment or isolation. In another specific embodiment, said OPACs
are not exposed to shear stress during collection, enrichment or
isolation.
[0195] The OPACs provided herein can be cryopreserved, e.g., in
cryopreservation medium in small containers, e.g., ampoules.
Suitable cryopreservation medium includes, but is not limited to,
culture medium including, e.g., growth medium, or cell freezing
medium, for example commercially available cell freezing medium,
e.g., C2695, C2639 or C6039 (Sigma). Cryopreservation medium
preferably comprises DMSO (dimethylsulfoxide), at a concentration
of, e.g., about 10% (v/v). Cryopreservation medium may comprise
additional agents, for example, methylcellulose and/or glycerol.
OPACs 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.
[0196] 5.1.8 Compositions Comprising OPACs
[0197] Provided herein are compositions comprising OPACs, or
biomolecules therefrom. The OPACs provided herein can be combined
with any physiologically-acceptable or medically-acceptable
compound, composition or device for use in, e.g., research or
therapeutics.
[0198] 5.1.8.1 Cryopreserved OPACs
[0199] The populations of OPACs provided herein can be preserved,
for example, cryopreserved for later use. Methods for
cryopreservation of cells, such as OPACs, are well known in the
art. OPACs populations can be prepared in a form that is easily
administrable to an individual. For example, provided herein is a
OPACs 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
OPACs 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 cell population.
[0200] The cryopreserved OPACs population can comprise OPACs
derived from a single donor, or from multiple donors. The
population of OPACs can be completely HLA-matched to an intended
recipient, or partially or completely HLA-mismatched.
[0201] Thus, in one embodiment, provided herein is a composition
comprising a population of OPACs in a container. In a specific
embodiment, the population is cryopreserved. In another specific
embodiment, the container is a bag, flask, or jar. In more specific
embodiment, said bag is a sterile plastic bag. In a more specific
embodiment, said bag is suitable for, allows or facilitates
intravenous administration of an OPAC population. The bag can
comprise multiple lumens or compartments that are interconnected to
allow mixing of the OPACs 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 cell population. In another
specific embodiment, said population of OPACs 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 population
of OPACs comprises placental cells that are HLA-matched to a
recipient of said population. In another specific embodiment, said
population of OPACs comprises cells that are at least partially
HLA-mismatched to a recipient of said population. In another
specific embodiment, said OPACs are derived from a plurality of
donors.
[0202] 5.1.8.2 Pharmaceutical Compositions
[0203] Populations of OPACs, or populations of cells comprising
OPACs, can be formulated into pharmaceutical compositions for use
in vivo. Such pharmaceutical compositions comprise a population of
OPACs, or a population of cells comprising OPACs, in a
pharmaceutically-acceptable carrier, e.g., a saline solution or
other accepted physiologically-acceptable solution for in vivo
administration. Pharmaceutical compositions provided herein can
comprise any of the OPAC populations described elsewhere herein.
The pharmaceutical compositions can comprise fetal, maternal, or
both fetal and maternal OPACs. The pharmaceutical compositions
provided herein can further comprise OPACs obtained from a single
individual or chorion, or from a plurality of individuals or
chorion.
[0204] The pharmaceutical compositions provided herein can comprise
any therapeutically useful number of OPACs. For example, a single
unit dose of OPACs 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 OPACs.
[0205] The pharmaceutical compositions provided herein can comprise
populations of cells that comprise 50% viable cells or more (that
is, at least about 50% of the cells in the population are
functional or living). Preferably, at least about 60% of the cells
in the population are viable. More preferably, at least about 70%,
80%, 90%, 95%, or 99% of the cells in the population in the
pharmaceutical composition are viable.
[0206] 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.
[0207] 5.1.8.3 OPAC Conditioned Media
[0208] The OPACs provided herein can be used to produce conditioned
medium, that is, medium comprising one or more biomolecules
secreted or excreted by the cells. In various embodiments, the
conditioned medium comprises medium in which OPACs 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 OPACs have grown to at least about 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 OPACs, or stem cells of another kind. In another
embodiment, the conditioned medium comprises medium in which OPACs
have been differentiated into a terminally differentiated cell
type, or a cell having one ore more characteristics of a terminally
differentiated cell. In another embodiment, the conditioned medium
provided herein comprises medium in which OPACs and non-OPACs have
been cultured.
[0209] 5.1.8.4 Matrices Comprising OPACs
[0210] Further provided herein are matrices, hydrogels, scaffolds,
and the like that comprise an OPAC, or a population of OPACs. In
certain embodiments, the matrix can be any substrate known to one
skilled in the art to be useful for treating bone defects. For
example, the matrix can be .beta.-tricalcium phosphate substrate, a
.beta.-tricalcium phosphate-collagen substrate, a collagen
substrate, a calcium phosphate substrate, a mineralized collagen
substrate, and a hyaluronic acid substrate. In some embodiments,
the collagen in the matrix can be placental collagen. Methods and
compositions for isolating and preparing placental collagen are
extensively described, for example, in U.S. Patent Application
Publication No. 2007/0020225, the disclosure of which is
incorporated by reference herein in its entirety.
[0211] OPACs can be seeded onto the matrix for treating bone prior
to or after a differentiation step. For example, OPACs can be
cultured in, e.g., osteogenic medium for, e.g., about 1-20 days,
then seeded onto the matrix. Alternately, OPACs can be isolated and
seeded onto the matrix, then cultured in osteogenic medium as
described herein for, e.g., about 1-20 days. In another embodiment,
OPACs are cultured in, e.g., osteogenic medium for, e.g., about
1-20 days, then seeded onto the matrix, then cultured in osteogenic
medium as described herein for, e.g., about 1-20 days.
[0212] OPACs 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 OPACs can be seeded are
described in Hariri, U.S. Application Publication No.
2004/0048796.
[0213] OPACs as provided herein can be suspended in a hydrogel
solution suitable for, e.g., injection. Suitable hydrogels for such
compositions include self-assembling peptides, such as RAD16. In
one embodiment, a hydrogel solution comprising the cells can be
allowed to harden, for instance in a mold, to form a matrix having
cells dispersed therein for implantation. OPACs in such a matrix
can also be cultured so that the cells are mitotically expanded
prior to implantation. The hydrogel is, e.g., an organic polymer
(natural or synthetic) that is cross-linked via covalent, ionic, or
hydrogen bonds to create a three-dimensional open-lattice structure
that entraps water molecules to form a gel. Hydrogel-forming
materials include polysaccharides such as alginate and salts
thereof, peptides, polyphosphazines, and polyacrylates, which are
crosslinked ionically, or block polymers such as polyethylene
oxide-polypropylene glycol block copolymers which are crosslinked
by temperature or pH, respectively. In some embodiments, the
hydrogel or matrix biodegradable.
[0214] 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).
[0215] 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.
[0216] The OPACs or populations thereof can be seeded onto a
three-dimensional framework or scaffold and implanted in vivo. Such
a framework can be implanted in combination with any one or more
growth factors, cells, drugs or other components that stimulate
tissue formation or otherwise enhance or improve repair of
tissue.
[0217] Examples of scaffolds that can be used include nonwoven
mats, porous foams, or self assembling peptides. Nonwoven mats can
be formed using fibers comprised of a synthetic absorbable
copolymer of glycolic and lactic acids (e.g., PGA/PLA) (VICRYL,
Ethicon, Inc., Somerville, N.J.). Foams, composed of, e.g.,
poly(.epsilon.-caprolactone)/poly(glycolic acid) (PCL/PGA)
copolymer, formed by processes such as freeze-drying, or
lyophilization (see, e.g., U.S. Pat. No. 6,355,699), can also be
used as scaffolds.
[0218] OPACs provided herein can also be seeded onto, or contacted
with, a physiologically-acceptable ceramic material including, but
not limited to, mono-, di-, tri-, alpha-tri-, beta-tri-, and
tetra-calcium phosphate, hydroxyapatite, fluoroapatites, calcium
sulfates, calcium fluorides, calcium oxides, calcium carbonates,
magnesium calcium phosphates, biologically active glasses such as
BIOGLASS.RTM., and mixtures thereof. Porous biocompatible ceramic
materials currently commercially available include SURGIBONE.RTM.
(CanMedica Corp., Canada), ENDOBON.RTM. (Merck Biomaterial France,
France), CEROS.RTM. (Mathys, A G, 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.
[0219] In another embodiment, OPACs 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.
[0220] The OPACs provided herein can, in another embodiment, be
seeded onto foam scaffolds that may be composite structures. Such
foam scaffolds can be molded into a useful shape, such as that of a
portion of a specific structure in the body to be repaired,
replaced or augmented. In some embodiments, the framework is
treated, e.g., with 0.1M acetic acid followed by incubation in
polylysine, PBS, and/or collagen, prior to inoculation of the OPACs
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.
[0221] 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 OPACs. The scaffold can further
comprise agents that stimulate bone growth and/or inhibit bone
resorption. For example, the scaffold can comprise bone morphogenic
proteins, e.g., BMP-2 and/or BMP-7, WNT inhibitors, and the
like.
[0222] 5.1.9 Immortalized OPAC Cell Lines
[0223] OPACs 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.
[0224] External regulation of the growth-promoting protein can be
achieved by placing the growth-promoting gene under the control of
an externally-regulatable promoter, e.g., a promoter the activity
of which can be controlled by, for example, modifying the
temperature of the transfected cells or the composition of the
medium in contact with the cells. In one embodiment, a tetracycline
(tet)-controlled gene expression system can be employed (see Gossen
et al., Proc. Natl. Acad. Sci. USA 89:5547-5551, 1992; Hoshimaru et
al., Proc. Natl. Acad. Sci. USA 93:1518-1523, 1996). In the absence
of tet, a tet-controlled transactivator (tTA) within this vector
strongly activates transcription from ph.sub.CMV*.sub.-1, a minimal
promoter from human cytomegalovirus fused to tet operator
sequences. tTA is a fusion protein of the repressor (tetR) of the
transposon-10-derived tet resistance operon of Escherichia coli and
the acidic domain of VP16 of herpes simplex virus. Low, non-toxic
concentrations of tet (e.g., 0.01-1.0 .mu.g/mL) almost completely
abolish transactivation by tTA.
[0225] 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 as described herein. 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.
[0226] Transfection can be achieved by any of a variety of means
known to those of ordinary skill in the art including, but not
limited to, retroviral infection. In general, a cell culture may be
transfected by incubation with a mixture of conditioned medium
collected from the producer cell line for the vector and DMEM/F12
containing N2 supplements. For example, a placental cell culture
prepared as described above may be infected after, e.g., five days
in vitro by incubation for about 20 hours in one volume of
conditioned medium and two volumes of DMEM/F12 containing N2
supplements. Transfected cells carrying a selectable marker may
then be selected as described above.
[0227] Following transfection, cultures are passaged onto a surface
that permits proliferation, e.g., allows at least about 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.m/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.
[0228] The conditionally-immortalized OPAC 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.
[0229] Clonal cell lines can be isolated from a
conditionally-immortalized human OPAC 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.
[0230] Conditionally-immortalized human OPAC 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.
[0231] 5.1.10 Assays
[0232] The OPACs provided 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 OPACs proliferation, expansion, and/or differentiation,
compared to OPACs not exposed to such conditions.
[0233] In a preferred embodiment, the OPACs provided herein are
assayed for changes in proliferation, expansion or differentiation
upon contact with a molecule. For example, osteogenic
differentiation can be assayed by monitoring alkaline phosphatase
activity and/or calcium mineralization.
[0234] In one embodiment, for example, provided herein is a method
of identifying a compound that modulates the proliferation of a
plurality of OPACs, comprising contacting said plurality of OPACs
with said compound under conditions that allow proliferation,
wherein if said compound causes a detectable change in
proliferation of said plurality of OPACs compared to a plurality of
OPACs not contacted with said compound, said compound is identified
as a compound that modulates proliferation of OPACs. 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.
[0235] In another embodiment, provided herein is a method of
identifying a compound that modulates the expansion of a plurality
of OPACs, comprising contacting said plurality of OPACs with said
compound under conditions that allow expansion, wherein if said
compound causes a detectable change in expansion of said plurality
of OPACs compared to a plurality of OPACs not contacted with said
compound, said compound is identified as a compound that modulates
expansion of OPACs. 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.
[0236] In another embodiment, provided herein is a method of
identifying a compound that modulates the differentiation of OPACs,
comprising contacting said OPACs with said compound under
conditions that allow differentiation, wherein if said compound
causes a detectable change in differentiation of said OPACs
compared to OPACs not contacted with said compound, said compound
is identified as a compound that modulates proliferation of OPACs.
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.
5.2 Uses of OPACS
[0237] 5.2.1 Treatment of Bone-Related Cancers Using OPACs
[0238] Provided herein are methods of treating individuals having a
bone-related cancer comprising administering to the individual a
therapeutically-effective amount of OPACs. Bone-related cancers
include, without limitation, multiple myeloma, bone cancer, breast
cancer, lung cancer, neuroblastoma, osteosarcoma, Ewing's sarcoma,
chondrosarcoma, chordoma, malignant fibrous histiocytoma of bone,
fibrosarcoma of bone, metastatic cancer, multiple myeloma, and any
form of metastatic cancer characterized by bone metastases. In
certain embodiments, the administration of OPACs is therapeutically
effective to reduce, ameliorate or reverse one or more symptoms
associated with the bone-related cancer, e.g., a symptom caused by
or related to an effect of the cancer on one or more bones in the
individual. As one skilled in the art will recognize, treatment of
bone defects caused by cancer may not necessarily abate the cancer
itself. Treatment of cancer-related bone defects as provided herein
can occur before, after, or concurrently with additional cancer
therapies, as discussed below. Accordingly, in one embodiment, bone
defects are treated before the cancer is treated with an
anti-cancer therapy. In another embodiment, bone defects are
treated at or near the same time that the cancer is treated with an
anti-cancer therapy. In another embodiment, bone defects are
treated after the cancer is treated with an anti-cancer
therapy.
[0239] In certain embodiments, treatment of bone-related cancers,
e.g., multiple myeloma, comprises administering a
therapeutically-effective amount of OPACs to an individual having
bone-related cancer cells, e.g., multiple myeloma cells, wherein at
least some of said OPACs directly contact at least some multiple
myeloma cells, e.g., there is direct cell-cell contact between at
least some of said OPACs and some of said bone-related cancer
cells. In certain other embodiments, treatment of bone-related
cancers, e.g., multiple myeloma, comprises administering a
therapeutically-effective amount of OPACs to an individual having
bone-related cancer cells, e.g., multiple myeloma cells, wherein
none, or substantially none, of said OPACs directly contact
multiple myeloma cells, e.g., there is no, or substantially no,
direct cell-cell contact between at least some of said OPACs and
bone-related cancer cells.
[0240] In certain embodiments, the OPACs are administered
intralesionally, e.g., directly into, or adjacent to (e.g., within
1-5 cm of) one or more bone lesions caused by the cancer. In
certain embodiments, the OPACs arc administered in combination with
a matrix, e.g., an injectable matrix.
[0241] In certain other embodiments, OPACs are administered to an
individual having a bone-related cancer in combination with a solid
matrix, e.g., a bone substitute, a matrix or bone substitute
described in Section 5.2.2, below.
[0242] In certain other embodiments, the OPACs are administered
intravenously to the individual. The OPACs can be administered from
any container, and by any delivery system, medically suitable for
the delivery of fluids, e.g., fluids comprising cells, to an
individual. Such containers can be, for example, a sterile plastic
bag, flask, jar, or other container from which the OPACs 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.
[0243] Intralesional or intravenous administration can comprise,
e.g., about, at least, or no more than 1.times.10.sup.5,
5.times.10.sup.5, 1.times.10.sup.6, 5.times.10.sup.6,
1.times.10.sup.7, 5.times.10.sup.7, 1.times.10.sup.8,
5.times.10.sup.8, 1.times.10.sup.9, 5.times.10.sup.9,
1.times.10.sup.10, 5.times.10.sup.10, 1.times.10.sup.11 or more
OPACs in a single dose. OPACs may be administered once, or more
than once, during a course of therapy. Preferably, the administered
OPACs comprise 50% viable cells or more (that is, at least about
50% of the cells in the population are functional or living).
Preferably, at least about 60% of the cells in the population are
viable. More preferably, at least about 70%, 80%, 90%, 95%, or 99%
of the cells in the population in the pharmaceutical composition
are viable.
[0244] 5.2.1.1 Treatment of Multiple Myeloma
[0245] Provided herein are methods of treating an individual having
multiple myeloma, comprising administering to said individual a
plurality of OPACs, wherein said OPACs have any combination of, or
all of, the characteristics described in Section 5.1, above. In a
specific embodiment, said plurality of OPACs is CD105.sup.+ and
CD200.sup.dim or CD105.sup.+ and CD200.sup.-. The methods of
treatment provided herein encompass the use of any of the OPACs,
populations of OPACs, or populations of cells comprising OPACs,
described in Section 5.1, above.
[0246] Multiple myeloma is a cancer of plasma cells, which are
antibody-producing cells of the immune system. The disease
typically presents with four main characteristics: elevated
calcium, renal failure, anemia, and bone lesions. These symptoms
and others are discussed below.
[0247] Bone Pain--Myeloma cells secrete IL-6, also known as
osteoclast activating factor (OAF), which is a cytokine that
activates osteoclasts to break down bone, creating painful bone
lesions. These bone lesions are lytic in nature and are visible in
radiographs, which may show "punched-out" resorptive lesions.
Myeloma bone pain usually involves the spine and ribs, and worsens
with activity. Persistent localized pain may be present, and can
indicate a pathological bone fracture. Involvement of the vertebrae
may lead to spinal cord compression. The breakdown of bone also
leads to release of calcium into the blood, leading to
hypercalcemia and its associated symptoms.
[0248] The areas of breakdown of bone, as viewed in a skeletal
survey, typically appears as one or more lytic lesions on the bone,
that is, regions in which the bone appears absent or "punched
out."
[0249] Infection--Another common symptom of multiple myeloma is
infection, as the immune system is disrupted. The increased risk of
infection is due to immune deficiency resulting from diffuse
hypogammaglobulinemia, which is due to decreased production and
increased destruction of normal antibodies. The most common
infections are pneumonias and pyelonephritis. Common pneumonia
pathogens causing disease in multiple myeloma patients include
Streptococcus pneumoniae, Staphylococcus aureus, and Klebsiella
pneumoniae, while common pathogens causing pyelonephritis include
Escherichia coli. Typically, infection occurs in the initial few
months after the start of chemotherapy.
[0250] Renal failure--Multiple myeloma also tends to result in
renal failure, which may develop both acutely and chronically.
Renal failure in multiple myeloma is largely attributable to
hypercalcemia, which develops as osteoclasts dismantle existing
bone. Renal failure is also caused by tubular damage from excretion
of light chains, also called Bence Jones proteins, which can
manifest as the Fanconi syndrome (type II renal tubular acidosis).
Other causes include glomerular deposition of amyloid,
hyperuricemia, recurrent infections (e.g., pyelonephritis), and
local infiltration of tumor cells. Renal failure can be associated
with elevated levels of serum creatinin.
[0251] Anemia--The anemia found in myeloma is usually normocytic
and normochromic, and results from the replacement of normal bone
marrow by infiltrating tumor cells and inhibition of normal red
blood cell production (hematopoiesis) by cytokines.
[0252] Neurological symptoms--Symptoms of multiple myeloma include
a spectrum of neurological conditions, including weakness,
confusion and fatigue due to hypercalcemial headache, visual
changes and retinopathy, which can be the result of hyperviscosity
of the blood depending on the properties of paraprotein (see
below). Other neurological symptoms include radicular pain, loss of
bowel or bladder control (for example, due to involvement of spinal
cord leading to cord compression), and carpal tunnel syndrome and
other neuropathies (for example, due to infiltration of peripheral
nerves by amyloid). Multiple myeloma may give rise to paraplegia in
late presenting cases.
[0253] Presence of Paraprotein--A diagnostic symptom of multiple
myeloma is the presence in the blood and/or urine of paraprotein,
which is a monoclonal protein (M protein), e.g., an immunoglobulin
light-chain that is produced by the clonal proliferation of plasma
cells, or immunoglobulin fragments.
[0254] Symptomatic multiple myeloma is typically diagnosed when the
following symptoms or signs are present: clonal plasma cells
constituting greater than 10% of cells on bone marrow biopsy or, in
any quantity in a biopsy from other tissues (e.g., plasmacytoma);
paraprotein in either serum or urine; evidence of end-organ damage
(related organ or tissue impairment), for example, hypercalcemia
(e.g., corrected calcium>2.75 mmol/L in the blood), renal
insufficiency attributable to myeloma, anemia defined as
hemoglobin<10 g/dL blood, bone lesions (e.g., lytic lesions or
osteoporosis with compression fractures, frequent severe infections
(>2 a year), amyloidosis (the deposition of amyloid protein) of
other organs, and hyperviscosity syndrome (increase in the
viscosity of blood).
[0255] Individuals having multiple myeloma fall into one of the
following groups. In one embodiment, the individual having multiple
myeloma has never been treated for the disease. In another
embodiment, the individual has responsive myeloma; that is,
multiple myeloma that is responding to therapy. In a specific
embodiment, such an individual exhibits a decrease in M protein
(paraprotein) of at least 50% as a result of treatment. In another
specific embodiment, the individual exhibits a decrease in M
protein of between 25% and 50% as a result of treatment. In another
embodiment, the individual has stable multiple myeloma, which
refers to myeloma that has not responded to treatment (for example,
the decrease in M protein has not reached 50%), but has not
progressed or gotten worse. In another embodiment, the individual
has progressive multiple myeloma, which refers to active myeloma
that is worsening (for example, increasing M protein and worsening
organ or tissue impairment or end organ damage). In another
embodiment, the individual has relapsed multiple myeloma, which
refers to myeloma disease that initially responded to therapy but
has then begun to progress again. In specific embodiments, the
individual has relapsed after initial therapy or has relapsed after
subsequent therapy. In another embodiment, the individual has
refractory multiple myeloma. In a specific embodiment, the
refractory multiple myeloma is multiple myeloma that has not
responded to initial therapy. In another specific embodiment, the
refractory multiple myeloma is relapsed multiple myeloma that has
not responded to subsequent treatment. In another specific
embodiment, the refractory multiple myeloma is non-responding
progressing refractory disease, which refers to refractory disease
that is progressing. In another specific embodiment, the refractory
multiple myeloma is non-responding non-progressing refractory
disease, which refers to refractory disease that is not
worsening.
[0256] Thus, in one embodiment, provided herein is a method of
treating an individual having multiple myeloma, comprising
administering to the individual OPACs, a population of OPACs or a
population of cells comprising OPACs, wherein said administration
results in the detectable reduction of progression, detectable
cessation of worsening, and/or detectable improvement, of one or
more symptoms of multiple myeloma. In specific embodiments, said
one or more symptoms comprise elevated blood or urine calcium
compared to normal, the presence of bone lesions, anemia, or renal
failure. In a more specific embodiment, said one or more symptoms
comprises clonal plasma cells constituting greater than 10% of
cells on bone marrow biopsy or, in any quantity in a biopsy from
other tissues (e.g., plasmacytoma); paraprotein in either serum or
urine; and/or evidence of end-organ damage. In a more specific
embodiment, said one or more symptoms is a concentration of calcium
in the blood of greater than 2.75 mmol/L, renal insufficiency, less
than 10 g hemoglobin per deciliter of blood, the presence of bone
lesions, or amyloidosis of one or more organs other than bone
marrow.
[0257] In another specific embodiment, said symptom is a
neurological symptom. In more specific embodiments, said
neurological symptoms are weakness, confusion, fatigue, headache,
visual changes, retinopathy, radicular pain, loss of bowel or
bladder control, carpal tunnel syndrome, and/or paraplegia.
[0258] In a specific embodiment, provided herein is a method of
treating an individual having multiple myeloma, comprising
administering to the individual OPACs, a population of OPACs or a
population of cells comprising OPACs, wherein said administration
results in the detectable reduction in number of multiple myeloma
cells, e.g., clonal multiple myeloma cells, in one or more organs
of the individual.
[0259] In a specific embodiment, provided herein is a method of
treating an individual having multiple myeloma, comprising
administering to the individual OPACs, a population of OPACs or a
population of cells comprising OPACs, wherein said administration
results in the detectable increase in hemoglobin in the blood of
the individual, e.g., an increase to within normal limits. Normal
hemoglobin levels vary by the age and sex of the individual, as
shown in Table 1, below:
TABLE-US-00001 TABLE 1 Newborns 17-22 gm/dl One (1) week of age
15-20 gm/dl One (1) month of age 11-15 gm/dl Children 11-13 gm/dl
Adult males 14-18 gm/dl Adult women 12-16 gm/dl Men after middle
age 12.4-14.9 gm/dl Women after middle age 11.7-13.8 gm/dl
[0260] Thus, in a more specific embodiment, provided herein is a
method of treating an individual having multiple myeloma,
comprising administering to the individual OPACs, a population of
OPACs or a population of cells comprising OPACs, wherein said
administration results in the increase of blood hemoglobin levels
in said individual to between 11 g/dL blood and 20 g/dL blood. In a
more specific embodiment, said administering results in the
increase of blood hemoglobin levels in said individual to between
11 g/dL blood and 13 g/dL blood. In another more specific
embodiment, said administering results in the increase of blood
hemoglobin levels in said individual to between 12 g/dL blood and
16 g/dL blood. In a more specific embodiment, said administering
results in the increase of blood hemoglobin levels in said
individual to between 14 g/dL blood and 18 g/dL blood.
[0261] In another embodiment, provided herein is a method of
treating an individual having multiple myeloma, comprising
administering to the individual OPACs, a population of OPACs or a
population of cells comprising OPACs, wherein said administration
results in detectable reduction in the level of paraprotein in
blood or urine from said individual. In a specific embodiment, said
administering results in the reduction of paraprotein in blood or
urine of said individual to an undetectable level.
[0262] In another embodiment, provided herein is a method of
treating an individual having multiple myeloma, comprising
administering to the individual OPACs, a population of OPACs or a
population of cells comprising OPACs, wherein said administration
results in detectable reduction in the severity and/or number of
bone lesions caused by multiple myeloma in said individual, as
determined by, e.g., bone scan or radiography. In another
embodiment, provided herein is a method of treating an individual
having multiple myeloma, comprising administering to the individual
OPACs, a population of OPACs or a population of cells comprising
OPACs, wherein said administration results in detectable reduction
in loss of bone mass or bone mineral content, cessation of loss of
bone mass or bone mineral content, or increase in bone mass or bone
mineral content, in said individual.
[0263] In another specific embodiment of the method of treatment,
said one or more symptoms of multiple myeloma are bone pain,
osteocytic lesions (e.g., visible by X-ray or magnetic resonance
imaging (MRI)), osteoporosis, anemia, hypercalcemia or a symptom
due to hypercalcemia, or renal failure. In other specific
embodiments, said individual has never been treated for multiple
myeloma; said individual has been treated for multiple myeloma and
responds to non-OPAC therapy; said individual has been treated for
multiple myeloma and has not responded to non-OPAC therapy, but the
course of multiple myeloma in said individual has not progressed;
or said individual has progressive multiple myeloma.
[0264] In another aspect, provided herein is a method of
suppressing the proliferation of multiple myeloma cells, comprising
contacting said multiple myeloma cells with a plurality of OPACs,
such that proliferation of said multiple myeloma cells is
detectably suppressed. In certain embodiments, provided herein is a
method of suppressing the proliferation of multiple myeloma cells
in vivo, comprising administering a therapeutically-effective
amount of OPACs to an individual comprising multiple myeloma cells,
wherein said administering detectably reduces proliferation of said
multiple myeloma cells. In a specific embodiment, said
administering detectably reduces (e.g., improves) one or more
symptoms or signs of multiple myeloma, or lessens the worsening of
said one or more symptoms or signs of multiple myeloma.
[0265] OPACs, useful in the methods provided herein, are adherent,
osteogenic cells from chorion (but not chorionic skirt (laeve))
that can be identified and selected by the morphological, marker,
and culture characteristics discussed at least in Section 5.1,
above.
[0266] 5.2.1.2 Combination Therapies
[0267] Treatment of a bone-related cancer, e.g., multiple myeloma,
can comprise administration of OPACs, in combination with another
therapy, to the individual having the cancer.
[0268] Thus, in another aspect, provided herein is a method of
treating an individual having a bone-related cancer, e.g., multiple
myeloma, comprising administering to the individual OPACs, a
population of OPACs or a population of cells comprising OPACs, in
combination with one or more other anticancer therapies, e.g., one
or more chemotherapies or chemotherapeutic compounds. Such other
anticancer therapies can be administered to the individual at the
same time as, during the same course of treatment as, or separately
from, said administration of OPACs. In a specific embodiment, the
administration of said other anticancer therapies is administered
sequentially with administration of said OPACs. In a more specific
embodiment, said other anticancer therapy or anticancer therapies
are administered to said individual before administration of said
OPACs; e.g., a course of such other anticancer therapies is
administered to the individual, and completed, prior to
administration to the individual of OPACs. In another more specific
embodiment, said OPACs are administered to the individual before
administration of said other anticancer therapies; e.g., a course
of OPACs is administered to said individual before administration
of said other anticancer therapies, and completed, prior to
administration to the individual said other anticancer therapy or
anticancer therapies.
[0269] In a specific embodiment, the anticancer agent is melphalan
(also known as L-phenylalanine mustard or L-PAM; trade name
Alkeran). Thus, in one embodiment, the method of treating an
individual having multiple myeloma comprises administering to said
individual melphalan, e.g., a therapeutically effective dose or
doses of melphalan. Administration is typically oral or
intravenous. In another specific embodiment, the anticancer agent
is thalidomide. In another specific embodiment, the anticancer
agent is pomalidomide (sold under the trade name ACTIMID.RTM.);
lenalidomide (sold under the trade name REVLIMID.RTM.); or
lenalidomide in combination with dexamethasone. In another specific
embodiment, the anticancer treatment is bortezomib (VELCADE.RTM.).
In another specific embodiment, the anticancer agent comprises a
combination of melphalan, prednisone, and thalidomide (administered
separately or together). In another specific embodiment, the
anticancer agent is bortezomib, melphalan and prednisone
(administered separately or together).
[0270] Other anticancer agents are well-known in the art. Thus, in
other specific embodiments, the anticancer agents include, but are
not limited to: acivicin; aclarubicin; acodazole hydrochloride;
acronine; adozelesin; aldesleukin; altretamine; ambomycin;
ametantrone acetate; amsacrine; anastrozole; anthramycin;
asparaginase; asperlin; azacitidine; azetepa; azotomycin;
batimastat; benzodepa; bicalutamide; bisantrene hydrochloride;
bisnafide dimesylate; hizelesin; bleomycin sulfate; brequinar
sodium; bropirimine; busulfan; cactinomycin; calusterone;
caracemide; carbetimer; carboplatin; carmustine; carubicin
hydrochloride; carzelesin; cedefingol; celecoxib (COX-2 inhibitor);
chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol
mesylate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin;
daunorubicin hydrochloride; decitabine; dexonnaplatin; dezaguanine;
dezaguanine mesylate; diaziquone; docetaxel; doxorubicin;
doxorubicin hydrochloride; droloxifene; droloxifene citrate;
dromostanolone propionate; duazomycin; edatrexate; eflomithine
hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine;
epirubicin hydrochloride; erbulozole; esorubicin hydrochloride;
estramustine; estramustine phosphate sodium; etanidazole;
etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride;
fazarabine; fenretinide; floxuridine; fludarabine phosphate;
fluorouracil; flurocitabine; fosquidone; fostriecin sodium;
gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin
hydrochloride; ifosfamide; ilmofosine; iproplatin; irinotecan;
irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide
acetate; liarozole hydrochloride; lometrexol sodium; lomustine;
losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine
hydrochloride; megestrol acetate; melengestrol acetate; melphalan;
menogaril; mercaptopurine; methotrexate; methotrexate sodium;
metoprine; meturedepa; mitindomide; mitocarcin; mitocromin;
mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone
hydrochloride; mycophenolic acid; nocodazole; nogalamycin;
ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin;
pentamustine; peplomycin sulfate; perfosfamide; pipobroman;
piposulfan; piroxantrone hydrochloride; plicamycin; plomestane;
porfimer sodium; porfiromycin; prednimustine; procarbazine
hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin;
riboprine; safingol; safingol hydrochloride; semustine; simtrazene;
sparfosate sodium; sparsomycin; spirogermanium hydrochloride;
spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur;
talisomycin; tecogalan sodium; taxotere; tegafur; teloxantrone
hydrochloride; temoporfin; teniposide; teroxirone; testolactone;
thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine;
toremifene citrate; trestolone acetate; triciribine phosphate;
trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole
hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin;
vinblastine sulfate; vincristine sulfate; vindesine; vindesine
sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine
sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine
sulfate; vorozole; zeniplatin; zinostatin; and zorubicin
hydrochloride.
[0271] Other anti-cancer drugs include, but are not limited to:
20-epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone;
aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin;
ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine;
aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole;
andrographolide; angiogenesis inhibitors; antagonist D; antagonist
G; antarelix; anti-dorsalizing morphogenetic protein-1;
antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston;
antisense oligonucleotides; aphidicolin glycinate; apoptosis gene
modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA;
arginine deaminase; asulacrine; atamestane; atrimustine;
axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin;
azatyrosine; baccatin III derivatives; balanol; batimastat; BCR/ABL
antagonists; benzochlorins; benzoylstaurosporine; beta lactam
derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF
inhibitor; bicalutamide; bisantrene; bisaziridinylspermine;
bisnafide; bistratene A; bizelesin; breflate; bropirimine;
budotitane; buthionine sulfoximine; calcipotriol; calphostin C;
camptothecin derivatives; capecitabine; carboxamide-amino-triazole;
carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived
inhibitor; carzelesin; casein kinase inhibitors (ICOS);
castanospermine; cecropin B; cetrorelix; chlorins;
chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin;
cladribine; clomifene analogues; clotrimazole; collismycin A;
collismycin B; combretastatin A4; combretastatin analogue;
conagenin; crambescidin 816; crisnatol; cryptophycin 8;
cryptophycin A derivatives; curacin A; cyclopentanthraquinones;
cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;
cytostatin; dacliximab; decitabine; dehydrodidenmin B; deslorelin;
dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;
diaziquone; didemnin B; didox; diethylnorspermine;
dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin; diphenyl
spiromustine; docetaxel; docosanol; dolasetron; doxifluridine;
doxorubicin; droloxifene; dronabinol; duocarmycin SA; ebselen;
ecomustine; edelfosine; edrecolomab; eflornithine; elemene;
emitefur; epirubicin; epristeride; estramustine analogue; estrogen
agonists; estrogen antagonists; etanidazole; etoposide phosphate;
exemestane; fadrozole; fazarabine; fenretinide; filgrastim;
finasteride; flavopiridol; flezelastine; fluasterone; fludarabine;
fluorodaunorunicin hydrochloride; forfenimex; formestane;
fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate;
galocitabine; ganirelix; gelatinase inhibitors; gemcitabine;
glutathione inhibitors; hepsulfam; heregulin; hexamethylene
bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene;
idramantone; ilmofosine; ilomastat; imatinib (e.g., GLEEVEC.RTM.),
imiquimod; immunostimulant peptides; insulin-like growth factor-1
receptor inhibitor; interferon agonists; interferons; interleukins;
iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine;
isobengazole; isohomohalicondrin B; itasetron; jasplakinolide;
kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin;
lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia
inhibiting factor; leukocyte alpha interferon;
leuprolide+estrogen+progesterone; leuprorelin; levamisole;
liarozole; linear polyamine analogue; lipophilic disaccharide
peptide; lipophilic platinum compounds; lissoclinamide 7;
lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone;
loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic
peptides; maitansine; mannostatin A; marimastat; masoprocol;
maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors;
menogaril; merbarone; meterelin; methioninase; metoclopramide; MIF
inhibitor; mifepristone; miltefosine; mirimostim; mitoguazone;
mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast
growth factor-saporin; mitoxantrone; mofarotene; molgramostim;
Erbitux, human chorionic gonadotrophin; monophosphoryl lipid
A+myobacterium cell wall sk; mopidamol; mustard anticancer agent;
mycaperoxide B; mycobacterial cell wall extract; myriaporone;
N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;
naloxone+pentazocine; napavin; naphterpin; nartograstim;
nedaplatin; nemorubicin; neridronic acid; nilutamide; nisamycin;
nitric oxide modulators; nitroxide antioxidant; nitrullyn;
oblimersen (GENASENSE.RTM.); O.sup.6-benzylguanine; octreotide;
okicenone; oligonucleotides; onapristone; ondansetron; ondansetron;
oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin;
oxaunomycin; paclitaxel; paclitaxel analogues; paclitaxel
derivatives; palauamine; palmitoylrhizoxin; pamidronic acid;
panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;
peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;
perflubron; perfosfamide; perillyl alcohol; phenazinomycin;
phenylacetate; phosphatase inhibitors; picibanil; pilocarpine
hydrochloride; pirarubicin; piritrexim; placetin A; placetin B;
plasminogen activator inhibitor; platinum complex; platinum
compounds; platinum-triamine complex; porfimer sodium;
porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2;
proteasome inhibitors; protein A-based immune modulator; protein
kinase C inhibitor; protein kinase C inhibitors, microalgal;
protein tyrosine phosphatase inhibitors; purine nucleoside
phosphorylase inhibitors; purpurins; pyrazoloacridine;
pyridoxylated hemoglobin polyoxyethylene conjugate; raf
antagonists; raltitrexed; ramosetron; ras farnesyl protein
transferase inhibitors; ras inhibitors; ras-GAP inhibitor;
retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin;
ribozymes; RII retinamide; rohitukine; romurtide; roquinimex;
rubiginone B1; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A;
sargramostim; Sdi 1 mimetics; semustine; senescence derived
inhibitor 1; sense oligonucleotides; signal transduction
inhibitors; sizofiran; sobuzoxane; sodium borocaptate; sodium
phenylacetate; solverol; somatomedin binding protein; sonermin;
sparfosic acid; spicamycin D; spiromustine; splenopentin;
spongistatin 1; squalamine; stipiamide; stromelysin inhibitors;
sulfinosine; superactive vasoactive intestinal peptide antagonist;
suradista; suramin; swainsonine; tallimustine; tamoxifen
methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur;
tellurapyrylium; telomerase inhibitors; temoporfin; teniposide;
tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline;
thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin
receptor agonist; thymotrinan; thyroid stimulating hormone; tin
ethyl etiopurpurin; tirapazamine; titanocene bichloride; topsentin;
toremifene; translation inhibitors; tretinoin; triacetyluridine;
triciribine; trimetrexate; triptorelin; tropisetron; turosteride;
tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex;
urogenital sinus-derived growth inhibitory factor; urokinase
receptor antagonists; vapreotide; variolin B; velaresol; veramine;
verdins; verteporfm; vinorelbine; vinxaltine; vitaxin; vorozole;
zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer.
[0272] In other embodiments, the combination therapy comprises
administration of OPACs to an individual in combination with an
inhibitor of osteoclasts. In a specific embodiment, the osteoclast
inhibitor is an inhibitor of RANKL, e.g., Denosumab. In another
specific embodiment, the osteoclast inhibitor is an integrin or
cathepsin K inhibitor.
[0273] In another embodiment, the combination therapy comprises
administration of OPACs in combination with bisphosphonates. In
specific embodiments, the bisphosphonates are clodronate and/or
pamidronate.
[0274] 5.2.2 Treatment of Bone Defects Using OPACs
[0275] Populations of OPACs can be used to treat bone defects,
e.g., bone defects arising from trauma, or from disease, e.g.,
disease other than a hone-relate cancer. OPACs can also be used to
treat any disease, disorder or condition that results in, or is
related to, loss of bone. As used herein, "treat" encompasses the
cure of remediation of, improvement of, lessening of the severity
of, or reduction in the time course of, a disease, disorder or
condition, or any parameter or symptom thereof.
[0276] Isolated populations of OPACs may also be used to treat bone
fractures, e.g., non-union bone fractures. Isolated populations of
OPACs may also be used to fuse vertebrae together in order to,
e.g., complete a spinal fusion in a subject in need thereof
Isolated populations of OPACs, in combination with stem or
progenitor cell populations, may also be used to treat the
foregoing.
[0277] OPACs can be combined with a substrate, e.g., a matrix, to
form an implantable composition. For example, provided herein is a
composition, e.g., an implantable composition, comprising OPACs. In
a specific embodiment, the implantable composition comprises a
matrix. 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 specific embodiment,
said matrix comprises hydroxyapatite. In a more specific
embodiment, said matrix comprises both collagen and hydroxyapatite,
e.g., the matrix is HEALOS.RTM.. 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. In
certain embodiments, the matrix is a synthetic degradable polymer
such as, for example, polylactic acid or polyglycolic acid. In
certain embodiments, the implantable scaffolding substrate is a
.beta.-tricalcium phosphate substrate, a .beta.-tricalcium
phosphate-collagen substrate, a collagen substrate, a calcium
phosphate substrate, a mineralized human placental collagen
substrate, a hyaluronic acid substrate, or a ceramic substrate. In
certain embodiments, the implantable scaffolding substrate is a
.beta.-tricalcium phosphate substrate. In certain embodiments, the
implantable scaffolding substrate is a .beta.-tricalcium
phosphate-collagen substrate. In certain embodiments, the
implantable scaffolding substrate is a collagen substrate. In
certain embodiments, the implantable scaffolding substrate is a
calcium phosphate substrate. In certain embodiments, the
implantable scaffolding substrate is a mineralized human placental
collagen substrate.
[0278] OPACs, and populations of OPACs, can also be induced to
differentiate into a particular cell type, either ex vivo or in
vivo, in preparation for administration to an individual in need of
such cells, or cells differentiated from such cells. For example,
OPACs can be injected into a damaged organ, e.g., a damaged bone,
for organ neogenesis and repair of injury in vivo. Such injury may
be due to such conditions and disorders including, but not limited
to, bone defects including lesions resulting from osteoporosis,
cancer, fractures, and spinal conditions treatable with, e.g.,
spinal fusion. The OPACs can be injected into the damaged bone
alone or can be introduced with an implantable substrate as
described herein.
[0279] When OPACs are administered as a suspension or liquid
injectable, the cells can be administered intravenously, or,
preferably, at the site of the bone defect, e.g., break.
[0280] In certain aspects, provided herein is a method for treating
bone defects in a subject, comprising administering to a subject in
need thereof an implantable or injectable composition comprising a
population of OPACs provided herein, thereby treating the bone
defect in the subject. In certain embodiments, the bone defect is
an osteolytic lesion associated with a cancer, a bone fracture, or
a spine, e.g., in need of fusion. In certain embodiments, the
osteolytic lesion is associated with multiple myeloma, bone cancer,
or metastatic cancer. In certain embodiments, the bone fracture is
a non-union fracture. In certain embodiments, an implantable
composition is surgically implanted, e.g., at the site of the bone
defect. In certain embodiments, an injectable composition is
surgically administered to the region of the bone defect. In
certain embodiments, the injectable composition is systemically
administered.
[0281] In a specific embodiment, the implantable composition
comprising OPACs comprises a matrix. 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, 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. In
certain embodiments, the matrix is a synthetic degradable polymer
such as, for example, polylactic acid or polyglycolic acid. In
certain embodiments, the implantable scaffolding substrate is
selected from the group consisting of a .beta.-tricalcium phosphate
substrate, a .beta.-tricalcium phosphate-collagen substrate, a
collagen substrate, a calcium phosphate substrate, a mineralized
human placental collagen substrate, a hyaluronic acid substrate,
and a ceramic substrate. In certain embodiments, the implantable
scaffolding substrate is a .beta.-tricalcium phosphate substrate.
In certain embodiments, the implantable scaffolding substrate is a
.beta.-tricalcium phosphate-collagen substrate. In certain
embodiments, the implantable scaffolding substrate is a collagen
substrate. In certain embodiments, the implantable scaffolding
substrate is a calcium phosphate substrate. In certain embodiments,
the implantable scaffolding substrate is a mineralized human
placental collagen substrate.
[0282] In another aspect, provided herein is a method for
formulating an injectable composition, comprising combining a
population of OPACs with injectable hyaluronic acid or collagen. In
another aspect, provided herein is an injectable composition
comprising OPACs and hyaluronic acid or collagen.
[0283] OPACs can be administered without being cultured under
conditions that cause the OPACs to differentiate. Alternately, the
OPACs can be cultured in, e.g., e.g., osteogenic medium for, e.g.,
about 1-20 days, prior to administration. Alternately, OPACs can be
isolated and seeded on a matrix, then cultured in osteogenic medium
for, e.g., about 1-20 days. In another embodiment, OPACs can be
cultured in, e.g., osteogenic medium for, e.g., about 1-20 days,
then seeded onto a matrix, then cultured in osteogenic medium as
described herein for, e.g., about 1-20 days.
[0284] In other embodiments, isolated populations of OPACs may be
used in autologous or heterologous tissue regeneration or
replacement therapies or protocols, including, but not limited to
treatment of corneal epithelial defects, cartilage repair, facial
dermabrasion, mucosal membranes, tympanic membranes, intestinal
linings, neurological structures (e.g., retina, auditory neurons in
basilar membrane, olfactory neurons in olfactory epithelium), burn
and wound repair for traumatic injuries of the skin, or for
reconstruction of other damaged or diseased organs or tissues.
[0285] In certain embodiments, an isolated population of OPACs is
used in hematopoietic reconstitution in an individual that has
suffered a partial or total loss of hematopoietic stem cells, e.g.,
individuals exposed to lethal or sub-lethal doses of radiation
(whether industrial, medical or military); individuals that have
undergone myeloablation as part of, e.g., cancer therapy, and the
like. Isolated populations of OPACs can be used in place of, or to
supplement, bone marrow or populations of stem cells derived from
bone marrow. Typically, approximately 1.times.10.sup.8 to
2.times.10.sup.8 bone marrow mononuclear cells per kilogram of
patient weight are infused for engraftment in a bone marrow
transplantation (i.e., about 70 ml of marrow for a 70 kg donor). To
obtain 70 ml requires an intensive donation and significant loss of
donor blood in the donation process. An isolated population of
OPACs for hematopoietic reconstitution 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
OPACs.
[0286] The OPACs provided herein, alone or in combination with
other stem cell or progenitor cell populations, can be used in the
manufacture of a tissue or organ in vivo. The methods provided
herein encompass using OPACs to seed a matrix and to be cultured
under the appropriate conditions to allow the cells to
differentiate and populate the matrix. The tissues and organs
obtained by the methods provided herein can be used for a variety
of purposes, including research and therapeutic purposes.
[0287] In a preferred embodiment, OPACs as provided herein, and
populations of OPACs, may be used for autologous and allogenic
transplants, including matched and mismatched HLA type
hematopoietic transplants. In one embodiment of the use of OPACs as
allogenic hematopoietic transplants, the host is treated to reduce
immunological rejection of the donor cells, or to create
immunotolerance (see, e.g., U.S. Pat. Nos. 5,800,539 and
5,806,529). In another embodiment, the host is not treated to
reduce immunological rejection or to create immunotolerance.
6. EXAMPLES
[0288] The following examples are intended to illustrate the
present embodiments and are not to be construed to be limiting in
any way. All references, whether patent references, literature
references, or otherwise, cited herein are hereby incorporated by
reference for all purposes.
6.1 Example 1
Isolation and Characterization of OPACS
[0289] 6.1.1 Materials And Methods
[0290] Isolation of OPACs: Term placenta was collected from healthy
donor mothers after informed consent was obtained. Chorionic tissue
was manually separated from amnion, and 12 g of chorionic tissue
was excised and minced into 1 mm.sup.3 pieces. Minced tissue was
then transferred to a 240 mL solution of dispase II at a
concentration of 2.4 U/mL; tissue was incubated with dispase II for
1 hour (hr) at 37 C with agitation at 80 RPM. After incubation with
dispase, digested tissues were aliquoted into 50 mL tubes to allow
for centrifugation; digested tissue sample was centrifuged at 220 g
for 5 minutes at room temperature (RT). After carefully decanting
supernatants, pelleted tissues were resuspended and pooled into a
warm collagenase II solution (270 U/mL in a 240 mL volume) and
incubated for 1 hr at 37 C with agitation at 80 RPM. Digestates
were aliquoted into 50 mL tubes to allow for centrifugation;
digested tissue sample was centrifuged at 220 g for 5 minutes at
RT. Enzyme present in digested tissues was neutralized with a 5%
FBS/PBS wash. Samples were again subject to centrifugation (220 g
for 5 minutes at RT). Pellets (containing liberated cells as well
as tissue) were then re-suspended in 20% FBS (Hyclone)/.alpha.-MEM
(Invitrogen) containing 1.times. penicillin-streptomycin and
1.times.1-glutamine or 10% FBS(Hyclone)/DMEM (Invitrogen) media
also containing 1.times. penicillin-streptomycin and
1.times.1-glutamine.
Selective Adhesion Isolation of OPACs
[0291] As part of the selective adhesion strategy, cell suspensions
(obtained as described above) were added to flasks that had been
precoated with fibronectin (FN, Sigma), collagen (COL, StemCell
Technologies), vitronectin (VN, Sigma), and laminin (LN, Sigma).
Flasks were coated by incubating flasks with 10 .mu.g/mL solutions
of each protein for 1 hr at room temperature for fibronectin and
collagen, 1 hr at 37.degree. C. for vitronectin, and 2 hours at 37
C for laminin; after these incubations flasks were washed 2.times.
with PBS.
[0292] Three hours, 24 hours, and 6 days after seeding of cell
suspensions onto coated flasks, non-adherent cells/tissues were
removed from establishment cultures. Cells remaining in tissue
culture flasks were then allowed to proliferate. Once cultures
achieved about 80-90% confluence, cells were cryopreserved.
[0293] Chorion derived adherent cells (OPACs) were separated based
on cell surface expression of CD200 using magnetic assisted cell
sorting (MACs) technique using anti-human CD200-PE antibody (BD
Biosciences, cat 552475), anti-PE microbeads (Miltentyi, cat
#130-048-801), and MACs columns (Miltenyi) according to Miltenyi's
MACs column protocol.
[0294] Culture of OPACs
[0295] Conditions from the selective adhesion strategy that
displayed the highest levels of alkaline phosphatase (AP) induction
(LN or VN coatings, 20% FBS/.alpha.-MEM, 6 day establishment
adhesion) were further expanded in conditions that encourage
osteogenic functionality. These conditions included subculturing on
fibronectin-coated surfaces, using commercially-available
mesenchymal stem cell media, or using mesenchymal stem
cell-qualified fetal bovine serum. Two cell lines from the original
selective adhesion matrix were subcultured according to the
following matrix on LN, VN, or FN coated surfaces, and with the
three media depicted in Table 2.
TABLE-US-00002 TABLE 2 Establishment coating LN VN media 1 LN FN VN
FN 2 LN FN VN FN 3 LN FN VN FN 1. Hyclone FBS/.alpha.MEM 2. Lonza
MSC media 3. MSC-qualifd FBS (Stem Cell Tech)/.alpha.MEM
[0296] The cells obtained using the selective adhesion matrix shown
in Table 2 were then analyzed by flow cytometry and for alkaline
phosphatase activity. Cells were characterized using the following
assays: flow cytometry, gene expression analysis, and alkaline
phosphatase (AP) activity, and protein secretion.
Gene Expression
[0297] Cells were trypsinized and nucleated cell counts were
performed to determine a minimum of 1.times.10.sup.6 to
1.times.10.sup.7 cells. Cells were then lysed using protocol for
lysis from Qiagen RNEasy kit; lysates were then passed through QIA
shredder to maximize cell lysis. RNA isolation was performed using
a Qiagen RNEasy kit.
[0298] RNA quantity was determined using Nanodrop ND 1000
spectrophotometer; a minimum of 28 ng/.mu.l of RNA analyzed by
Nanodrop ND1000. RNA quality was measured by Agilent 2100
bioanalyzer; an rRNA ratio 28S/18S of 2.0 was used as determinant
of good quality RNA. cDNA reactions were generated from RNA using
High capacity cDNA archive kit protocol. Real time PCR reactions
were performed using TAQMAN.RTM. universal PCR master mix from
Applied Biosystems. The PCR reaction uses the 5' activity of
Amplitaq Gold DNA polymerase to cleave a TAQMAN.RTM. probe during
PCR. The TAQMAN.RTM. probe contains a reporter dye at 5'' end and a
quencher dye a the 3'' end of the probe. Accumulation of the PCR
products is detected directly by monitoring the increase in
fluorescence of the reporter dye.
[0299] RNA was isolated from OPACs, placental stem cells,
mesenchymal stem cells (ScienCell Research Labs., Carlsbad,
Calif.), and human dermal fibroblasts (ScienCell Research Labs.,
Carlsbad, Calif.) for the osteogenesis superarray, and from OPACs
and placental stem cells (for the TGF-BMP array). Cells were
cultured in basal media (growth conditions for each respective cell
type) for 3 days and cultured in osteogenic media (osteogenic
conditions) for one week. Samples were isolated in quadruplicate
using Qiagen's RNeasy Plus Mini Kit. All RNA isolation was of good
quality and sufficient yield was achieved to run (as measured by
Nanodrop for concentration and Agilent chip for purity). Arrays
were run according to the manufacturer's protocol and results
quantified on an ABI 7900.
Alkaline Phosphatase Activity
[0300] To induce osteogenesis, cells were seeded in growth medium
at 5.times.10.sup.3 cells/cm.sup.2 for about 3 days, and then
maintained in growth medium or induced with OS medium (10%
FBS/DMEM) containing ascorbic acid (50 .mu.g/mL), dexamethasone
(0.1 .mu.M), and .alpha.-glycerophosphate (10 mM) for up to 2
weeks; cells were fed bi-weekly with fresh growth or osteogenic
medium.
[0301] Alkaline phosphatase (AP) activity in cell lysates was
determined using a colorimetric assay (Cell Biolabs, San Diego,
Calif.), which measures the formation of p-nitrophenol product; AP
activity was normalized to .mu.g of DNA (to account for any
differences in cell number) using the PicoGreen dsDNA fluorescent
assay. The amount of p-nitrophenol formed by cell lysates was
determined by linear regression analysis from a standard curve
generated using known amounts of p-nitrophenol (provided by the
kit). Alkaline phosphatase activity was also assessed
histochemically per manufacturer's instructions using a kit (#85)
from Sigma.
Colony-Forming Unit Assay--Alkaline Phosphatase Activity
[0302] Pre-sorted OPACs containing a mixture of CD200.sup.- and
CD200.sup.+ cells, CD200.sup.+ populations, and flow-through
fractions (which contained a relatively high concentration of CD200
negative/dim populations) were seeded in osteogenic differentiation
media (10% FBS/DMEM/50 .mu.g/ml ascorbic acid/0.1 .mu.M
dexamethasone) at 22.5 cells/cm.sup.2 in 35 mm gridded dishes.
Cells were fed bi-weekly with osteogenic media, and after 10 days
cells were assessed histochemically for alkaline phosphatase
activity per manufacturer's instructions using a kit (#85) from
Sigma. The number of alkaline phosphatase positive colonies was
quantified visually using a stereomicroscope.
Immunofluorescent Staining
[0303] Cells were cultured on Labtek slides at 5000 cells/cm.sup.2
and cultured for 2-3 days. Cells were fixed with 3.7% formaldehyde
for 9 minutes, then washed 3.times. with PBS. After blocking for 20
minutes at room temperature with blocking buffer (10% goat serum,
2.times. casein, 0.3% triton), cells were stained for NG2
(chondroitin sulfate) and .alpha.-smooth muscle actin using rabbit
anti-human NG2 (Chemicon, 1:150 dilution), mouse anti-human
.alpha.-smooth muscle actin (Dako, 1:30 dilution).antibodies.;
cells were incubated with primary antibodies overnight at 4 C. Next
samples were then washed with PBS and incubated with fluorescently
labeled secondary antibodies, either AlexaFluor488 anti-rabbit
(Invitrogen, 1:400 dilution) or AlexaFluor488 anti-mouse
(Invitrogen, 1:400 dilution); cells were incubated with secondary
antibodies for 30 minutes at room temperature. Next cells were
washed 3.times. with PBS and mounted using mounting media
containing DAPI in order to counterstain for nuclei. Cells were
imaged using an epifluorescence microscope.
Luminex Assay
[0304] Secreted factors in conditioned medium samples from cells
were analyzed using the human bone panel 1B-11plex (cat
#HBN1B-51K-11) from Millipore, according to manufacturer's
instructions.
Antibody Arrays
[0305] OPACs, mesenchymal stem cells, placental stem cells, and
fibroblasts were cultured in basal media (growth conditions) for 3
days and then switched to serum free media for 24 hours. Media
samples were run on RayBiotech RAYBIO.RTM. Biotin Label-based
Antibody Array per the manufacturer's instructions. The
chemiluminescence arrays were imaged and dot sizes were analyzed
using a GelLogic 2200 Imaging System and a Kodak MI imaging
program, respectively.
[0306] 6.1.2 Results
Selective Adhesion and Gene Expression
[0307] All 6 day adhesion conditions yielded sufficient numbers of
cells for analysis, however the 3 hr and 24 hr adhesion conditions
in 10% FBS/DMEM did not proliferate sufficiently to yield enough
cells for analysis. The genes chosen for mRNA expression analysis
included: alkaline phosphatase (AP), DLX5 (transcription factor),
bone sialoprotein, RUNX2 (transcription factor), collagenase III,
osterix (transcription factor), and osteocalcin. Of these genes,
cells derived from the 20% FBS/.alpha.MEM media, 6-day adhesion,
LN, VN, and COL coating showed increased expression in AP mRNA
levels compared to bone marrow-derived mesenchymal stem cells (FIG.
1). All other genes analyzed demonstrated lower levels of gene
expression in newly isolated placental cells compared to
mesenchymal stem cells.
[0308] Results from immunophenotyping showed significant increases
in the CD200.sup.dim/CD200.sup.- populations in the 6 day adhesion
conditions compared to placental stem cells although the highest
increases were detected in the LN, VN, COL coated conditions in 20%
FBS/.alpha.MEM. (FIG. 2). Flow cytometry also confirmed that OPACs
are CD34.sup.- and CD105.sup.+ (FIG. 2).
[0309] Culture permutations, namely the LN, VN, COL coated
conditions in 20% FBS/.alpha.MEM, which yielded the increased
CD200.sup.dim/CD200.sup.- populations, also demonstrated
significant increases in AP.sup..dbd. and Stro-1.sup.+ populations
by flow cytometry (FIG. 3); these conditions also displayed high
levels of AP gene expression vs. MSC controls. These same
conditions also demonstrated significant increases in SSEA3 and
SSEA4 positive populations (FIG. 4).
[0310] Functional analysis, as determined by AP activity,
demonstrated inducible AP activity under osteogenic differentiation
conditions (10 day induction) in the FN, LN, VN, COL coated
substrates in 20% FBS/.alpha.MEM conditions, although the FN coated
condition demonstrated a more moderate induction (FIG. 5).
Characterization of Cultured Cells
[0311] Immunophenotyping of cells resulting from propagation in
various growth conditions demonstrated that, in the CD200.sup.+
populations, between 40-60% of the cells were CD200.sup.+, vs
.about.100% for placental stem cells (PDAC.TM.s) described, e.g.,
in US Application Publication No. 2007/0275362. There was also a
modest increase in the percentage of AP positive cells relative to
PDAC.TM.s, whereas the percentage of CD105 positive cells remained
the same as for PDAC.TM.s (FIG. 6).
[0312] To better define chorion derived cells (OPACs),
immunofluorescence staining was conducted to stain for markers
associated with cells that display osteogenic activity. Pericytes,
which are cells associated with vasculature and known to harbor
osteogenic activity stain positively for NG2 and .alpha.-smooth
muscle actin. Therefore immunofluorescence staining studies were
conducted for these markers. OPACs displayed two differences in
staining compared to PDAC.TM.s: (1) a lack of staining for
.alpha.-smooth muscle actin compared to .alpha.-smooth muscle
actin-positive PDAC.TM.s, and (2) diffuse cellular localization of
NG2 compared to focal adhesion localized NG2 staining for standard
PDAC.TM.s; localization of NG2 to subcellular structures such as
focal adhesions carries implications in the activity of NG2 (data
not shown).
[0313] After a 10 day induction under osteogenic differentiation
conditions, the LN-LN medium 1 and VN-VN medium 1 conditions, as
shown in FIG. 7, showed highest induction (osteogenic/basal) of AP
activity (black arrow) while LN-LN medium 3 and VN-FN medium 1
demonstrated highest overall AP activity (open arrow).
[0314] To address whether the functional activity, such as
osteogenic activity and T cell suppression, derived from the CD200
low/negative fraction or in the CD200.sup.+ fraction of these
selective adhesion cell preparations, MACs (magnetic assisted cell
separation) using an anti-human CD200 antibody was used to separate
these 2 cell populations. The 4 cell lines identified above as
having either highest AP induction or highest overall AP activity
were processed for magnetic separation based on expression of CD200
and analyzed by flow cytometry, AP activity, and AP positive
colony-forming unit formation.
[0315] The immunophenotyping displayed>85% pure CD200.sup.+
populations for 2 out 4 samples and that the flow-through fraction
still contained CD200.sup.+ cells (FIG. 8).
[0316] Cells resulting from the separation were induced with
osteogenic media for 10 days and analyzed for AP activity. The
results show that the CD200.sup.+ fraction had decreased AP
inducibility compared to the CD200.sup.dim/CD200.sup.- fractions
(FIG. 9).
[0317] CFU (colony forming unit assay) assays measure progenitor
cells in a population; for example, CFU-F assays are commonly used
to measure the number of progenitors in bone marrow aspirate. The
CFU-AP assay estimates the number of potential osteoblastic
precursors in a population. The CD200 fractions were seeded in 35
mm gridded dishes for 10 days in osteogenic media and stained for
alkaline phosphates. The number of colonies per dish and the number
of AP positive colonies were quantified. CD200.sup.+ fractions
showed no CFU-AP and the lowest total CFU activity, whereas the
pre-sort, which contain the highest CD200 dim/negative populations
showed the greatest number of AP positive CFU and CFU activity.
Comparatively, CD200.sup.+ populations had much lower levels of
CFU-AP and total CFU activity (FIGS. 10 and 11).
[0318] An 11-plex Luminex assay for bone-related secreted proteins
was performed on conditioned media samples from OPACs, CD200.sup.+
placental stem cells (PDAC.TM.s) and mesenchymal stem cells (MSCs).
Cells were cultured for 3 days in their respective media, then
incubated with serum free DMEM for 24 hours. To ascertain behavior
of cells under osteogenic conditions, cells were cultured for 3
days in growth conditions, subjected to osteogenic differentiation
media (media supplemented with ascorbic acid and dexamethasone) for
7 days, then incubated with serum free DMEM overnight. The results
show that of the 3 cell types, OPACs constitutively secreted the
highest levels of levels osteoprotegerin. Furthermore, when the
three cell types were cultured under osteogenic differentiation
conditions, only OPACs displayed upregulation of osteoprotegerin
secretion, whereas MSCs and PDAC.TM.s showed downregulated
osteoprotegerin. This implies that in a bone microenvironment,
OPACs may be better suited to inhibit osteoclast activity via
release of high levels of osteoprotegerin.
[0319] Analysis of expression of 84 genes relating to osteogenesis
in OPACs, PDAC.TM.s, MSCs, and fibroblasts cultured in growth
medium and in osteogenic medium was performed using a SuperArray
RT2 Profiler PCR Array-Human Osteogenesis (SABiosciences,
Frederick, Md.). Growth medium used in the experiment was
.alpha.MEM/20% Fetal Bovine Serum comprising 1.times.
penicillin-streptomycin, and 1.times. L-glutamine, and osteogenic
medium used in the experiment was osteogenic medium is
.alpha.MEM/20% Fetal Bovine Serum comprising 1.times.
penicillin-streptomycin, 1.times. L-glutamine, 50 .mu.g/mL ascorbic
acid, and 100 nM dexamethasone. Expression of the
osteogenesis-related genes was measured in OPACs, PDAC.TM.s, MSCs,
and fibroblasts cultured under growth and osteogenic
differentiation conditions.
[0320] Gene expression (as measured by Ct value, the PCR cycle at
which a statistically significant increase of fluorescence signal
is first detected) was found for most of the osteogenic genes
measured in all cell types. OPACs showed a unique gene expression
profile when compared to PDAC.TM.s, MSCs, and fibroblasts (Tables
3A-3C, respectively), and a unique gene expression induction
profile, when cells were shifted from growth to osteogenic medium,
when compared to PDAC.TM.s, MSCs, and fibroblasts (Tables 3D-3F,
respectively). Ct values (Tables 3A-3C) are numbers obtained
directly from the PCR cycler and reported as a range; as such,
these values were converted to one, two, three or four plus signs,
as explained below. To calculate fold-change in gene expression in
growth medium compared to osteogenic medium, a correction factors
is applied to the Ct values based on the expression of housekeeping
genes. This correction factor normalizes for slight differences in
amount of RNA used in the PCR.
[0321] Tables 3A-3C: Osteogenic gene expression (Ct values) of
OPACS vs. PDACs.TM. (Table 3A), OPACs vs. MSCs (Table 3B) and OPACs
vs. fibroblasts (Table 3C) in growth or osteogenic media, as
defined above (n=2). The expression level of the genes was
classified as ++++ (very high, Ct<20). +++ (high,
20<Ct<25), ++ (medium, 25<Ct<30), + (low
30<Ct<35), - (low 35<Ct<40) or blank (no signal
detected).
TABLE-US-00003 TABLE 3A OPAC PDAC .TM. Symbol G O G O AHSG - + ALPL
+ + ++ + AMBN + AMELY + + + + ANXA5 ++++ ++++ ++++ ++++ BGLAP ++ ++
++ ++ BGN +++ ++ +++ +++ BMP1 +++ +++ +++ +++ BMP2 ++ ++ ++ BMP3 +
BMP4 +++ +++ +++ +++ BMP5 + + BMP6 ++ ++ ++ ++ CALCR + ++ + + CD36
++ + ++ + CDH11 ++++ ++++ +++ +++ COL10A1 ++ + + + COL11A1 ++ ++ ++
+++ COL12A1 ++++ +++ ++++ +++ COL14A1 +++ ++++ ++ +++ COL15A1 ++++
++++ ++ ++ COL1A1 ++++ ++++ ++++ ++++ COL1A2 ++++ ++++ ++++ ++++
COL2A1 + + + COL3A1 ++++ ++++ ++++ ++++ COL4A3 + + + + COL5A1 ++++
++++ ++++ ++++ COMP + ++ + +++ CSF2 + + + CSF3 + ++ + - CTSK +++
++++ +++ ++++ DMP1 - DSPP + + EGF + + ++ + EGFR +++ +++ +++ +++
ENAM + + FGF1 ++ + ++ ++ FGF2 +++ +++ +++ +++ FGF3 + FGFR1 ++ ++ ++
++ FGFR2 ++ + + + FLT1 ++ ++ +++ ++ FN1 ++++ ++++ ++++ ++++ GDF10 -
ICAM1 +++ +++ +++ +++ IGF1 + ++ + + IGF1R +++ +++ +++ +++ IGF2 + ++
++ ++ ITGA1 +++ ++++ +++ +++ ITGA2 ++ +++ +++ ++ ITGA3 +++ +++ +++
+++ ITGAM + ITGB1 ++++ ++++ ++++ ++++ MINPP1 +++ +++ +++ +++ MMP10
+ ++ - + MMP2 ++++ ++++ ++++ ++++ MMP8 + - + MMP9 + ++++ + + MSX1
++ ++ ++ ++ NFKB1 +++ +++ +++ +++ PDGFA +++ +++ +++ ++ PHEX + + + +
RUNX2 +++ ++ +++ ++ SCARB1 ++ ++ +++ ++ SERPINH1 ++++ ++++ ++++
++++ SMAD1 +++ +++ +++ ++ SMAD2 +++ +++ +++ +++ SMAD3 +++ +++ +++
+++ SMAD4 +++ +++ +++ +++ SOX9 + + ++ + STATH + + + + TFIP11 +++
+++ +++ +++ TGFB1 +++ ++++ +++ +++ TGFB2 +++ +++ +++ +++ TGFB3 +++
++++ ++ +++ TGFBR1 +++ +++ ++ ++ TGFBR2 ++ ++ ++ + TNF + + ++ +
TUFT1 +++ ++ +++ ++ TWIST1 +++ +++ ++++ +++ VCAM1 +++ ++ ++++ +++
VDR ++ +++ +++ ++ VEGFA +++ +++ +++ +++ VEGFB +++ +++ +++ +++ G:
Relative expression vs. control in growth medium. O: Relative
expression v. control in osteogenic medium.
TABLE-US-00004 TABLE 3B OPAC MSC Symbol G O G O AHSG - - ++ ALPL +
+ ++ +++ AMBN + AMELY + + + + ANXA5 ++++ ++++ ++++ ++++ BGLAP ++ ++
+++ +++ BGN +++ ++ +++ ++++ BMP1 +++ +++ +++ +++ BMP2 ++ ++ ++ ++
BMP3 + + + BMP4 +++ +++ ++ +++ BMP5 + + + BMP6 ++ ++ ++ ++ CALCR +
++ - + CD36 ++ + + ++ CDH11 ++++ ++++ +++ ++++ COL10A1 ++ + ++ +++
COL11A1 ++ ++ +++ ++++ COL12A1 ++++ +++ +++ ++++ COL14A1 +++ ++++
++ +++ COL15A1 ++++ ++++ ++ ++ COL1A1 ++++ ++++ ++++ ++++ COL1A2
++++ ++++ ++++ ++++ COL2A1 + + + COL3A1 ++++ ++++ +++ ++++ COL4A3 +
+ + ++ COL5A1 ++++ ++++ +++ ++++ COMP + ++ + +++ CSF2 + + + + CSF3
+ ++ + - CTSK +++ ++++ +++ ++++ DMP1 - DSPP + + EGF + + + ++ EGFR
+++ +++ +++ +++ ENAM + + + + FGF1 ++ + ++ ++ FGF2 +++ +++ +++ +++
FGF3 + + FGFR1 ++ ++ ++ ++ FGFR2 ++ + + ++ FLT1 ++ ++ + ++ FN1 ++++
++++ ++++ ++++ GDF10 - - ICAM1 +++ +++ +++ +++ IGF1 + ++ + ++ IGF1R
+++ +++ +++ +++ IGF2 + ++ +++ +++ ITGA1 +++ ++++ +++ +++ ITGA2 ++
+++ +++ ++ ITGA3 +++ +++ +++ +++ ITGAM + ITGB1 ++++ ++++ ++++ ++++
MINPP1 +++ +++ +++ +++ MMP10 + ++ + + MMP2 ++++ ++++ ++++ ++++ MMP8
+ - + + MMP9 + ++++ + MSX1 ++ ++ + + NFKB1 +++ +++ +++ +++ PDGFA
+++ +++ ++ ++ PHEX + + + ++ RUNX2 +++ ++ +++ +++ SCARB1 ++ ++ +++
+++ SERPINH1 ++++ ++++ ++++ ++++ SMAD1 +++ +++ +++ +++ SMAD2 +++
+++ +++ +++ SMAD3 +++ +++ +++ +++ SMAD4 +++ +++ +++ +++ SOX9 + + ++
+++ STATH + + + + TFIP11 +++ +++ +++ +++ TGFB1 +++ ++++ +++ +++
TGFB2 +++ +++ +++ +++ TGFB3 +++ ++++ ++ +++ TGFBR1 +++ +++ ++ ++
TGFBR2 ++ ++ ++ + TNF + + + + TUFT1 +++ ++ ++ ++ TWIST1 +++ +++ +++
+++ VCAM1 +++ ++ +++ +++ VDR ++ +++ ++ +++ VEGFA +++ +++ +++ +++
VEGFB +++ +++ +++ ++++ G: Relative expression vs. control in growth
medium. O: Relative expression v. control in osteogenic medium.
TABLE-US-00005 TABLE 3C OPAC Fibroblast Symbol G O G O AHSG - -
ALPL + + ++ ++ AMBN + AMELY + + + + ANXA5 ++++ ++++ ++++ ++++ BGLAP
++ ++ ++ ++ BGN +++ ++ +++ +++ BMP1 +++ +++ +++ +++ BMP2 ++ ++ + +
BMP3 + - BMP4 +++ +++ + ++ BMP5 + + BMP6 ++ ++ + ++ CALCR + ++ - +
CD36 ++ + ++ ++ CDH11 ++++ ++++ +++ ++++ COL10A1 ++ + ++ +++
COL11A1 ++ ++ ++ +++ COL12A1 ++++ +++ +++ ++++ COL14A1 +++ ++++ +
++ COL15A1 ++++ ++++ + ++ COL1A1 ++++ ++++ +++ ++++ COL1A2 ++++
++++ ++++ ++++ COL2A1 + + + COL3A1 ++++ ++++ +++ ++++ COL4A3 + + +
++ COL5A1 ++++ ++++ +++ +++ COMP + ++ + +++ CSF2 + + + + CSF3 + ++
++ + CTSK +++ ++++ ++++ ++++ DMP1 - DSPP + + EGF + + + + EGFR +++
+++ +++ +++ ENAM + + + + FGF1 ++ + +++ +++ FGF2 +++ +++ +++ +++
FGF3 + FGFR1 ++ ++ ++ ++ FGFR2 ++ + + FLT1 ++ ++ ++ ++ FN1 ++++
++++ ++++ ++++ GDF10 - ICAM1 +++ +++ +++ +++ IGF1 + ++ + IGF1R +++
+++ ++ +++ IGF2 + ++ + ++ ITGA1 +++ ++++ +++ +++ ITGA2 ++ +++ ++ ++
ITGA3 +++ +++ +++ +++ ITGAM + + ITGB1 ++++ ++++ ++++ ++++ MINPP1
+++ +++ ++ +++ MMP10 + ++ - + MMP2 ++++ ++++ +++ ++++ MMP8 + - + ++
MMP9 + ++++ + + MSX1 ++ ++ ++ ++ NFKB1 +++ +++ +++ +++ PDGFA +++
+++ ++ ++ PHEX + + + ++ RUNX2 +++ ++ ++ +++ SCARB1 ++ ++ ++ ++
SERPINH1 ++++ ++++ ++++ ++++ SMAD1 +++ +++ ++ ++ SMAD2 +++ +++ +++
+++ SMAD3 +++ +++ ++ +++ SMAD4 +++ +++ +++ +++ SOX9 + + + + STATH +
+ + + TFIP11 +++ +++ +++ +++ TGFB1 +++ ++++ +++ +++ TGFB2 +++ +++
++ +++ TGFB3 +++ ++++ ++ +++ TGFBR1 +++ +++ ++ ++ TGFBR2 ++ ++ + ++
TNF + + + + TUFT1 +++ ++ ++ ++ TWIST1 +++ +++ +++ ++++ VCAM1 +++ ++
+ + VDR ++ +++ +++ +++ VEGFA +++ +++ ++ +++ VEGFB +++ +++ +++ +++
G: Relative expression vs. control in growth medium. O: Relative
expression v. control in osteogenic medium.
[0322] Tables 3D-3F: Fold-difference in expression of OPACs and
PDACs.TM., MSCs and fibroblasts of selected genes in growth medium
compared to osteogenic medium. A fold difference of 10 for a
particular gene, for example, indicates that the gene is induced by
ten-fold in osteogenic medium compared to growth medium. Only
results in which fold-induction in osteogenic medium in OPACs is at
least ten-fold higher or lower than fold induction in PDACs.TM.
(Table 3D), MSCs (Table 3E) or fibroblasts (Table 3F) are shown
(blank results were assigned a value of 0 for selection).
TABLE-US-00006 TABLE 3D OPAC PDAC .TM. Symbol Fold difference Fold
difference BMP2 11.65 COL11A1 3.64 84.86 COL1A1 0.38 9.31 COL4A3
1.26 12.91 COL5A1 0.99 6.84 COMP 17.90 4095.78 CSF3 59.08 CTSK 6.88
79.18 FGF1 0.12 1.81 FGFR2 0.27 76.81 MMP10 279.30 MMP9 7079936.55
12.25 TGFB2 15.77 0.72 Fold difference: Difference between
expression in growth medium and osteogenic medium. Blank: Fold
difference could not be calculated because fluorescence from one
cell type was too low.
TABLE-US-00007 TABLE 3E OPAC MSC Symbol Fold difference Fold
difference ALPL 2.02 29.13 CD36 1.08 84.08 COL10A1 0.08 19.86
COL11A1 3.64 43.73 COL12A1 0.42 5.02 COL1A1 0.38 7.23 COL4A3 1.26
15.62 COMP 17.90 1629.84 CSF3 59.08 CTSK 6.88 148.38 FGF1 0.12 1.93
FGFR2 0.27 88.30 IGF1R 4.21 1.53 IGF2 53.38 0.60 ITGA2 8.10 0.16
ITGA3 2.70 0.24 MMP10 279.30 1.44 MMP9 7079936.55 TGFB2 15.77 0.64
Fold difference: Difference between expression in growth medium and
osteogenic medium. Blank: Fold difference could not be calculated
because fluorescence from one cell type was too low.
TABLE-US-00008 TABLE 3F OPAC Fibroblast Symbol Fold difference Fold
difference BMP2 11.65 0.95 BMP4 0.23 72.12 COL12A1 0.42 6.55 COMP
17.90 431.41 CSF2 2.16 0.12 CSF3 59.08 0.10 FGF1 0.12 3.26 IGF1
31.46 ITGA2 8.10 0.54 MMP10 279.30 MMP8 38.76 MMP9 7079936.55 0.40
Fold difference: Difference between expression in growth medium and
osteogenic medium. Blank: Fold difference could not be calculated
because fluorescence from one cell type was too low.
[0323] When OPACs and PDAC.TM.s cultured in growth conditions were
compared using a set of 84 genes in the TGF.beta./BMP superfamily,
OPACs generally showed higher expression of genes related to bone
formation (Table 4). OPACs had greater expression of genes which
related to trophic support of bone formation including BMPs
(especially BMP-2) and TGF-.beta.'s. OPACs also had greater
expression of genes which could be related to the trophic support
of the inhibition of bone formation or bone resorption including
inhibins and TGF-.beta.'s (TGF-.beta. is implicated in both bone
formation and resorption). The TGF.beta./BMP superfamily represents
a complex feedback system of regulation of multiple different organ
systems. The fact that genes of this superfamily are expressed in
OPACs at greater levels then in PDAC.TM.s indicate that OPACs have
a greater capacity to regulate bone metabolism than PDAC.TM.s.
TABLE-US-00009 TABLE 4 Up/down regulation Symbol OPAC PDAC (fold)
CHRD ++ - 53.25 GDF7 + + 12.18 IGFBP3 ++++ ++ 1187.56 INHA ++ +
16.38 TGFB2 +++ +++ -11.02 Up/down regulation: Fold difference
OPAC/PDAC .TM. Table 4: TGF.beta./BMP superfamily members gene
expression (Ct values) of OPACS, PDACs .TM., MSCs and fibroblasts
in growth media (n = 3). The expression level of the genes is
classified as ++++ (very high, Ct < 20). +++ (high, 20 < Ct
< 25), ++ (medium, 25 < Ct < 30), + (low 30 < Ct <
35), - (low 35 < Ct < 40) or blank (no signal detected). Fold
difference represents the difference in expression level when OPACs
are compared to PDACs .TM.. Only genes for which expression in
OPACs exceeded expression in PDACs .TM., or for which expression in
PDACs .TM. exceeded expression in OPACs, are shown.
[0324] In addition to gene expression, analysis of protein
secretion (RayBiotech's RayBio.RTM. Biotin Label-based Antibody
Array) from OPACs, PDAC.TM.s, MSCs, and fibroblasts grown under
growth conditions was also performed (FIG. 13). A total of 46
secreted proteins were identified between the 4 cell types.
Proteins secreted by OPACs, but not PDACs.TM., included coagulation
factor II/tissue factor, decorin, epiregulin, follistatin-like 1,
IGFBP6, IGF-IIR, IL-2R.alpha. (interleukin 2 receptor .alpha.),
IL-12R.beta.2 (interleukin 12 receptor subunit .beta.2), IL-17RC
(interleukin receptor C), IL-27 (interleukin 27), Latent TGF-beta
binding protein 1, NCAM-1/CD56 (neural cell adhesion molecule 1),
sFRP-4 (secreted frizzled-related protein 4), SMAD4, spinesin, TFPI
(tissue factor pathway inhibitor), TGF-.beta. RI/ALK5 (transforming
growth factor beta receptor 1), TIMP-2 (tissue inhibitor of
metalloproteases 2), and TSG-6 (tumor necrosis factor
(TNF)-stimulated gene 6). Proteins secreted by OPACs but not MSCs
included decorin, epiregulin, FGF-7/KGF, IGFBP-3, IL-2R.alpha.
(interleukin-2 receptor alpha), IL-3R.alpha. (interleukin 3
receptor alpha), IL-5R.alpha. (interleukin 5 receptor alpha),
IL-17RC, IL-27, NCAM-1/CD56, SMAD4, TFPI, TGF-.beta.R1/ALK-5,
TGF-.beta.RIII (transforming growth factor beta receptor 3), and
TIMP2. Proteins, the expression of which was unique to OPACs in
comparison to PDACs.TM. and MSCs, were decorin, epiregulin,
IGFBP-3, IGFBP-6, IL-2 R alpha, IL-17RC, IL-27, Latent TGF-beta
binding protein 1 (LTBP), NCAM-1, Smad4, TFPI, TGF-beta R1/ALK5 and
TIMP-2. Of these decorin, IGFBP-6, and IL-27 are implicated in bone
regulation. Of these, follistatin-like-1, sFRP-4, and TSG-6 are
implicated in bone regulation.
[0325] OPACs, in a separate experiment, were also shown not to
express RANKL, as assessed by quantitative RT-PCR; bone
marrow-derived MSCs, however, produced significant amounts of RNA
for RANKL.
6.2 Example 2
In Vivo Bone Forming Capacity of OPACS
[0326] To evaluate the in vivo bone forming capacity of OPACs, an
in vivo study using a cranial defect model of bone repair was
performed. Experimentally, forty-eight (48) male Hsd:RH-Foxn1rnu
athymic rats (Harlan Laboratories, Indianapolis, Ind.),
approximately 6 weeks old at the commencement of the study, were
used. All rats had a 3 mm.times.5 mm defect created on each side of
the calvaria. The left defect of each was treated with a negative
control (HEALOS.RTM. bone graft replacement, alone) and the right
defect of each was treated with either with a positive control
(HEALOS.RTM.+BMP-2), a negative control (empty defect; bone is
removed and not replaced with anything), or cells (PDAC.TM.s or
OPACs) loaded onto the HEALOS.RTM. carrier matrix. Eight animals
were assigned to the cell treatment groups and four animals were
assigned to BMP-2 and empty defect groups. The defects were treated
with Healos containing the following dosages: 5 .mu.g BMP-2, or
3.times.10.sup.5-4.times.10.sup.5 cells.
[0327] Rats were sacrificed at seven (7) weeks following
implantation. At necropsy, the skulls were collected and placed in
10% formaldehyde. The calvariae were scanned with a PIXI,
radiographed, and then decalcified for paraffin embedding and
sectioning. The coronal histological sections of the calvariae were
stained with toluidine blue and H&E stain (hematoxylin and
eosin). Amount of bone ingrowth to the defect was assessed by a 0
to 4 scoring system, with 4 as the largest amount.
Surgery Details
[0328] Total of forty-eight (48) plus four (4) spare male athymic
rats Hsd:RH-Foxn1rnu was ordered from Harlan, Indianapolis, Ind.
USA. The animals were specific pathogen free and approximately 6
weeks old upon arrival at MDS-PS-Efficacy Pharmacology. The rats
were anesthetized using ketamine/xylazine delivered via
intraperitoneal injection, as per standard operating procedures
(SOP). General anesthesia was accomplished in approximately 3-5
minutes and was noted by a lack of response to a toe pinch.
Sedation was maintained throughout surgery with isoflurane, as
needed.
[0329] The skull area was shaved using an electric clipper and
prepared with alcohol and chlorhexadine scrub. The animal was
positioned to firmly hold the head in a forward stable position and
a local anesthetic injection (approximately 0.2 ml xylocaine) was
administered subcutaneously in the central cranial area between the
ears. A transverse skin incision was made at the xylocaine
injection site and a tissue expander placed into the central region
of the rostral margin of the incision (skin flap). The expander
opened up the incision and exposed the cranium. The xylocaine, that
becomes gel-like, was excised and a transverse incision made in the
periosteum at the parietal/interparietal suture using the scalpel
blade. The periosteum was removed from the parietal bones after the
incision was made. A Dremel drill at a medium speed was used to
gently carve out the margin of both defects, approximately 3 mm by
5 mm area in each parietal bone, until the central piece of bone
was completely free from attachment. The area was irrigated with a
sterile saline drip during the drilling to prevent the bone from
becoming overheated. When the piece of bone was completely detached
it was removed with the Adson forceps. The edges of the defect were
checked and gently smoothed using forceps if necessary. To remove
bone dust and chips, the cranium was flushed with approximately 3
mL of sterile saline, which was absorbed with a piece of sterile
gauze. Once clean and excess fluid removed, the defect was treated
with the assigned test article. The dermis was then pulled back
over the cranium and the dermal incision closed using 3.0 or 4.0
Vicryl sutures. The animal facility personnel made post-operative
checks to document complete recovery of the animal. On Day 49
post-surgery, the remaining animals were euthanized by CO.sub.2
asphyxiation.
Analysis Details
[0330] The calvariae were collected and placed in 10% formaldehyde.
Following fixation, a Lunar dual energy x-ray absorptiometry (PIXI)
was used to determine the bone mineral density (BMD) of both
calvaria defects. An ROI smaller than the margins of the defect
area was set and the same size ROI was used for both defects in all
samples. The PIXI measured bone mineral area (BMA) and bone mineral
content (BMC; g). BMC was then divided by the BMA (mm.sup.2) to
determine the area bone mineral density (BMD; g/mm.sup.2). After
completion of the PIXI densitometry the calvariae were radiographed
and processed for Histology. The calvariae were radiographed,
processed through decalcified tissue processing and grossed into
two pieces. After grossing, the calvariae were embedded in
paraffin. Three coronal sections through the defect areas were cut,
each approximately 4-6 .mu.m in thickness, and mounted on slides.
One section was stained with toluidine blue and one was stained
with H&E for histopathological evaluation of bone ingrowth.
Determination of defect closure using x-ray scans was performed by
designating a region of interest surrounding the cranial defect,
use thresholding to define radio-lucent (dark residual defect) area
from radio-opaque areas, quantifying these areas of residual defect
using imaging software.
[0331] The results demonstrated a 20% increase in bone formation,
as seen by increases in BMC and BMD, and by H&E staining, for
OPACs compared to PDAC.TM.s in this model. Based on analysis of the
in vivo data, OPACS exhibited at least 20% greater bone formation
than PDAC.TM.s by histological (FIG. 14) and densitometric (PIXI)
analysis (FIG. 15). Furthermore, analysis of residual defect area
using x-ray scans obtained at the time of sacrifice indicated that
63% of animals in the OPAC group showed greater than 50% closure of
the defect as compared to 38% of animals in the PDAC group (FIG.
16).
6.3 Example 3
Treatment of Multiple Myeloma Using OPACS
[0332] 6.3.1 Materials & Methods
[0333] 6.3.1.1 Establishment of OPACs Stably Transduced with
Enhanced Green Fluorescent Protein (EGFP)
[0334] The pLEGFP retroviral vector containing an Enhanced Green
Fluorescent Protein (EGFP) coding sequence (Clontech, Palo Alto,
Calif., USA) was used to transiently transfect the packaging cell
line Phoenix Eco (ecotropic) using SuperFect (QIAGEN Inc.,
Valencia, Calif., USA). EGFP is a red-shifted variant of wild-type
Aequorea victoria green fluorescent protein that has been optimized
for brighter fluorescence and higher expression in mammalian cells.
Supernatants containing retroviral particles were collected 24-48
hours after transfection. OPACs were infected with the retroviral
particles in the presence of 8 .mu.g/ml polybrene for 12 hours at
which time the media were replaced with fresh culture medium. In
some experiments, cells were exposed to the supernatants containing
the viral particles once more before being selected by culturing
them in the presence of 200-400 .mu.g/ml of G418 for 2-3 weeks.
[0335] 6.3.1.2 Engraftment of OPACs in Myelomatous SCID-Rab
Mice
[0336] As an alternative to using human bone tissue in a SCID-hu
model of primary human myeloma, a system in which rabbit bones were
implanted into SCID mice (SCID-rab mice), followed by introduction
of myeloma cells directly into the implanted bone, was used
instead. Myelomatous SCID-rab mice were constructed as previously
described. See Yata, K. and Yaccoby, S., et al, Leukemia 2004;
18:1891-1897. CB.17/Icr-SCID mice (6-8-week old) were obtained from
Harlan Sprague Dawley (Indianapolis, Ind., USA) and pregnant New
Zealand rabbits from Myrtle Rabbitry (Thompson Station, Tenn.,
USA). The 3-4-week-old rabbits were deeply anesthetized with a high
dose of pentobarbital sodium and euthanized by cervical
dislocation. The rabbit femora and tibiae were cut into two pieces,
with the proximal and distal ends kept closed, while the vertebrae
were cut into small fragments (1.times.2 cm.sup.2).
[0337] For bone implantation, the right or left side of the SCID
mouse was rinsed with alcohol and blotted with sterile gauze. The
rabbit bone was inserted subcutaneously through a small (5 mm)
incision. The incision was then closed with sterile surgical
staples, and engraftment of the bones was allowed to take place for
6-8 weeks. In some experimental mice, two bones were simultaneously
implanted contralaterally in the same mouse. For each experiment,
10-50.times.10.sup.6 unseparated human patient-derived myeloma bone
marrow cells containing 17+/-8% plasma cells (PCs) or
3.3+/-1.6.times.10.sup.6 PCs in 50 .mu.l of phosphate-buffered
saline (PBS) were injected directly into the implanted rabbit bone.
At least two mice were used for each experiment. Mice were
periodically bled from the tail vein to measure changes in levels
of circulating human immunoglobulin (Ig) of the M-protein
isotype.
[0338] Establishment of myeloma growth was demonstrated by
increased levels of human monoclonal immunoglobulins (hIg) in mouse
sera, as seen by ELISA, and by radiographic evaluation of lytic
bone lesions. 5.times.10.sup.5 EGFP-expressing OPACs were collected
with the use of trypsin-EDTA and resuspended in 50 .mu.l PBS. The
OPACs were injected directly into the implanted bones in the
SCID-rab mice. Experiments were continued for 8-16 weeks
post-injection. Changes in the bone mineral density (BMD) of the
implanted bones were determined using a PIXImus DEXA densitometer
(GE Medical Systems LUNAR, Madison, Wis.).
[0339] 6.3.1.3 Immunohistochemistry of Tissue Harvested from
SCID/Rab Mice
[0340] Decalcified bone sections from primary myeloma-bearing
SCID-rab mice were deparaffinized in xylene, rehydrated with
ethanol, rinsed in PBS, and antigen retrieved using microwave as
previously described (see Yata, K., supra). Cultured OPACs were
trypsinized, cytospin slides prepared and fixed with 10%
phosphate-buffered formalin for 20 min. After peroxidase quenching
with 3% hydrogen peroxide for 10 min, the slides were incubated
with monoclonal antibodies against EGFP, and human CD166,
osteocalcin and BMP-2 (5-10 .mu.g/ml) for 30-60 min and developed
using Dako's immunoperoxidase kit and counterstaining with
haematoxylin.
[0341] 6.3.1.4 Von Kossa and Alizarin Red Staining
[0342] For detection of calcium deposition (von Kossa staining),
OPACs were fixed in 10% phosphate-buffered formalin for 10 min.
Freshly prepared 5% silver nitrate was added and the specimens left
in the dark for 10 min, rinsed with distilled water and then
exposed to UV light for 15 min while covered with water. The
reaction was stopped by rinsing thoroughly with distilled
water.
[0343] 6.3.1.5 Statistical Analysis
[0344] Unless indicated otherwise, all values are expressed as
mean.+-.standard error of the mean (SEM). Student's paired t-test
was used to test the effect of different culture conditions on
myeloma cell numbers, viability, apoptosis, and proliferation, and
to test the effect of OPACs on tumor growth and human bone mineral
density (BMD) in SCID-rab mice.
[0345] 6.3.1.6 Lentivirus-Mediated Transduction Of OPACs
[0346] All recombinant lentiviruses were produced by transient
transfection of 293T cells according to a standard protocol.
Briefly, 293T cells were incubated overnight with transfection
precipitate; afterward, culture medium was replaced and incubation
for an additional 2 days followed. The media of the transfected
cells were harvested, centrifuged at 3,000 rpm at 4.degree. C. for
15 minutes, and filtered through a 0.22-.mu.m-pore-size filter. The
filtrate was layered on top of a 20% sucrose cushion and spun at
26,000 rpm at 4.degree. C. for 100 minutes in a Beckman
ultracentrifuge using an SW28 rotor. The pelleted virus-like
particles were suspended in DMEM and stored at -80.degree. C.
Titers of the virus stocks (titer units [TU]/ml) were determined by
adding aliquots of virus suspension on monolayers of 293T cells or
other appropriate cell types and then assessing the percentage of
GFP-positive cells by fluorescence-activated cell sorting (FACScan,
Becton Dickinson). Titers greater than 10.sup.9 TU/ml were
routinely obtained.
[0347] OPACs were transfected with the retroviral particles in the
presence of 8 .mu.g/ml polybrene for 12 hours at which time the
media were replaced with fresh culture medium. In some experiment,
cells were exposed to the supernatants containing the viral
particles once more before being selected by culturing them in the
presence of 200-400 .mu.g/ml of G418 for 2-3 weeks.
[0348] 6.3.2 Results
[0349] 6.3.2.1 Matrix Mineralization by OPACs
[0350] OPACs were cultured in presence or absence of osteogenic
media (alpha MEM supplemented with 10% FBS, dexamethasone (100 nM),
ascorbate (0.05 mM) and beta GP (10 mM)) for approximately 2 weeks.
For detection of calcium deposition cells were fixed in 10%
phosphate buffered formalin for 10 minutes. Fixed cells were
stained with alizarin red dye. The reaction was stopped by rinsing
thoroughly with distilled water. OPACs were cultured in osteogenic
media displayed signs of matrix mineralization as indicated by
binding alizarin red to calcium which is deposited on the
extracellular matrix produced by the cells. Matrix mineralization
is one marker of osteogenic differentiation (data not shown).
[0351] 6.3.2.2 OPAC Inhibition of Osteoclast Maturation
[0352] Coculture experiments: Using transwell devices, OPACs or
MSCs were first cultured on the backside of the insert membrane
with osteoblastic media; osteoclast precursors were then incubated
in the upper chamber. To allow differentiation of osteoblasts and
osteoclasts simultaneously, the inserts were then incubated with
.alpha.-MEM supplemented with 10% PBS, RANKL (50 ng/ml), M-CSF (25
ng/ml), dexamethasone (100 nM), ascorbate (0.05 mM) and .beta.GP
(10 mM) for approximately 2 weeks. This procedure resulted in
simultaneous growth of multinucleated osteoclasts expressing
tartrate-resistant acidic phosphatase (TRAP) and osteoblasts
expressing alkaline phosphate. Osteoclast precursors cultured in
the absence of OPACs or mesenchymal stem cells produced an average
of 120 osteoclasts (see FIG. 17). In contrast, osteoclast
precursors produced an average of 40 osteoclasts when cultured in
the presence of placental stem cells, and an average of 60
osteoclasts when cultured in the presence of MSCs. Thus, OPACs
appear to suppress the formation of osteoclasts more effectively
than do mesenchymal stem cells.
[0353] The effect of OPACs on osteoclast differentiation was
significantly reduced, and significantly more osteoclasts formed,
when osteoclasts and OPACs were cultured in the presence of an
antibody to osteoprotegerin (anti-OPG; p<0.04). Additionally,
significantly (p<0.004) more osteoclasts formed when OPACs,
alone, were cultured with osteoclast precursors than when
osteoclast precursors were cultured in the presence of anti-OPG.
Thus, without wishing to be bound by any particular mechanism or
theory, the suppression of osteoclast differentiation by OPACs
appears to be mediated by OPAC-secreted osteoprotegerin.
[0354] 6.3.2.3 Suppression of Multiple Myeloma Cell Growth by
OPACs
[0355] Multiple myeloma cells were obtained from heparinized bone
marrow (BM) aspirates from 27 patients with active myeloma during
scheduled clinic visits. The bone marrow samples were separated by
density centrifugation using Ficoll-Paque (specific gravity 1.077
g/ml) and the proportion of multiple myeloma plasma cells in the
light-density cell fractions determined by CD38/CD45 flow
cytometry. Plasma cells (PCs) were isolated using CD138
immunomagnetic bead selection and the autoMACs automated separation
system (Miltenyi-Biotec, Auburn, Calif.). PC purity was determined
by CD38/CD45 flow cytometry to be routinely.gtoreq.94%.
[0356] OPACs and MSCs were treated under standard conditions or
osteogenic conditions and cocultured with multiple myeloma cells.
For co-culture experiments transwell inserts with 1-.mu.m pores
were used. In this system, osteoblasts (i.e., MSCs or OPACs grown
under osteogenic conditions), MSCs or OPACs were grown on the
backside of the inserts' membranes and multiple myeloma (Con MM)
cells were cultured in the upper chamber of the inserts. For
culturing, 6-well inserts were flipped upside down and placed in a
sterile deep dish. MSCs or OPACs were collected with trypsin-EDTA
and resuspended in MSC medium (approximately
0.5.times.10.sup.6/ml). Approximately 600 .mu.l of cells were
placed onto the center of the inverted insert. The dish was then
covered with parafilm and placed in the incubator for 1 hour,
allowing cells to adhere to the membrane. Following incubation, the
inserts were flipped back and placed in 6-plate wells. When MSCs or
OPACs were approximately 80% confluent, they were cultured with MSC
medium or with osteoblastic medium for 2-3 weeks. The viability of
the multiple myeloma cells was assessed using an MTT assay after 72
hours. The MTT assay is a colorimetric assay for measuring the
activity of enzymes that reduce MTT to formazan, giving a purple
color. This reduction takes place only when mitochondrial reductase
enzymes are active, and therefore conversion is often used as a
measure of viable cells.
[0357] The results show that undifferentiated OPACs inhibited the
viability of Con MM cells by about 70% while MSCs only inhibited
Con MM viability by about 40% (see FIG. 18). Under osteogenic
conditions OPACs inhibited the viability of Con MM cells even more
than under non-osteogenic conditions; MSCs also showed more
inhibition under osteogenic conditions, although not to the same
level as OPACs (see FIG. 18).
[0358] OPACs were also tested for their ability to suppress
proliferation of multiple myeloma cell lines compared to fetal bone
marrow-derived mesenchymal stem cells (FB MSC) in a co-culture
environment that allowed for cell-cell contact. OPACs (10,000
cells/well) were cultured with multiple myeloma cell lines BN and
JB (see Li et al., Br. J Haematology 138(6):802-811 (2007)), ARP1
(dexamethasone-sensitive IgA multiple myeloma-derived cell line),
U266 (an IgE-producing plasma cell line), Dn and Hale (10,000
cells/well), all of which expressed luciferase, for 7 days in RPMI
medium comprising 10% fetal bovine serum and antibiotics. Growth of
multiple myeloma cells was assessed by detection of luciferase
activity. OPACs suppressed growth of the multiple myeloma cell
lines approximately as follows: BN (0.4); JB (0.5); ARP1 (0.15);
U266 (0.28); Dn (0.32); and hale (0.75), wherein the number in
parentheses indicates the growth of the MM cell lines in fold of
growth of the MM cell lines co-cultured with FB-MSC.
[0359] In a further experiment, multiple myeloma cells from six
different human patients were obtained and co-cultured at 400,000
cells/well with fetal bone marrow-derived mesenchymal stem cells or
OPACs for 6-10 days. Viability of the multiple myeloma cells was
significantly reduced in the presence of OPACs as compared to fetal
MSCs (p<0.03).
[0360] 6.3.2.4 Biodistribution of Labeled, Intralesionally
Administered OPACs in Animals
[0361] This example demonstrates the biodistribution of transfected
OPACs in mice. OPACs were transfected with a luciferase reporter.
The pLEGFP retroviral vector containing the EGFP (Clontech, Palo
Alto, Calif., USA) was used to transiently transfect the packaging
cell line Phoenix Eco using SuperFect (QIAGEN Inc., Valencia,
Calif., USA). Supernatants containing retroviral particles were
collected 24-48 hours after transfection. OPACs were transfected
with the retroviral particles in the presence of 8 .mu.g/ml
polybrene for 12 hours at which time the medium was replaced with
fresh culture medium. In some experiments, cells were exposed to
the supernatants containing the viral particles once more before
being selected by culturing them in the presence of 200-400
.mu.g/ml of G418 for 2-3 weeks.
[0362] SCID-rab animals were implanted with rabbit bone as
previously described. Animals having bone that showed progressive
lesions were injected with labeled cells (1.times.10.sup.6 cells)
directly into the lesions. Biodistribution of the OPACs in the mice
was monitored by bioluminescence imaging, a real-time, non-invasive
tool, at different time points. OPACs expressing EGFP could be
detected in the mice at 2 weeks and 5 weeks after infection in
areas where the exogenous bone was implanted into the SCID-rab
animals (data not shown).
[0363] 6.3.2.5 OPACs Increase Bone Mineral Density in Primary
Myelomatous SCID-rab Mice
[0364] Primary myelomatous SCID-rab mice were constructed as
described above. Upon establishment of myeloma growth as assessed
by increased level of human monoclonal immunoglobulins (hIg) in the
mice sera using ELISA and by radiographic evaluation of lytic bone
lesions, 1.times.10.sup.6 EGFP-expressing OPACs were collected with
the use of trypsin-EDTA and resuspended in 100 .mu.l PBS. The OPACs
and PBS were injected directly into the implanted bones, comprising
myelomatous lesions. Changes in the bone mineral density (BMD) of
the implanted bones were determined using a PIXImus DEXA (GE
Medical Systems LUNAR, Madison, Wis.) at 5 week intervals for 8-16
weeks post-injection. Intralesionally-administered OPACs were found
to increase the bone mineral density (BMD) of the implanted bone
compared to controls in which only medium was injected. (p=0.0006.)
(See FIG. 19)
[0365] 6.3.2.6 OPACs Increase Bone Mass in Multiple
Myeloma-Affected Bones
[0366] Myelomatous SCID-rab mice constructed as described above and
were injected with primary myeloma cells from a human patient.
Following the development of osteolytic lesions as demonstrated by
increased level of human monoclonal immunoglobulins (hIg) in the
mice sera, as assessed by ELISA and by radiographic evaluation of
lytic bone lesions, 5-10.times.10.sup.5 EGFP-expressing OPACs were
administered as described in the previous example. Experiments were
continued for 8-16 weeks post-injection. The level of human myeloma
cells was determined using human monoclonal immunoglobulins (hIg)
in the mice sera by ELISA assay post injection of the OPACS (see
FIG. 20). Changes in the bone mineral density (BMD) of the
implanted bones were determined using a PIXImus DEXa (BE Medical
Systems LUNAR, Madison, Wis.). Over the course of the study,
implanted bones in myelomatous mice receiving
intralesionally-administered OPACs showed a significant increase
(0.10 gm/cm.sup.2) in bone mass after 35 days post injection as
compared to mice receiving only buffer (p=0.006).
[0367] 6.3.2.7 OPACs Inhibit Bone Destruction in SCID-Rab Animals
Administered an Aggressive Multiple Myeloma Cell Line
[0368] Myelomatous SCID-rab mice were constructed as described
above. Upon establishment of myeloma using an aggressive BN myeloma
cell line (a non hyperdiploid cell line isolated from a human
patient), the effect of buffer or OPACs on the myeloma cells'
growth was assessed by increased level of human monoclonal
immunoglobulins (hIg) in the mice sera using ELISA and by
radiographic evaluation of lytic bone lesions. 0.5.times.10.sup.6
EGFP-expressing OPACs (0.5.times.10.sup.6 cells) were collected
with the use of trypsin-EDTA and resuspended in 50 .mu.l PBS. The
OPACs and PBS were injected directly into the implanted bones in
SCID-rab mice. Experiments were continued for 8-16 weeks
post-injection. Changes in the bone mineral density (BMD) of the
implanted bones were determined using a PlXImus DEXA (GE Medical
Systems LUNAR, Madison, Wis.). The data show that OPACs inhibited
bone loss that would be attributable to this aggressive multiple
myeloma cell line as measured by bone mineral density (BMD) and
bone mineral content (BMC). See FIG. 21.
[0369] The reduction/elimination of loss of bone mass due to the
OPACs was confirmed by X-ray radiography (data not shown).
OPAC-treated animals had higher bone mass as compared to control
animals as demonstrated by a significant increase in the
radio-dense areas of the new bone.
6.4 Example 4
Treatment of Multiple Myeloma Using OPACS in Combination with
Melphalan
[0370] This example demonstrates the effectiveness of
administration of OPACs in combination with melphalan to treat
osteolytic lesions associated with multiple myeloma.
[0371] Myelomatous SCID-rab mice were implanted with bone as
described in Section 6.3.1.2, above. Mice were separated into
groups receiving OPACS, at a dose of about 1 million in phosphate
buffered saline, and controls receiving no OPACs. Mice were
injected at week zero with an EGFP/Luciferase-expressing multiple
myeloma cell line (BN), directly into the implanted bone, and
treated with 10 mg/kg melphalan subcutaneously twice a week for
four weeks, starting at week 3 (weeks 3-7 post-myeloma cell
injection). At week 7, melphalan treatment was discontinued, and
approximately 1 million cells/mouse unlabeled OPACs were injected
intralesionally into the implanted bone. Progress of the disease
was followed for an additional eleven weeks. Live animal imaging
was performed at weeks 3, 7 and 18. Mice receiving OPACs showed a
reduced multiple myeloma tumor cell burden, as evidenced by EGFP
and luciferase fluorescence, compared to controls. Moreover, mice
receiving OPACs retained bone mass better than control mice,
showing an approximately 28% increase in bone mass post-melphalan
as compared to a loss of approximately 4% post-melphalan for
control mice.
6.5 Example 5
Production of Cryopreserved OPAC Product and Cell Bank
[0372] This Example demonstrates production of a frozen OPACs-based
product.
[0373] Cryopreservation: OPACs are obtained as described in Example
1. Cells to be frozen down are harvested from culture with
Trypsin-EDTA, quenched with 2% FBS in PBS, and counted on a
hemacytometer. After centrifugation, cells are resuspended with 10%
DMSO in FBS to a concentration of about 1 million cells/ml for
cells to be used for assembly of a cell bank, and 10 million
cells/ml for individual frozen cell doses. The cell solution is
transferred to a freezing container, which is placed in an
isopropyl alcohol bath in a -80.degree. C. freezer. The following
day, cells are transferred to liquid nitrogen.
[0374] 6.5.1 Design of an OPAC Bank
[0375] A "lot" is defined as all cell doses derived from a single
donor chorion. Cells maintained normal growth, karyotype, and cell
surface maker phenotype for over 8 passages and 30 doublings during
expansion culture. Given this limitation, doses comprise cells from
5 passages and about 20 doublings. To generate a supply of
equivalent cells, a single lot is expanded in culture and is stored
in a two-tiered cell bank and frozen doses. In particular, cells
harvested from the primary culture, which are defined as Passage 0
cells having undergone 0 doublings, are used to initiate an
expansion culture. After the first passage, approximately 4
doublings occur, and cells are frozen in a Master Cell Bank (MCB).
Vials from the MCB are used to seed additional expansion cultures.
After two additional passages of cells thawed from the MCB, cells
are frozen down in a Working Cell Bank (WCB), approximately 12
cumulative doublings. Vials from the WCB are used to seed an
expansion culture for another 2 passages, resulting in Passage 5
cells at approximately 20 doublings that are frozen down into
individual doses.
[0376] 6.5.2 Thawing Cells for Culture
[0377] Frozen containers of cells are placed into a sealed plastic
bag and immersed in a 37.degree. C. water bath. Containers are
gently swirled until all of the contents are melted except for a
small piece of ice. Containers are removed from the sealed plastic
bag and a 10.times. volume of culture medium is slowly added to the
cells with gentle mixing. A sample is counted on the hemacytometer
and seeded into expansion cultures.
[0378] 6.5.3 Thawing Cells for Injection
[0379] Frozen containers of cells are transferred to the
administration site in a dry nitrogen shipper. Prior to
administration, containers are placed into a sealed plastic bag and
immersed in a 37.degree. C. water bath. Containers are gently
swirled until all of the contents are melted except for a small
piece of ice. Containers are removed from the sealed plastic bag
and an equal volume of 2.5% HSA/5% Dextran is added. Cells are
injected with no further washing.
[0380] 6.5.4 Testing and Specifications
[0381] A maternal blood sample accompanies all donor placentas. The
sample is screened for Hepatitis B core antibody and surface
antigen, Hepatitis C Virus antibody and nucleic acid, and HIV I and
II antibody and nucleic acid. Placental processing and primary
culture begins prior to the receipt of test results, but continues
only for placentas associated with maternal blood samples testing
negative for all viruses. A lot is rejected if the donor tests
positive for any pathogen. In addition, the tests described in
Table 3 are performed on the MCB, the WCB, and a sample of the cell
dose material derived from a vial of the WCB. A lot is released
only when all specifications are met.
TABLE-US-00010 TABLE 3 Cell testing and specifications Test Methods
Required Result Sterility BD BACTEC PEDS Negative PLUS/F and BACTEC
Myco/F Lytic Endotoxin LAL gel clot .ltoreq.5 EU/ml* Viability
Trypan Blue >70% viable Mycoplasma Direct culture, DNA- Negative
fluorochrome (FDA PTC 1993) Identity Flow cytometry CD105.sup.+;
CD200.sup.dim/CD200.sup.- Cell Purity Microsatellite No
contaminating cell detected Karyotype G-banding and Normal
chromosome count on metaphase cells *For the product designed to be
40 ml of frozen cells/dose and a maximum of 5 EU/ml, the cell
product is below the upper limit of 5 EU/kg/dose for recipients
over 40 kg in body weight.
6.6 Example 6
Treatment of Multiple Myeloma by Administration of OPACS
[0382] 6.6.1 Intralesional Administration of OPACs in Solution
[0383] An individual presents with multiple myeloma, with symptoms
of bone pain and hypercalcemia (in this case, blood calcium levels
of between 3 and 4 mmol/L). X-ray imaging confirms the presence of
multiple lesions in the tibia, fibula, radius and ulna. About
1.times.10.sup.7 to about 1.times.10.sup.8 OPACs in 1.0-2.0 mL
phosphate buffered saline (PBS) per lesion are administered to the
individual by injection directly into the lesion. The individual is
assessed every two weeks following injection by X-ray to determine
the extent of the bone lesions, and blood calcium levels are
assessed every week until bone lesions are visibly reduced by
X-ray, or blood calcium levels are detectably reduced. OPACs are
optionally re-administered within four weeks of initial
administration.
[0384] 6.6.2 Intralesional Administration of OPACs in Collagen
Gel
[0385] An individual presents with multiple myeloma, with symptoms
of bone pain and hypercalcemia (in this case, blood calcium levels
of between 3 and 4 mmol/L). X-ray imaging confirms the presence of
multiple lesions in the tibia, fibula, radius and ulna. About
1.times.10.sup.7 to about 1.times.10.sup.8 OPACs, in 1.0-2.0 mL
phosphate buffered saline (PBS) comprising collagen sufficient to
form an injectable gel, per lesion, are administered to the
individual by injection directly into the lesion. The individual is
assessed every two weeks following injection by X-ray to determine
the extent of the bone lesions, and blood calcium levels are
assessed every week until bone lesions are visibly reduced by
X-ray, or blood calcium levels are reduced to 3 mmol/L or less.
OPACs are optionally re-administered within four weeks of initial
administration.
[0386] 6.6.3 Intralesional Administration of OPACs with Bone Graft
Replacement
[0387] An individual presents with multiple myeloma, with symptoms
of bone pain and hypercalcemia (in this case, blood calcium levels
of between 3 and 4 mmol/L). X-ray imaging confirms the presence of
multiple lesions in the tibia, fibula, radius and ulna. At bedside,
an injectable bone graft substitute (e.g., HEALOS.RTM.) is premixed
with about 1.times.10.sup.7 to about 1.times.10.sup.8 OPACs, in
1.0-2.0 mL phosphate buffered saline (PBS), then injected into the
individual at the site of the lesions. The individual is assessed
every two weeks following injection by X-ray to determine the
extent of the bone lesions, and blood calcium levels are assessed
every week until bone lesions are visibly reduced by X-ray, or
blood calcium levels are reduced to 3 mmol/L or less. OPACs are
optionally re-administered within four weeks of initial
administration.
[0388] 6.6.4 Intravenous Administration of OPACs
[0389] An individual presents with multiple myeloma, with symptoms
of bone pain and hypercalcemia (in this case, blood calcium levels
of between 3 and 4 mmol/L). X-ray imaging confirms the presence of
multiple lesions in the tibia, fibula, radius and ulna. About
1.times.10.sup.9 to about 1.times.10.sup.10 OPACs, in about 750 mL
phosphate buffered saline (PBS) are administered to the individual
by intravenous infusion. The individual is assessed every two weeks
following injection by X-ray to determine the extent of the bone
lesions, and blood calcium levels are assessed every week until
bone lesions are visibly reduced by X-ray, or blood calcium levels
are reduced to 3 mmol/L or less. OPACs are optionally
re-administered within four weeks of initial administration.
Equivalents:
[0390] The compositions and methods provided herein are not to be
limited in scope by the specific embodiments described herein.
Indeed, various modifications of the embodiments 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.
[0391] Various publications, patents and patent applications are
cited herein, the disclosures of which are incorporated by
reference in their entireties.
* * * * *