U.S. patent application number 11/321864 was filed with the patent office on 2006-07-13 for cartilage and bone repair and regeneration using postpartum-derived cells.
This patent application is currently assigned to Ethicon, Incorporated. Invention is credited to Sridevi Dhanaraj, Alexander M. Harmon, Darin J. Messina.
Application Number | 20060153818 11/321864 |
Document ID | / |
Family ID | 38456561 |
Filed Date | 2006-07-13 |
United States Patent
Application |
20060153818 |
Kind Code |
A1 |
Dhanaraj; Sridevi ; et
al. |
July 13, 2006 |
Cartilage and bone repair and regeneration using postpartum-derived
cells
Abstract
Cells derived from postpartum tissue and methods for their
isolation and induction to differentiate to cells of a chondrogenic
or osteogenic phenotype are provided by the invention. The
invention further provides cultures and compositions of the
postpartum-derived cells and products related thereto. The
postpartum-derived cells of the invention and products related
thereto have a plethora of uses, including but not limited to
research, diagnostic, and therapeutic applications, for example, in
the treatment of bone and cartilage conditions.
Inventors: |
Dhanaraj; Sridevi; (Raritan,
NJ) ; Harmon; Alexander M.; (Clinton, NJ) ;
Messina; Darin J.; (Somerville, NJ) |
Correspondence
Address: |
WOODCOCK WASHBURN LLP
ONE LIBERTY PLACE, 46TH FLOOR
1650 MARKET STREET
PHILADELPHIA
PA
19103
US
|
Assignee: |
Ethicon, Incorporated
Somerville
NJ
|
Family ID: |
38456561 |
Appl. No.: |
11/321864 |
Filed: |
December 29, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10876998 |
Jun 25, 2004 |
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11321864 |
Dec 29, 2005 |
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60483264 |
Jun 27, 2003 |
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Current U.S.
Class: |
424/93.7 ;
435/366 |
Current CPC
Class: |
A61K 38/204 20130101;
C12N 2500/34 20130101; A61K 38/27 20130101; A61P 19/00 20180101;
A61P 19/04 20180101; A61P 19/08 20180101; A61K 38/1866 20130101;
A61P 1/00 20180101; A61P 1/02 20180101; A61P 17/02 20180101; A61K
38/185 20130101; A61P 1/16 20180101; A61P 1/18 20180101; C12N
5/0606 20130101; A61P 39/06 20180101; A61P 43/00 20180101; A61K
38/1858 20130101; C12N 2533/50 20130101; A61K 38/1841 20130101;
A61K 35/12 20130101; A61P 21/00 20180101; C12N 2501/21 20130101;
C12N 2501/23 20130101; A61K 35/50 20130101; C12N 2509/00 20130101;
A61P 13/12 20180101; A61P 37/02 20180101; A61P 37/06 20180101; C12N
2506/03 20130101; A61K 38/1825 20130101; A61K 38/19 20130101; C12N
2500/90 20130101; A61K 38/18 20130101; A61K 38/1808 20130101; A61P
9/10 20180101; C12N 5/0605 20130101; C12N 5/0607 20130101; A61P
25/00 20180101; A61P 25/02 20180101; C12N 2501/12 20130101; A61P
27/02 20180101; A61P 27/06 20180101; C12N 2500/44 20130101; A61K
35/51 20130101; A61P 25/14 20180101; A61P 25/16 20180101; C12N
2500/95 20130101; A61P 19/10 20180101; A61P 7/02 20180101; A61K
38/2053 20130101; A61P 9/00 20180101; A61P 9/04 20180101; C12N
2502/02 20130101; A61P 35/00 20180101; C12N 2506/02 20130101; A61K
38/1833 20130101; A61K 38/1891 20130101; A61P 25/28 20180101; C12N
2500/32 20130101; A61P 29/00 20180101 |
Class at
Publication: |
424/093.7 ;
435/366 |
International
Class: |
A61K 35/12 20060101
A61K035/12; C12N 5/08 20060101 C12N005/08 |
Claims
1-85. (canceled)
86. A conditioned medium generated by the growth of a culture of
cells in a culture medium wherein said culture of cells comprises
at least one postpartum-derived cell (PPDC) wherein said PPDC is
derived from human postpartum tissue substantially free of blood,
wherein said PPDC is capable of self-renewal and expansion in
culture and has the potential to differentiate into a cell of an
osteogenic or chondrogenic phenotype; wherein said PPDC requires
L-valine for growth; wherein said PPDC is capable of growth in
about 5% to about 20% oxygen; wherein said PPDC further comprises
at least one of the following characteristics: (a) production of at
least one of granulocyte chemotactic protein 2 (GCP-2), reticulon
1, tissue factor, vimentin, and alpha-smooth muscle actin; (b) lack
of production of at least one of GRO-alpha or oxidized low density
lipoprotein receptor, as detected by flow cytometry; (c) production
of at least one of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, PD-L2
and HLA-A,B,C; (d) lack of production of at least one of CD31,
CD34, CD45, CD80, CD86, CD117, CD141, CD178, B7-H2, HLA-G, and
HLA-DR,DP,DQ, as detected by flow cytometry; (e) expression, which
relative to a human cell that is a fibroblast, a mesenchymal stem
cell, or an ileac crest bone marrow cell, is increased for at least
one of interleukin 8; reticulon 1; chemokine (C--X--C motif) ligand
1 (melanoma growth stimulating activity, alpha); chemokine (C--X--C
motif) ligand 6 (granulocyte chemotactic protein 2); chemokine
(C--X--C motif) ligand 3; and tumor necrosis factor, alpha-induced
protein 3 or expression, which relative to a human cell that is a
fibroblast, a mesenchymal stem cell, or an ileac crest bone marrow
cell, is increased for at least one of C-type lectin superfamily
member A2, Wilms tumor 1, aldehyde dehydrogenase 1 family member
A2, renin, oxidized low density lipoprotein receptor 1, protein
kinase C zeta, clone IMAGE:4179671, hypothetical protein
DKFZp564F013, downregulated in ovarian cancer 1, and clone
DKFZp547K1113; (f) expression, which relative to a human cell that
is a fibroblast, a mesenchymal stem cell, or an ileac crest bone
marrow cell, is reduced for at least one of: short stature homeobox
2; heat shock 27 kDa protein 2; chemokine (C--X--C motif) ligand 12
(stromal cell-derived factor 1); elastin; cDNA DKFZp586M2022 (from
clone DKFZp586M2022); mesenchyme homeobox 2; sine oculis homeobox
homolog 1; crystallin, alpha B; dishevelled associated activator of
morphogenesis 2; DKFZP586B2420 protein; similar to neuralin 1;
tetranectin; src homology three (SH3) and cysteine rich domain;
B-cell translocation gene 1, anti-proliferative; cholesterol
25-hydroxylase; runt-related transcription factor 3; hypothetical
protein FLJ23191; interleukin 11 receptor, alpha; procollagen
C-endopeptidase enhancer; frizzled homolog 7; hypothetical gene
BC008967; collagen, type VIII, alpha 1; tenascin C; iroquois
homeobox protein 5; hephaestin; integrin, beta 8; synaptic vesicle
glycoprotein 2; cDNA FLJ12280 fis, clone MAMMA1001744; cytokine
receptor-like factor 1; potassium intermediate/small conductance
calcium-activated channel, subfamily N, member 4; integrin, alpha
7; DKFZP586L151 protein; transcriptional co-activator with
PDZ-binding motif (TAZ); sine oculis homeobox homolog 2; KIAA1034
protein; early growth response 3; distal-less homeobox 5;
hypothetical protein FLJ20373; aldo-keto reductase family 1, member
C3 (3-alpha hydroxysteroid dehydrogenase, type II); biglycan;
fibronectin 1; proenkephalin; integrin, beta-like 1 (with EGF-like
repeat domains); cDNA clone EUROIMAGE 1968422; EphA3; KIAA0367
protein; natriuretic peptide receptor C/guanylate cyclase C
(atrionatriuretic peptide receptor C); hypothetical protein
FLJ14054; cDNA DKFZp564B222 (from clone DKFZp564B222);
vesicle-associated membrane protein 5; EGF-containing fibulin-like
extracellular matrix protein 1; BCL2/adenovirus E1B 19 kDa
interacting protein 3-like; AE binding protein 1; cytochrome c
oxidase subunit VIIa polypeptide 1 (muscle); neuroblastoma,
suppression of tumorigenicity 1; and insulin-like growth factor
binding protein 2, 36 kDa; (g) secretion of at least one of
monocyte chemotactic protein-1, interleukin(IL)-6, IL-8,
granulocyte chemotactic protein-2, hepatocyte growth factor,
keratinocyte growth factor, fibroblast growth factor, heparin
binding-epidermal growth factor, brain derived neurotrophic factor,
thrombopoietin, macrophage inflammatory protein (MIP)-1a, RANTES,
and tissue inhibitor of matrix metalloprotease 1; (h) lack of
secretion of at least one of transforming growth factor-beta2,
angiopoetin-2, platelet derived growth factor-bb, MIP 1b, I309,
macrophage-derived chemokine, and vascular endothelial growth
factor, as detected by ELISA; and (i) the ability to undergo at
least 40 population doublings in culture.
87-108. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This claims benefit of U.S. Provisional Application Ser. No.
60/483,264, filed Jun. 27, 2003, the entire contents of which are
incorporated by reference herein. Other related applications
include the following commonly-owned, co-pending applications, the
entire contents of each of which are incorporated by reference
herein: U.S. application No. [ETH-5073 NP1], filed Jun. 25, 2004,
U.S. application No. [ETH-5073 NP2], filed Jun. 25, 2004, U.S.
application No. [ETH-5073 NP3], filed Jun. 25, 2004, U.S.
application No. [ETH-5073 NP4], filed Jun. 25, 2004, U.S.
application No. [ETH-5073 NP5], filed Jun. 25, 2004, U.S.
application No. [ETH-5073 NP7], filed Jun. 25, 2004, and U.S.
Provisional Application No. 60/555,908 [ETH 5127], filed Mar. 24,
2004. [ETH 5127], filed Mar. 24, 2004.
FIELD OF THE INVENTION
[0002] This invention relates to the field of mammalian cell
biology and cell culture. In particular, the invention relates to
cells derived from postpartum tissue having the potential to
differentiate into chondrogenic and osteogenic lineages, and
methods of preparation and use of those postpartum tissue-derived
cells, including cell-based therapies for conditions of bone and
cartilage.
BACKGROUND OF THE INVENTION
[0003] Diseases and conditions of bone and cartilage affect a large
portion of the population. Three types of cartilage are present in
mammals and include: hyaline cartilage, fibrocartilage, and elastic
cartilage. Hyaline cartilage consists of a gristly mass having a
firm, elastic consistency, is translucent and pearly blue in color.
Hyaline cartilage is predominantly found on the articulating
surfaces of articulating joints. It is found also in epiphyseal
plates, costal cartilage, tracheal cartilage, bronchial cartilage
and nasal cartilage. Fibrocartilage is essentially the same as
hyaline cartilage except that it contains fibrils of type I
collagen that add tensile strength to the cartilage. The
collagenous fibers are arranged in bundles, with the cartilage
cells located between the bundles. Fibrocartilage is found commonly
in the annulus fibrosis of the invertebral disc, tendonous and
ligamentous insertions, menisci, the symphysis pubis, and
insertions of joint capsules. Elastic cartilage also is similar to
hyaline cartilage except that it contains fibers of elastin. It is
more opaque than hyaline cartilage and is more flexible and pliant.
These characteristics are defined in part by the elastic fibers
embedded in the cartilage matrix. Typically, elastic cartilage is
present in the pinna of the ears, the epiglottis, and the
larynx.
[0004] The surfaces of articulating bones in mammalian joints are
covered with articular cartilage. The articular cartilage prevents
direct contact of the opposing bone surfaces and permits the near
frictionless movement of the articulating bones relative to one
another. Two types of articular cartilage defects are commonly
observed in mammals and include full-thickness and
partial-thickness defects. The two types of defects differ not only
in the extent of physical damage but also in the nature of repair
response each type of lesion elicits.
[0005] Full-thickness articular cartilage defects include damage to
the articular cartilage, the underlying subchondral bone tissue,
and the calcified layer of cartilage located between the articular
cartilage and the subchondral bone. Full-thickness defects
typically arise during severe trauma of the joint or during the
late stages of degenerative joint diseases, for example, during
osteoarthritis. Since the subchondral bone tissue is both
innervated and vascularized, damage to this tissue is often
painful. The repair reaction induced by damage to the subchondral
bone usually results in the formation of fibrocartilage at the site
of the full-thickness defect. Fibrocartilage, however, lacks the
biomechanical properties of articular cartilage and fails to
persist in the joint on a long term basis.
[0006] Partial-thickness articular cartilage defects are restricted
to the cartilage tissue itself. These defects usually include
fissures or clefts in the articulating surface of the cartilage.
Partial-thickness defects are caused by mechanical arrangements of
the joint which in turn induce wearing of the cartilage tissue
within the joint. In the absence of innervation and vasculature,
partial-thickness defects do not elicit repair responses and
therefore tend not to heal. Although painless, partial-thickness
defects often degenerate into full-thickness defects.
[0007] Cartilage may develop abnormally or may be damaged by
disease, such as rheumatoid arthritis or osteoarthritis, or by
trauma, each of which can lead to physical deformity and
debilitation. Whether cartilage is damaged from trauma or
congenital anomalies, its successful clinical regeneration is often
poor at best, as reviewed by Howell, et al. Osteoarthritis:
Diagnosis and Management, 2nd ed., (Philadelphia, W.B. Saunders,
1990) and Kelley, et al. Textbook of Rheumatology, 3rd ed.,
(Philadelphia, W.B. Saunders, 1989).
[0008] Bone conditions also are widespread. For example, there
generally are two types of bone conditions: non-metabolic bone
conditions, such as bone fractures, bone/spinal deformation,
osteosarcoma, myeloma, bone dysplasia and scoliosis, and metabolic
bone conditions, such as osteoporosis, osteomalacia, rickets,
fibrous osteitis, renal bone dystrophy and Paget's disease of bone.
Osteoporosis, a metabolic bone condition, is a systemic disease
characterized by increased bone fragility and fracturability due to
decreased bone mass and change in fine bone tissue structure, its
major clinical symptoms including spinal kyphosis, and fractures of
dorsolumbar bones, vertebral centra, femoral necks, lower end of
radius, ribs, upper end of humerus, and others. In bone tissue,
bone formation and destruction due to bone resorption occur
constantly. Upon deterioration of the balance between bone
formation and bone destruction due to bone resorption, a
quantitative reduction in bone occurs. Traditionally, bone
resorption suppressors such as estrogens, calcitonin and
bisphosphonates have been mainly used to treat osteoporosis.
[0009] Bone grafting is often used for the treatment of bone
conditions. Indeed, more than 1.4 million bone grafting procedures
are performed in the developed world annually. Most of these
procedures are administered following joint replacement surgery or
during trauma surgical reconstruction. The success or failure of
bone grafting is dependent upon a number of factors including the
vitality of the site of the graft, the graft processing, and the
immunological compatibility of the engrafted tissue.
[0010] In view of the prevalence of bone and cartilage conditions,
novel sources of bone and cartilage tissue for therapeutic,
diagnostic, and research uses are in high demand.
SUMMARY OF THE INVENTION
[0011] The invention is generally directed to postpartum-derived
cells which are derived from postpartum tissue which is
substantially free of blood and which is capable of self-renewal
and expansion in culture and having the potential to differentiate
into a cell of osteocyte or chondrocyte phenotypes.
[0012] In some embodiments, the present invention provides cells
derived from human postpartum tissue substantially free of blood,
capable of self-renewal and expansion in culture, having the
potential to differentiate into a cell of an osteogenic or
chondrogenic phenotype; requiring L-valine for growth; capable of
growth in about 5% to about 20% oxygen; and further having at least
one of the following characteristics:
[0013] production of at least one of GCP-2, tissue factor,
vimentin, and alpha-smooth muscle actin;
[0014] lack of production of at least one of NOGO-A, GRO-alpha or
oxidized low density lipoprotein receptor, as detected by flow
cytometry;
[0015] production of at least one of CD10, CD13, CD44, CD73, CD90,
PDGFr-alpha, PD-L2 and HLA-A,B,C;
[0016] lack of production of at least one of CD31, CD34, CD45,
CD80, CD86, CD117, CD141, CD178, B7-H2, HLA-G, and HLA-DR,DP,DQ, as
detected by flow cytometry;
[0017] expression, which relative to a human cell that is a
fibroblast, a mesenchymal stem cell, or an ileac crest bone marrow
cell, is increased for at least one of interleukin 8; reticulon 1;
chemokine (C--X--C motif) ligand 1 (melanoma growth stimulating
activity, alpha); chemokine (C--X--C motif) ligand 6 (granulocyte
chemotactic protein 2); chemokine (C--X--C motif) ligand 3; tumor
necrosis factor, alpha-induced protein 3 or expression, which
relative to a human cell that is a fibroblast, a mesenchymal stem
cell, or an ileac crest bone marrow cell, is increased for at least
one of C-type lectin superfamily member A2, Wilms tumor 1, aldehyde
dehydrogenase 1 family member A2, renin, oxidized low density
lipoprotein receptor 1, protein kinase C zeta, clone IMAGE:4179671,
hypothetical protein DKFZp564F013, downregulated in ovarian cancer
1, and clone DKFZp547K1113;
[0018] expression, which relative to a human cell that is a
fibroblast, a mesenchymal stem cell, or an ileac crest bone marrow
cell, is reduced for at least one of: short stature homeobox 2;
heat shock 27 kDa protein 2; chemokine (C--X--C motif) ligand 12
(stromal cell-derived factor 1); elastin; cDNA DKFZp586M2022 (from
clone DKFZp586M2022); mesenchyme homeobox 2; sine oculis homeobox
homolog 1; crystallin, alpha B; dishevelled associated activator of
morphogenesis 2; DKFZP586B2420 protein; similar to neuralin 1;
tetranectin; src homology three (SH3) and cysteine rich domain;
B-cell translocation gene 1, anti-proliferative; cholesterol
25-hydroxylase; runt-related transcription factor 3; hypothetical
protein FLJ23191; interleukin 11 receptor, alpha; procollagen
C-endopeptidase enhancer; frizzled homolog 7; hypothetical gene
BC008967; collagen, type VIII, alpha 1; tenascin C; iroquois
homeobox protein 5; hephaestin; integrin, beta 8; synaptic vesicle
glycoprotein 2; cDNA FLJ12280 fis, clone MAMMA1001744; cytokine
receptor-like factor 1; potassium intermediate/small conductance
calcium-activated channel, subfamily N, member 4; integrin, alpha
7; DKFZP586L151 protein; transcriptional co-activator with
PDZ-binding motif (TAZ); sine oculis homeobox homolog 2; KIAA1034
protein; early growth response 3; distal-less homeobox 5;
hypothetical protein FLJ20373; aldo-keto reductase family 1, member
C3 (3-alpha hydroxysteroid dehydrogenase, type II); biglycan;
fibronectin 1; proenkephalin; integrin, beta-like 1 (with EGF-like
repeat domains); cDNA clone EUROIMAGE 1968422; EphA3; KIAA0367
protein; natriuretic peptide receptor C/guanylate cyclase C
(atrionatriuretic peptide receptor C); hypothetical protein
FLJ14054; cDNA DKFZp564B222 (from clone DKFZp564B222);
vesicle-associated membrane protein 5; EGF-containing fibulin-like
extracellular matrix protein 1; BCL2/adenovirus E1B 19 kDa
interacting protein 3-like; AE binding protein 1; cytochrome c
oxidase subunit VIIa polypeptide 1 (muscle); neuroblastoma,
suppression of tumorigenicity 1; and insulin-like growth factor
binding protein 2, 36 kDa;
[0019] secretion of at least one of MCP-1, IL-6, IL-8, GCP-2, HGF,
KGF, FGF, HB-EGF, BDNF, TPO, MIP1a, RANTES, and TIMP1;
[0020] lack of secretion of at least one of TGF-beta2, ANG2,
PDGFbb, MIP1b, I309, MDC, and VEGF, as detected by ELISA; and
[0021] the ability to undergo at least 40 population doublings in
culture.
[0022] In certain embodiments, the postpartum-derived cell is an
umbilical cord-derived cell. In other embodiments, it is a
placenta-derived cell. In specific embodiments, the cell has all
identifying features of any one of: cell type PLA 071003 (P8) (ATCC
Accession No. PTA-6074); cell type PLA 071003 (P11) (ATCC Accession
No. PTA-6075); cell type PLA 071003 (P16) (ATCC Accession No.
PTA-6079); cell type UMB 022803 (P7) (ATCC Accession No. PTA-6067);
or cell type UMB 022803 (P17) (ATCC Accession No. PTA-6068). The
postpartum-derived cells of the invention are preferably human
cells.
[0023] The cells may be induced to differentiate to an osteogenic
or chondrogenic phenotype. Methods for inducing differentiation of
postpartum-derived cells of the invention are contemplated. For
example, the cells may be induced to differentiate to a cell having
an osteogenic or chondrogenic phenotype. Methods of inducing
differentiation of the cells of the invention preferably involve
exposing the cells to one or more differentiation-inducing agents.
Osteogenesis inducing agents of the invention include bone
morphogenic proteins (e.g., BMP-2, BMP-4) and transforming growth
factor (TGF)-beta1, and combinations thereof. Chondrogenesis
inducing agents of the invention include TGF-beta3, GDF-5, and a
combination thereof. The invention includes the
differentiation-induced cells and populations, compositions, and
products thereof. Differentiation-induced cells of an osteogenic
lineage preferably express at least one osteogenic lineage marker
(e.g., osteocalcin, bone sialoprotein, alkaline phosphatase).
Differentiation of PPDCs to an osteogenic lineage may be assessed
by any means known in the art, for example but not limited to,
measurement of mineralization (e.g., von Kossa staining).
Differentiation-induced cells of a chondrogenic lineage preferably
express at least one chondrogenic lineage marker (e.g.,
glycosaminoglycan, type II collagen). Differentiation of PPDCs to a
chondrogenic lineage may be assessed by any means known in the art,
for example but not limited to, Safranin-O or hematoxylin/eosin
staining.
[0024] Populations of PPDCs are provided by the invention. The
PPDCs may be differentiation-induced or undifferentiated. In some
embodiments, a population of postpartum-derived cells is mixed with
another population of cells. In some embodiments, the cell
population is heterogeneous. A heterogeneous cell population of the
invention may comprise at least about 5%, 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, or 95% undifferentiated or
differentiation-induced PPDCs of the invention. The heterogeneous
cell populations of the invention may further comprise bone marrow
cells, stem cells, chondroblasts, chondrocytes, osteoblasts,
osteocytes, osteoclasts, bone lining cells, or other bone or
cartilage cell progenitors. Cell populations of the invention may
be substantially homogeneous, i.e., comprises substantially only
PPDCs (preferably at least about 96%, 97%, 98%, 99% or more PPDCs).
The homogeneous cell population of the invention may comprise
umbilical cord- or placenta-derived cells. Homogeneous populations
of placenta-derived cells may be of neonatal or maternal lineage.
Homogeneity of a cell population may be achieved by any method
known in the art, for example, by cell sorting (e.g., flow
cytometry), bead separation, or by clonal expansion.
[0025] The invention also provides heterogeneous and homogeneous
cell cultures containing undifferentiated or
differentiation-induced postpartum-derived cells of the
invention.
[0026] Some embodiments of the invention provide a matrix for
implantation into a patient seeded with one or more
postpartum-derived cells of the invention or containing or treated
with a cell lysate, conditioned medium, or extracellular matrix
thereof. The PPDCs may be differentiation-induced or
undifferentiated. The matrix may contain or be treated with one or
more bioactive factors including anti-apoptotic agents (e.g., EPO,
EPO mimetibody, TPO, IGF-I and IGF-II, HGF, caspase inhibitors);
anti-inflammatory agents (e.g., p38 MAPK inhibitors, TGF-beta
inhibitors, statins, IL-6 and IL-1 inhibitors, PEMIROLAST,
TRANILAST, REMICADE, SIROLIMUS, and NSAIDs (non-steroidal
anti-inflammatory drugs; e.g., TEPOXALIN, TOLMETIN, SUPROFEN);
immunosupressive/immunomodulatory agents (e.g., calcineurin
inhibitors, such as cyclosporine, tacrolimus; mTOR inhibitors
(e.g., SIROLIMUS, EVEROLIMUS); anti-proliferatives (e.g.,
azathioprine, mycophenolate mofetil); corticosteroids (e.g.,
prednisolone, hydrocortisone); antibodies such as monoclonal
anti-IL-2Ralpha receptor antibodies (e.g., basiliximab,
daclizumab), polyclonal anti-T-cell antibodies (e.g.,
anti-thymocyte globulin (ATG); anti-lymphocyte globulin (ALG);
monoclonal anti-T cell antibody OKT3)); anti-thrombogenic agents
(e.g., heparin, heparin derivatives, urokinase, PPack
(dextrophenylalanine proline arginine chloromethylketone),
antithrombin compounds, platelet receptor antagonists,
anti-thrombin antibodies, anti-platelet receptor antibodies,
aspirin, dipyridamole, protamine, hirudin, prostaglandin
inhibitors, and platelet inhibitors); and anti-oxidants (e.g.,
probucol, vitamin A, ascorbic acid, tocopherol, coenzyme Q-10,
glutathione, L-cysteine, N-acetylcysteine.
[0027] Also encompassed within the scope of the invention are
PPDC-products, including extracellular matrices of PPDCs, cell
lysates (e.g., soluble cell fractions) of PPDCs, and
PPDC-conditioned medium.
[0028] In some embodiments the invention provides compositions of
PPDCs and one or more bioactive factors, for example, but not
limited to growth factors, chondrogenic or osteogenic
differentiation inducing factors, anti-apoptotic agents (e.g., EPO,
EPO mimetibody, TPO, IGF-I and IGF-II, HGF, caspase inhibitors);
anti-inflammatory agents (e.g., p38 MAPK inhibitors, TGF-beta
inhibitors, statins, IL-6 and IL-1 inhibitors, PEMIROLAST,
TRANILAST, REMICADE, SIROLIMUS, and NSAIDs (non-steroidal
anti-inflammatory drugs; e.g., TEPOXALIN, TOLMETIN, SUPROFEN);
immunosupressive/immunomodulatory agents (e.g., calcineurin
inhibitors, such as cyclosporine, tacrolimus; mTOR inhibitors
(e.g., SIROLIMUS, EVEROLIMUS); anti-proliferatives (e.g.,
azathioprine, mycophenolate mofetil); corticosteroids (e.g.,
prednisolone, hydrocortisone); antibodies such as monoclonal
anti-IL-2Ralpha receptor antibodies (e.g., basiliximab,
daclizumab), polyclonal anti-T-cell antibodies (e.g.,
anti-thymocyte globulin (ATG); anti-lymphocyte globulin (ALG);
monoclonal anti-T cell antibody OKT3)); anti-thrombogenic agents
(e.g., heparin, heparin derivatives, urokinase, PPack
(dextrophenylalanine proline arginine chloromethylketone),
antithrombin compounds, platelet receptor antagonists,
anti-thrombin antibodies, anti-platelet receptor antibodies,
aspirin, dipyridamole, protamine, hirudin, prostaglandin
inhibitors, and platelet inhibitors); and anti-oxidants (e.g.,
probucol, vitamin A, ascorbic acid, tocopherol, coenzyme Q-10,
glutathione, L-cysteine, N-acetylcysteine).
[0029] Pharmaceutical compositions of the postpartum-derived cells,
extracellular matrix produced thereby, cell lysates thereof, and
PPDC-conditioned medium are included within the scope of the
invention. The pharmaceutical compositions preferably include a
pharmaceutically acceptable carrier or excipient. The
pharmaceutical compositions are preferably for treating bone or
cartilage conditions as defined herein.
[0030] The invention further provides in some aspects methods of
regenerating bone or cartilage tissue in a patient in need thereof
by administering cells, matrices, or PPDC-products of the invention
into a patient are provided.
[0031] Further provided by the invention are methods for treating a
condition such as a bone or cartilage condition in a patient by
administering one or more postpartum-derived cells, PPDC
populations, matrices, cell lysates, a combination of cell lysate
and extracellular matrix, conditioned medium, or compositions of
the invention. The PPDCs, whether differentiated or
undifferentiated, or a combination thereof, extracellular matrix
produced thereby, cell lysates thereof, a combination of cell
lysate and extracellular matrix, matrices (e.g., scaffolds),
conditioned medium, and compositions of the invention may be used
in the treatment of bone or cartilage tissue conditions, for
example, but not limited to congenital defects, bone fractures,
meniscal injuries or defects, bone/spinal deformation,
osteosarcoma, myeloma, bone dysplasia and scoliosis, osteoporosis,
periodontal disease, dental bone loss, osteomalacia, rickets,
fibrous osteitis, renal bone dystrophy, spinal fusion, spinal disc
reconstruction or removal, Paget's disease of bone, meniscal
injuries, rheumatoid arthritis, osteoarthritis, or a traumatic or
surgical injury to cartilage or bone.
[0032] Also provided by the invention are kits comprising the
postpartum-derived cells, PPDC-conditioned medium, lysate, and/or
extracellular matrices of the invention. The kits of the invention
may further contain at least one component of a matrix, a second
cell type, a hydrating agent, a ell culture substrate, a
differentiation-inducing agent, cell culture media, and
instructions, for example, for culture of the cells or
administration of the cells and/or cell products.
[0033] In some embodiments, the invention provides methods for
identifying compounds that modulate chondrogenesis or osteogenesis
of a postpartum-derived cell comprising contacting a cell of the
invention with said compound and monitoring the cell for a marker
of chondrogenesis or osteogenesis. Also provided are methods for
identifying compound toxic to a postpartum-derived cell of the
invention by contacting said cell with said compound and monitoring
survival of said cell.
[0034] Other features and advantages of the invention will be
apparent from the detailed description and examples that
follow.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
Definitions
[0035] Various terms used throughout the specification and claims
are defined as set forth below.
[0036] Stem cells are undifferentiated cells defined by their
ability at the single cell level to both self-renew and
differentiate to produce progeny cells, including self-renewing
progenitors, non-renewing progenitors and terminally differentiated
cells. Stem cells are also characterized by their ability to
differentiate in vitro into functional cells of various cell
lineages from multiple germ layers (endoderm, mesoderm and
ectoderm), as well as to give rise to tissues of multiple germ
layers following transplantation and to contribute substantially to
most, if not all, tissues following injection into blastocysts.
[0037] Stem cells are classified by their developmental potential
as: (1) totipotent--able to give rise to all embryonic and
extraembryonic cell types; (2) pluripotent--able to give rise to
all embryonic cell types; (3) multipotent--able to give rise to a
subset of cell lineages, but all within a particular tissue, organ,
or physiological system (for example, hematopoietic stem cells
(HSC) can produce progeny that include HSC (self-renewal), blood
cell-restricted oligopotent progenitors, and all cell types and
elements (e.g., platelets) that are normal components of the
blood); (4) oligopotent--able to give rise to a more restricted
subset of cell lineages than multipotent stem cells; and (5)
unipotent--able to give rise to a single cell lineage (e.g.,
spermatogenic stem cells).
[0038] Stem cells are also categorized on the basis of the source
from which they may be obtained. An adult stem cell is generally a
multipotent undifferentiated cell found in tissue comprising
multiple differentiated cell types. The adult stem cell can renew
itself and, under normal circumstances, differentiate to yield the
specialized cell types of the tissue from which it originated, and
possibly other tissue types. An embryonic stem cell is a
pluripotent cell from the inner cell mass of a blastocyst-stage
embryo. A fetal stem cell is one that originates from fetal tissues
or membranes. A postpartum stem cell is a multipotent or
pluripotent cell that originates substantially from extraembryonic
tissue available after birth, namely, the placenta and the
umbilical cord. These cells have been found to possess features
characteristic of pluripotent stem cells, including rapid
proliferation and the potential for differentiation into many cell
lineages. Postpartum stem cells may be blood-derived (e.g., as are
those obtained from umbilical cord blood) or non-blood-derived
(e.g., as obtained from the non-blood tissues of the umbilical cord
and placenta).
[0039] Embryonic tissue is typically defined as tissue originating
from the embryo (which in humans refers to the period from
fertilization to about six weeks of development. Fetal tissue
refers to tissue originating from the fetus, which in humans refers
to the period from about six weeks of development to parturition.
Extraembryonic tissue is tissue associated with, but not
originating from, the embryo or fetus. Extraembryonic tissues
include extraembryonic membranes (chorion, amnion, yolk sac and
allantois), umbilical cord and placenta (which itself forms from
the chorion and the maternal decidua basalis).
[0040] Differentiation is the process by which an unspecialized
("uncommitted") or less specialized cell acquires the features of a
specialized cell, such as a nerve cell or a muscle cell, for
example. A differentiated or differentiation-induced cell is one
that has taken on a more specialized ("committed") position within
the lineage of a cell. The term committed, when applied to the
process of differentiation, refers to a cell that has proceeded in
the differentiation pathway to a point where, under normal
circumstances, it will continue to differentiate into a specific
cell type or subset of cell types, and cannot, under normal
circumstances, differentiate into a different cell type or revert
to a less differentiated cell type. De-differentiation refers to
the process by which a cell reverts to a less specialized (or
committed) position within the lineage of a cell. As used herein,
the lineage of a cell defines the heredity of the cell, i.e., which
cells it came from and what cells it can give rise to. The lineage
of a cell places the cell within a hereditary scheme of development
and differentiation. A lineage-specific marker refers to a
characteristic specifically associated with the phenotype of cells
of a lineage of interest and can be used to assess the
differentiation of an uncommitted cell to the lineage of
interest.
[0041] In a broad sense, a progenitor cell is a cell that has the
capacity to create progeny that are more differentiated than itself
and yet retains the capacity to replenish the pool of progenitors.
By that definition, stem cells themselves are also progenitor
cells, as are the more immediate precursors to terminally
differentiated cells. When referring to the cells of the present
invention, as described in greater detail below, this broad
definition of progenitor cell may be used. In a narrower sense, a
progenitor cell is often defined as a cell that is intermediate in
the differentiation pathway, i.e., it arises from a stem cell and
is intermediate in the production of a mature cell type or subset
of cell types. This type of progenitor cell is generally not able
to self-renew. Accordingly, if this type of cell is referred to
herein, it will be referred to as a non-renewing progenitor cell or
as an intermediate progenitor or precursor cell.
[0042] As used herein, the phrase differentiates into a mesodermal,
ectodermal or endodermal lineage refers to a cell that becomes
committed to a specific mesodermal, ectodermal or endodermal
lineage, respectively. Examples of cells that differentiate into a
mesodermal lineage or give rise to specific mesodermal cells
include, but are not limited to, cells that are adipogenic,
chondrogenic, cardiogenic, dermatogenic, hematopoetic,
hemangiogenic, myogenic, nephrogenic, urogenitogenic, osteogenic,
pericardiogenic, or stromal. Examples of cells that differentiate
into ectodermal lineage include, but are not limited to epidermal
cells, neurogenic cells, and neurogliagenic cells. Examples of
cells that differentiate into endodermal lineage include, but are
not limited to pleurigenic cells, and hepatogenic cells, cell that
give rise to the lining of the intestine, and cells that give rise
to pancreogenic and splanchogenic cells.
[0043] The cells of the invention are referred to herein as
postpartum-derived cells (PPDCs). Subsets of the cells of the
present invention are referred to as placenta-derived cells (PDCs)
or umbilical cord-derived cells (UDCs). PPDCs of the invention
encompass undifferentiated and differentiation-induced cells. In
addition, the cells may be described as being stem or progenitor
cells, the latter term being used in the broad sense. The term
derived is used to indicate that the cells have been obtained from
their biological source and grown or otherwise manipulated in vitro
(e.g., cultured in a growth medium to expand the population and/or
to produce a cell line). The in vitro manipulations of
postpartum-derived cells and the unique features of the
postpartum-derived cells of the present invention are described in
detail below.
[0044] Various terms are used to describe cells in culture. Cell
culture refers generally to cells taken from a living organism and
grown under controlled condition ("in culture"). A primary cell
culture is a culture of cells, tissues or organs taken directly
from organisms and before the first subculture. Cells are expanded
in culture when they are placed in a growth medium under conditions
that facilitate cell growth and/or division, resulting in a larger
population of the cells. When cells are expanded in culture, the
rate of cell proliferation is sometimes measured by the amount of
time needed for the cells to double in number. This is referred to
as doubling time.
[0045] A cell line is a population of cells formed by one or more
subcultivations of a primary cell culture. Each round of
subculturing is referred to as a passage. When cells are
subcultured, they are referred to as having been passaged. A
specific population of cells, or a cell line, is sometimes referred
to or characterized by the number of times it has been passaged.
For example, a cultured cell population that has been passaged ten
times may be referred to as a P10 culture. The primary culture,
i.e., the first culture following the isolation of cells from
tissue, is designated P0. Following the first subculture, the cells
are described as a secondary culture (P1 or passage 1). After the
second subculture, the cells become a tertiary culture (P2 or
passage 2), and so on. It will be understood by those of skill in
the art that there may be many population doublings during the
period of passaging; therefore the number of population doublings
of a culture is greater than the passage number. The expansion of
cells (i.e., the number of population doublings) during the period
between passaging depends on many factors, including but not
limited to the seeding density, substrate, medium, and time between
passaging.
[0046] A conditioned medium is a medium in which a specific cell or
population of cells has been cultured, and then removed. While the
cells are cultured in the medium, they secrete cellular factors
that can provide trophic support to other cells. Such trophic
factors include, but are not limited to hormones, cytokines,
extracellular matrix (ECM), proteins, vesicles, antibodies, and
granules. The medium containing the cellular factors is the
conditioned medium.
[0047] Generally, a trophic factor is defined as a substance that
promotes survival, growth, proliferation, maturation,
differentiation, and/or maintenance of a cell, or stimulates
increased activity of a cell.
[0048] When referring to cultured vertebrate cells, the term
senescence (also replicative senescence or cellular senescence)
refers to a property attributable to finite cell cultures; namely,
their inability to grow beyond a finite number of population
doublings (sometimes referred to as Hayflick's limit). Although
cellular senescence was first described using fibroblast-like
cells, most normal human cell types that can be grown successfully
in culture undergo cellular senescence. The in vitro lifespan of
different cell types varies, but the maximum lifespan is typically
fewer than 100 population doublings (this is the number of
doublings for all the cells in the culture to become senescent and
thus render the culture unable to divide). Senescence does not
depend on chronological time, but rather is measured by the number
of cell divisions, or population doublings, the culture has
undergone. Thus, cells made quiescent by removing essential growth
factors are able to resume growth and division when the growth
factors are re-introduced, and thereafter carry out the same number
of doublings as equivalent cells grown continuously. Similarly,
when cells are frozen in liquid nitrogen after various numbers of
population doublings and then thawed and cultured, they undergo
substantially the same number of doublings as cells maintained
unfrozen in culture. Senescent cells are not dead or dying cells;
they are actually resistant to programmed cell death (apoptosis),
and have been maintained in their nondividing state for as long as
three years. These cells are very much alive and metabolically
active, but they do not divide. The nondividing state of senescent
cells has not yet been found to be reversible by any biological,
chemical, or viral agent.
[0049] As used herein, the term Growth medium refers to a culture
medium sufficient for expansion of postpartum-derived cells. The
culture medium of Growth medium preferably contains Dulbecco's
Modified Essential Media (DMEM). More preferably, Growth medium
contains glucose. Growth medium preferably contains DMEM-low
glucose (DMEM-LG) (Invitrogen, Carlsbad, Calif.). Growth medium
preferably contains about 15% (v/v) serum (e.g., fetal bovine
serum, defined bovine serum). Growth medium preferably contains at
least one antibiotic agent and/or antimycotic agent (e.g.,
penicillin, streptomycin, amphotericin B, gentamicin, nystatin;
preferably, 50 units/milliliter penicillin G sodium and 50
micrograms/milliliter streptomycin sulfate). Growth medium
preferably contains 2-mercaptoethanol (Sigma, St. Louis Mo.). Most
preferably, Growth medium contains DMEM-low glucose, serum,
2-mercaptoethanol, and an antibiotic agent.
[0050] As used herein, standard growth conditions refers to
standard atmospheric conditions comprising about 5% CO.sub.2, a
temperature of about 35-39.degree. C., more preferably 37.degree.
C., and a relative humidity of about 100%.
[0051] The term isolated refers to a cell, cellular component, or a
molecule that has been removed from its native environment.
[0052] The term about refers to an approximation of a stated value
within a range of .+-.10%.
[0053] Bone condition (or injury or disease) is an inclusive term
encompassing acute and chronic and metabolic and non-metabolic
conditions, disorders or diseases of bone. The term encompasses
conditions caused by disease or trauma or failure of the tissue to
develop normally. Examples of bone conditions include but are not
limited to congenital bone defects, bone fractures, meniscal
injuries or defects, bone/spinal deformation, osteosarcoma,
myeloma, bone dysplasia and scoliosis, osteoporosis, periodontal
disease, dental bone loss; osteomalacia, rickets, fibrous osteitis,
renal bone dystrophy, spinal fusion, spinal disc reconstruction or
removal, and Paget's disease of bone.
[0054] Cartilage condition (or injury or disease) is an inclusive
term encompassing acute and chronic conditions, disorders, or
diseases of cartilage. The term encompasses conditions including
but not limited to congenital defects, meniscal injuries,
rheumatoid arthritis, osteoarthritis, or a traumatic or surgical
injury to cartilage.
[0055] The term treating (or treatment of) a bone or cartilage
condition refers to ameliorating the effects of, or delaying,
halting or reversing the progress of, or delaying or preventing the
onset of, a bone or cartilage condition as defined herein.
[0056] The term effective amount refers to a concentration of a
reagent or pharmaceutical composition, such as a growth factor,
differentiation agent, trophic factor, cell population or other
agent, that is effective for producing an intended result,
including cell growth and/or differentiation in vitro or in vivo,
or treatment of a bone or cartilage condition as described herein.
With respect to growth factors, an effective amount may range from
about 1 nanogram/milliliter to about 1 microgram/milliliter. With
respect to PPDCs as administered to a patient in vivo, an effective
amount may range from as few as several hundred or fewer to as many
as several million or more. In specific embodiments, an effective
amount may range from 10.sup.3-10.sup.11. It will be appreciated
that the number of cells to be administered will vary depending on
the specifics of the disorder to be treated, including but not
limited to size or total volume/surface area to be treated, as well
as proximity of the site of administration to the location of the
region to be treated, among other factors familiar to the medicinal
biologist.
[0057] The terms effective period (or time) and effective
conditions refer to a period of time or other controllable
conditions (e.g., temperature, humidity for in vitro methods),
necessary or preferred for an agent or pharmaceutical composition
to achieve its intended result.
[0058] The term patient or subject refers to animals, including
mammals, preferably humans, who are treated with the pharmaceutical
compositions or in accordance with the methods described
herein.
[0059] The term matrix as used herein refers to a support for the
PPDCs of the invention, for example, a scaffold (e.g., VICRYL,
PCL/PGA, or RAD16) or supporting medium (e.g., hydrogel,
extracellular membrane protein (e.g., MATRIGEL (BD Discovery
Labware, Bedford, Mass.)).
[0060] The term pharmaceutically acceptable carrier (or medium),
which may be used interchangeably with the term biologically
compatible carrier or medium, refers to reagents, cells, compounds,
materials, compositions, and/or dosage forms which are, within the
scope of sound medical judgment, suitable for use in contact with
the tissues of human beings and animals without excessive toxicity,
irritation, allergic response, or other complication commensurate
with a reasonable benefit/risk ratio. As described in greater
detail herein, pharmaceutically acceptable carriers suitable for
use in the present invention include liquids, semi-solid (e.g.,
gels) and solid materials (e.g., cell scaffolds). As used herein,
the term biodegradable describes the ability of a material to be
broken down (e.g., degraded, eroded, dissolved) in vivo. The term
includes degradation in vivo with or without elimination (e.g., by
resorption) from the body. The semi-solid and solid materials may
be designed to resist degradation within the body
(non-biodegradable) or they may be designed to degrade within the
body (biodegradable, bioerodable). A biodegradable material may
further be bioresorbable or bioabsorbable, i.e., it may be
dissolved and absorbed into bodily fluids (water-soluble implants
are one example), or degraded and ultimately eliminated from the
body, either by conversion into other materials or by breakdown and
elimination through natural pathways.
[0061] Several terms are used herein with respect to cell
replacement therapy. The terms autologous transfer, autologous
transplantation, autograft and the like refer to treatments wherein
the cell donor is also the recipient of the cell replacement
therapy. The terms allogeneic transfer, allogeneic transplantation,
allograft and the like refer to treatments wherein the cell donor
is of the same species as the recipient of the cell replacement
therapy, but is not the same individual. A cell transfer in which
the donor's cells have been histocompatibly matched with a
recipient is sometimes referred to as a syngeneic transfer. The
terms xenogeneic transfer, xenogeneic transplantation, xenograft
and the like refer to treatments wherein the cell donor is of a
different species than the recipient of the cell replacement
therapy.
[0062] The following abbreviations are used herein:
[0063] ANG2 (or Ang2) for angiopoietin 2;
[0064] APC for antigen-presenting cells;
[0065] BDNF for brain-derived neurotrophic factor;
[0066] bFGF for basic fibroblast growth factor;
[0067] bid (BID) for "bis in die" (twice per day);
[0068] BSP for bone sialoprotein;
[0069] CK18 for cytokeratin 18;
[0070] CXC ligand 3 for chemokine receptor ligand 3;
[0071] DAPI for 4'-6-Diamidino-2-phenylindole-2HCl;
[0072] DMEM for Dulbecco's Minimal Essential Medium;
[0073] DMEM:lg (or DMEM:Lg, DMEM:LG) for DMEM with low glucose;
[0074] EDTA for ethylene diamine tetraacetic acid;
[0075] EGF (or E) for epidermal growth factor;
[0076] EPO for erythropoietin;
[0077] FACS for fluorescent activated cell sorting;
[0078] FBS for fetal bovine serum;
[0079] FGF (or F) for fibroblast growth factor;
[0080] GCP-2 for granulocyte chemotactic protein-2;
[0081] GDF-5 for growth and differentiation factor 5;
[0082] GFAP for glial fibrillary acidic protein;
[0083] HB-EGF for heparin-binding epidermal growth factor;
[0084] HCAEC for Human coronary artery endothelial cells;
[0085] HGF for hepatocyte growth factor;
[0086] hMSC for Human mesenchymal stem cells;
[0087] HNF-1alpha for hepatocyte-specific transcription factor;
[0088] HUVEC for Human umbilical vein endothelial cells;
[0089] I309 for a chemokine and the ligand for the CCR8 receptor
and is responsible for chemoattraction of TH2 type T-cells;
[0090] IGF for insulin-like growth factor;
[0091] IL-6 for interleukin-6;
[0092] IL-8 for interleukin 8;
[0093] K19 for keratin 19;
[0094] K8 for keratin 8;
[0095] KGF for keratinocyte growth factor;
[0096] MCP-1 for monocyte chemotactic protein 1;
[0097] MDC for macrophage-derived chemokine;
[0098] MIP1alpha for macrophage inflammatory protein 1alpha;
[0099] MIP1beta for macrophage inflammatory protein 1beta;
[0100] MMP for matrix metalloprotease (MMP);
[0101] MSC for mesenchymal stem cells;
[0102] NHDF for Normal Human Dermal Fibroblasts;
[0103] NPE for Neural Progenitor Expansion media;
[0104] OxLDLR for oxidized low density lipoprotein receptor;
[0105] PBMC for peripheral blood mononuclear cell;
[0106] PBS for phosphate buffered saline;
[0107] PDC for placenta-derived cell;
[0108] PDGFbb for platelet derived growth factor;
[0109] PDGFr-alpha for platelet derived growth factor receptor
alpha;
[0110] PD-L2 for programmed-death ligand 2;
[0111] PE for phycoerythrin;
[0112] PO for "per os" (by mouth);
[0113] PPDC for postpartum-derived cell;
[0114] Rantes (or RANTES) for regulated on activation, normal T
cell expressed and secreted;
[0115] rb for rabbit;
[0116] rh for recombinant human;
[0117] SC for subcutaneously;
[0118] SCID for severe combined immunodeficiency;
[0119] SDF-1alpha for stromal-derived factor 1alpha;
[0120] SHH for sonic hedgehog;
[0121] SMA for smooth muscle actin;
[0122] SOP for standard operating procedure;
[0123] TARC for thymus and activation-regulated chemokine;
[0124] TCP for tissue culture plastic;
[0125] TGFbeta2 for transforming growth factor beta2;
[0126] TGFbeta-3 for transforming growth factor beta-3;
[0127] TIMP1 for tissue inhibitor of matrix metalloproteinase
1;
[0128] TPO for thrombopoietin;
[0129] TuJ1 for BIII Tubulin;
[0130] UDC for umbilical cord-derived cell;
[0131] VEGF for vascular endothelial growth factor;
[0132] vWF for von Willebrand factor; and
[0133] alphaFP for alpha-fetoprotein.
Description
[0134] Various patents and other publications are cited herein and
throughout the specification, each of which is incorporated by
reference herein in its entirety.
[0135] In one aspect, the invention provides postpartum-derived
cells (PPDCs) derived from postpartum tissue substantially free of
blood. The PPDCs may be derived from placenta of a mammal including
but not limited to human. The cells are capable of self-renewal and
expansion in culture. The postpartum-derived cells have the
potential to differentiate into cells of other phenotypes. The
invention provides, in one of its several aspects cells that are
derived from umbilical cord, as opposed to umbilical cord blood.
The invention also provides, in one of its several aspects, cells
that are derived from placental tissue.
[0136] The cells have been characterized as to several of their
cellular, genetic, immunological, and biochemical properties. For
example, the cells have been characterized by their growth by their
cell surface markers, by their gene expression, by their ability to
produce certain biochemical trophic factors, and by their
immunological properties.
[0137] Derivation and Expansion of Postpartum-Derived Cells
(PPDCs)
[0138] According to the methods described herein, a mammalian
placenta and umbilical cord are recovered upon or shortly after
termination of either a full-term or pre-term pregnancy, for
example, after expulsion after birth. Postpartum tissue can be
obtained from any completed pregnancy, full-term or less than
full-term, whether delivered vaginally, or through other means, for
example, Cessarian section. The postpartum tissue may be
transported from the birth site to a laboratory in a sterile
container such as a flask, beaker, culture dish, or bag. The
container may have a solution or medium, including but not limited
to a salt solution, such as, for example, Dulbecco's Modified
Eagle's Medium (DMEM) or phosphate buffered saline (PBS), or any
solution used for transportation of organs used for
transplantation, such as University of Wisconsin solution or
perfluorochemical solution. One or more antibiotic and/or
antimycotic agents, such as but not limited to penicillin,
streptomycin, amphotericin B, gentamicin, and nystatin, may be
added to the medium or buffer. The postpartum tissue may be rinsed
with an anticoagulant solution such as heparin-containing solution.
It is preferable to keep the tissue at about 4-10.degree. C. prior
to extraction of PPDCs. It is even more preferable that the tissue
not be frozen prior to extraction of PPDCs.
[0139] Isolation of PPDCs preferably occurs in an aseptic
environment. Blood and debris are preferably removed from the
postpartum tissue prior to isolation of PPDCs. For example, the
postpartum tissue may be washed with buffer solution, such as but
not limited to phosphate buffered saline. The wash buffer also may
comprise one or more antimycotic and/or antibiotic agents, such as
but not limited to penicillin, streptomycin, amphotericin B,
gentamicin, and nystatin.
[0140] In some aspects of the invention, the different cell types
present in postpartum tissue are fractionated into subpopulations
from which the PPDCs can be isolated. This may be accomplished
using techniques for cell separation including, but not limited to,
enzymatic treatment to dissociate postpartum tissue into its
component cells, followed by cloning and selection of specific cell
types, for example but not limited to selection based on
morphological and/or biochemical markers; selective growth of
desired cells (positive selection), selective destruction of
unwanted cells (negative selection); separation based upon
differential cell agglutinability in the mixed population as, for
example, with soybean agglutinin; freeze-thaw procedures;
differential adherence properties of the cells in the mixed
population; filtration; conventional and zonal centrifugation;
centrifugal elutriation (counter-streaming centrifugation); unit
gravity separation; countercurrent distribution; electrophoresis;
and flow cytometry, for example, fluorescence activated cell
sorting (FACS).
[0141] In a preferred embodiment, postpartum tissue comprising a
whole placenta or a fragment or section thereof is disaggregated by
mechanical force (mincing or shear forces), enzymatic digestion
with single or combinatorial proteolytic enzymes, such as a matrix
metalloprotease and/or neutral protease, for example, collagenase,
trypsin, dispase, LIBERASE (Boehringer Mannheim Corp.,
Indianapolis, Ind.), hyaluronidase, and/or pepsin, or a combination
of mechanical and enzymatic methods. For example, the cellular
component of the postpartum tissue may be disaggregated by methods
using collagenase-mediated dissociation. Enzymatic digestion
methods preferably employ a combination of enzymes, such as a
combination of a matrix metalloprotease and a neutral protease. The
matrix metalloprotease is preferably a collagenase. The neutral
protease is preferably thermolysin or dispase, and most preferably
is dispase. More preferably, enzymatic digestion of postpartum
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. Collagenase may be type 1, 2, 3, or 4.
Other enzymes known in the art for cell isolation include papain,
deoxyribonucleases, serine proteases, such as trypsin,
chymotrypsin, or elastase, that may be used either on their own or
in combination with other enzymes such as matrix metalloproteases,
mucolytic enzymes, and neutral proteases. Serine proteases are
preferably used consecutively following use of other enzymes. The
temperature and period of time tissues or cells are in contact with
serine proteases is particularly important. 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 degree of dilution of the
digestion may also greatly affect the cell yield as cells may be
trapped within the viscous digest. The LIBERASE (Boehringer
Mannheim Corp., Indianapolis, Ind.) Blendzyme (Roche) series of
enzyme combinations are very useful and may be used in the instant
methods. Other sources of enzymes are known, and the skilled
artisan may also obtain such enzymes directly from their natural
sources. The skilled artisan is also well-equipped to assess new,
or additional enzymes or enzyme combinations for their utility in
isolating the cells of the invention. Preferred enzyme treatments
are 0.5, 1, 1.5, or 2 hours long or longer. In more preferred
embodiments, the tissue is incubated at 37.degree. C. during the
enzyme treatment of the disintegration step.
[0142] Postpartum tissue comprising the umbilical cord and placenta
may be used without separation. Alternatively, the umbilical cord
may be separated from the placenta by any means known in the art.
In some embodiments of the invention, postpartum tissue is
separated into two or more sections, such as umbilical cord and
placenta. In some embodiments of the invention, placental tissue is
separated into two or more sections, each section consisting
predominantly of either neonatal, neonatal and maternal, or
maternal aspect. The separated sections then are dissociated by
mechanical and/or enzymatic dissociation according to the methods
described herein. Cells of neonatal or maternal lineage may be
identified by any means known in the art, for example, by karyotype
analysis or in situ hybridization for the Y-chromosome. Karyotype
analysis also may be used to identify cells of normal
karyotype.
[0143] Isolated cells or postpartum tissue from which PPDCs grow
out may be used to initiate, or seed, cell cultures. Cells are
transferred to sterile tissue culture vessels either uncoated or
coated with extracellular matrix or ligands such as laminin,
collagen, gelatin, fibronectin, ornithine, vitronectin, and
extracellular membrane protein (e.g., MATRIGEL (BD Discovery
Labware, Bedford, Mass.)). PPDCs are cultured in any culture medium
capable of sustaining growth of the cells such as, but not limited
to, 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), DMEM/F12, RPMI
1640, advanced DMEM (Gibco), DMEM/MCDB201 (Sigma), and CELL-GRO
FREE. The culture medium may be supplemented with one or more
components including, for example, serum (e.g., fetal bovine serum
(FBS), preferably about 2-15% (v/v); equine 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), insulin-like growth factor-1 (IGF-1), leukemia
inhibitory factor (LIF), epidermal growth factor (EGF), basic
fibroblast growth factor (bFGF), 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. The culture medium preferably
comprises Growth medium (DMEM-low glucose, serum, BME, an
antimycotic agent, and an antibiotic agent).
[0144] The cells are seeded in culture vessels at a density to
allow cell growth. 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 to about 40.degree. C., more preferably about 35.degree. C. to
about 39.degree. C., and more preferably are cultured at 37.degree.
C. The cells are preferably cultured in an incubator. The medium in
the culture vessel can be static or agitated, for example, using a
bioreactor. PPDCs preferably are grown under low oxidative stress
(e.g., with addition of glutathione, ascorbic acid, catalase,
tocopherol, N-acetylcysteine). "Low oxidative stress", as used
herein, refers to conditions of no or minimal free radical damage
to the cultured cells.
[0145] Methods for the selection of the most appropriate culture
medium, medium preparation, and cell culture techniques are well
known in the art and are described in a variety of sources,
including Doyle et al., (eds.), 1995, CELL & TISSUE CULTURE:
LABORATORY PROCEDURES, John Wiley & Sons, Chichester; and Ho
and Wang (eds.), 1991, ANIMAL CELL BIOREACTORS,
Butterworth-Heinemann, Boston, which are incorporated herein by
reference.
[0146] The culture medium is changed as necessary, for example, by
carefully aspirating the medium from the dish, for example, with a
pipette, and replenishing with fresh medium. Incubation is
continued until a sufficient number or density of cells accumulate
in the dish. The original explanted tissue sections may be removed
and the remaining cells trypsinized using standard techniques or
using a cell scraper. After trypsinization, the cells are
collected, removed to fresh medium and incubated as above. In some
embodiments, the medium is changed at least once at approximately
24 hours post-trypsinization to remove any floating cells. The
cells remaining in culture are considered to be PPDCs.
[0147] After culturing the cells or tissue fragments for a
sufficient period of time, PPDCs will have grown out, either as a
result of migration from the postpartum tissue or cell division, or
both. In some embodiments of the invention, PPDCs are passaged, or
removed to a separate culture vessel containing fresh medium of the
same or a different type as that used initially, where the
population of cells can be mitotically expanded. PPDCs are
preferably passaged up to about 100% confluence, more preferably
about 70 to about 85% confluence. The lower limit of confluence for
passage is understood by one skilled in the art. The
placenta-derived cells of the invention may be utilized from the
first subculture (passage 0) to senescence. The preferable number
of passages is that which yields a cell number sufficient for a
given application. In certain embodiments, the cells are passaged 2
to 25 times, preferably 4 to 20 times, more preferably 8 to 15
times, more preferably 10 or 11 times, and most preferably 11
times. Cloning and/or subcloning may be performed to confirm that a
clonal population of cells has been isolated.
[0148] Cells of the invention may be cryopreserved and/or stored
prior to use.
Characterization of PPDCs
[0149] PPDCs may be characterized, for example, by growth
characteristics (e.g., population doubling capability, doubling
time, passages to senescence), karyotype analysis (e.g., normal
karyotype; maternal or neonatal lineage), flow cytometry (e.g.,
FACS analysis), immunohistochemistry and/or immunocytochemistry
(e.g., for detection of epitopes including but not limited to
vimentin, desmin, alpha-smooth muscle actin, cytokeratin 18, von
Willebrand factor, CD34, GROalpha, GCP-2, oxidized low density
lipoprotein receptor 1, and NOGO-A), gene expression profiling
(e.g., gene chip arrays; polymerase chain reaction (for example,
reverse transcriptase PCR, real time PCR, and conventional PCR)),
protein arrays, protein secretion (e.g., by plasma clotting assay
or analysis of PPDC-conditioned medium, for example, by Enzyme
Linked ImmunoSorbent Assay (ELISA)), antibody analysis (e.g.,
ELISA; antibody staining for cell surface markers including but not
limited to CD10, CD13, CD31, CD34, CD44, CD45, CD73, CD80, CD86,
CD90, CD117, CD141, CD178, platelet-derived growth factor receptor
alpha (PDGFr-alpha), HLA class I antigens (HLA-A, HLA-B, HLA-C),
HLA class II antigens (HLA-DP, HLA-DQ, HLA-DR), B7-H2, and PD-L2),
mixed lymphocyte reaction (e.g., as measure of stimulation of
allogeneic PBMCs), and/or other methods known in the art.
[0150] PPDCs can undergo at least 40 population doublings in
culture. Population doubling may be calculated as [ln(cell
final/cell initial)/ln 2]. Doubling time may be calculated as (time
in culture (h)/population doubling).
[0151] Undifferentiated PPDCs preferably produce of at least one of
NOGO-A, GCP-2, tissue factor, vimentin, and alpha-smooth muscle
actin; more preferred are cells which produce each of GCP-2, tissue
factor, vimentin, and alpha-smooth muscle actin. In some
embodiments, two, three, four, or five of these factors are
produced by the PPDCs.
[0152] In some embodiments, PPDCs lack production of at least one
of NOGO-A, GRO-alpha, or oxidized low density lipoprotein receptor,
as detected by flow cytometry. In some embodiments, PPDCs lack
production of at least two or three of these factors.
[0153] PPDCs may comprise at least one cell surface marker of CD10,
CD13, CD44, CD73, CD90, PDGFr-alpha, PD-L2 and HLA-A,B,C. PPDCs
preferably produce each of these surface markers. PPDCs may be
characterized in their lack of production of at least one of CD31,
CD34, CD45, CD80, CD86, CD117, CD141, CD178, B7-H2, HLA-G, and
HLA-DR,DP,DQ, as detected by flow cytometry. PPDCs preferably lack
production of each of these surface markers.
[0154] In some embodiments, PPDCs exhibit expression, which
relative to a human cell that is a fibroblast, a mesenchymal stem
cell, or an ileac crest bone marrow cell, is increased for at least
one of interleukin 8; reticulon 1; chemokine (C--X--C motif) ligand
1 (melanoma growth stimulating activity, alpha); chemokine (C--X--C
motif) ligand 6 (granulocyte chemotactic protein 2); chemokine
(C--X--C motif) ligand 3; and tumor necrosis factor, alpha-induced
protein 3; or at least one of C-type lectin superfamily member A2,
Wilms tumor 1, aldehyde dehydrogenase 1 family member A2, renin,
oxidized low density lipoprotein receptor 1, protein kinase C zeta,
clone IMAGE:4179671, hypothetical protein DKFZp564F013,
downregulated in ovarian cancer 1, and clone DKFZp547K1113.
Preferred PPDCs express, relative to a human cell that is a
fibroblast, a mesenchymal stem cell, or an ileac crest bone marrow
cell, increased levels of interleukin 8; reticulon 1; chemokine
(C--X--C motif) ligand 1 (melanoma growth stimulating activity,
alpha); chemokine (C--X--C motif) ligand 6 (granulocyte chemotactic
protein 2); chemokine (C--X--C motif) ligand 3; and tumor necrosis
factor, alpha-induced protein 3; or increased levels of C-type
lectin superfamily member A2, Wilms tumor 1, aldehyde dehydrogenase
1 family member A2, renin, oxidized low density lipoprotein
receptor 1, protein kinase C zeta, clone IMAGE:4179671,
hypothetical protein DKFZp564F013, downregulated in ovarian cancer
1, and clone DKFZp547K1113. In PPDCs wherein expression, relative
to a human cell that is a fibroblast, a mesenchymal stem cell, or
an ileac crest bone marrow cell, is increased for at least one of
interleukin 8; reticulon 1; chemokine (C--X--C motif) ligand 1
(melanoma growth stimulating activity, alpha); chemokine (C--X--C
motif) ligand 6 (granulocyte chemotactic protein 2); chemokine
(C--X--C motif) ligand 3; and tumor necrosis factor, alpha-induced
protein 3, increased relative levels of at least one of C-type
lectin superfamily member A2, Wilms tumor 1, aldehyde dehydrogenase
1 family member A2, renin, oxidized low density lipoprotein
receptor 1, protein kinase C zeta, clone IMAGE:4179671,
hypothetical protein DKFZp564F013, downregulated in ovarian cancer
1, and clone DKFZp547K1113 are preferably not present. In PPDCs
wherein expression, relative to a human cell that is a fibroblast,
a mesenchymal stem cell, or an ileac crest bone marrow cell, is
increased for at least one of C-type lectin superfamily member A2,
Wilms tumor 1, aldehyde dehydrogenase 1 family member A2, renin,
oxidized low density lipoprotein receptor 1, protein kinase C zeta,
clone IMAGE:4179671, hypothetical protein DKFZp564F013,
downregulated in ovarian cancer 1, and clone DKFZp547K1113,
increased relative levels of at least one of interleukin 8;
reticulon 1; chemokine (C--X--C motif) ligand 1 (melanoma growth
stimulating activity, alpha); chemokine (C--X--C motif) ligand 6
(granulocyte chemotactic protein 2); chemokine (C--X--C motif)
ligand 3; and tumor necrosis factor, alpha-induced protein 3 are
preferably not present.
[0155] PPDCs may have expression, which relative to a human cell
that is a fibroblast, a mesenchymal stem cell, or an ileac crest
bone marrow cell, is reduced for at least one of: short stature
homeobox 2; heat shock 27 kDa protein 2; chemokine (C--X--C motif)
ligand 12 (stromal cell-derived factor 1); elastin; cDNA
DKFZp586M2022 (from clone DKFZp586M2022); mesenchyme homeobox 2;
sine oculis homeobox homolog 1; crystallin, alpha B; dishevelled
associated activator of morphogenesis 2; DKFZP586B2420 protein;
similar to neuralin 1; tetranectin; src homology three (SH3) and
cysteine rich domain; B-cell translocation gene 1,
anti-proliferative; cholesterol 25-hydroxylase; runt-related
transcription factor 3; hypothetical protein FLJ23191; interleukin
11 receptor, alpha; procollagen C-endopeptidase enhancer; frizzled
homolog 7; hypothetical gene BC008967; collagen, type VIII, alpha
1; tenascin C; iroquois homeobox protein 5; hephaestin; integrin,
beta 8; synaptic vesicle glycoprotein 2; cDNA FLJ12280 fis, clone
MAMMA1001744; cytokine receptor-like factor 1; potassium
intermediate/small conductance calcium-activated channel, subfamily
N, member 4; integrin, alpha 7; DKFZP586L151 protein;
transcriptional co-activator with PDZ-binding motif (TAZ); sine
oculis homeobox homolog 2; KIAA1034 protein; early growth response
3; distal-less homeobox 5; hypothetical protein FLJ20373; aldo-keto
reductase family 1, member C3 (3-alpha hydroxysteroid
dehydrogenase, type II); biglycan; fibronectin 1; proenkephalin;
integrin, beta-like 1 (with EGF-like repeat domains); cDNA clone
EUROIMAGE 1968422; EphA3; KIAA0367 protein; natriuretic peptide
receptor C/guanylate cyclase C (atrionatriuretic peptide receptor
C); hypothetical protein FLJ14054; cDNA DKFZp564B222 (from clone
DKFZp564B222); vesicle-associated membrane protein 5;
EGF-containing fibulin-like extracellular matrix protein 1;
BCL2/adenovirus E1B 19 kDa interacting protein 3-like; AE binding
protein 1; cytochrome c oxidase subunit VIIa polypeptide 1
(muscle); neuroblastoma, suppression of tumorigenicity 1; and
insulin-like growth factor binding protein 2, 36 kDa; the skilled
artisan will appreciate that the expression of a wide variety of
genes is conveniently characterized on a gene array, for example on
a Affymetrix GENECHIP.
[0156] PPDCs may secrete a variety of biochemically active factors,
such as growth factors, chemokines, cytokines and the like.
Preferred cells secrete at least one of MCP-1, IL-6, IL-8, GCP-2,
HGF, KGF, FGF, HB-EGF, BDNF, TPO, MIP1a, RANTES, and TIMP1. PPDCs
may be characterized in their lack of secretion of at least one of
TGF-beta2, ANG2, PDGFbb, MIP1b, I309, MDC, and VEGF, as detected by
ELISA. These and other characteristics are available to identify
and characterize the cells, and distinguish the cells of the
invention from others known in the art.
[0157] In preferred embodiments, the cell comprises two or more of
the foregoing characteristics. More preferred are those cells
comprising, three, four, or five or more of the characteristics.
Still more preferred are those postpartum-derived cells comprising
six, seven, or eight or more of the characteristics. Still more
preferred presently are those cells comprising all nine of the
claimed characteristics.
[0158] Also presently preferred are cells that produce at least two
of GCP-2, NOGO-A, tissue factor, vimentin, and alpha-smooth muscle
actin. More preferred are those cells producing three, four, or
five of these proteins.
[0159] The skilled artisan will appreciate that cell markers are
subject to vary somewhat under vastly different growth conditions,
and that generally herein described are characterizations in Growth
Medium, or variations thereof. Postpartum-derived cells that
produce of at least one, two, three, or four of CD10, CD13, CD44,
CD73, CD90, PDGFr-alpha, PD-L2 and HLA-A,B,C are preferred. More
preferred are those cells producing five, six, or seven of these
cell surface markers. Still more preferred are postpartum-derived
cells that can produce eight, nine, or ten of the foregoing cell
surface marker proteins.
[0160] PPDCs that lack of production of at least one, two, three,
or four of the proteins CD31, CD34, CD45, CD80, CD86, CD117, CD141,
CD178, B7-H2, HLA-G, and HLA-DR,DP,DQ, as detected by flow
cytometry are preferred. PPDCs lacking production of at least five,
six, seven, or eight or more of these markers are preferred. More
preferred are cells which lack production of at least nine or ten
of the cell surface markers. Most highly preferred are those cells
lacking production of eleven, twelve, or thirteen of the foregoing
identifying proteins.
[0161] Presently preferred cells produce each of CD10, CD13, CD44,
CD73, CD90, PDGFr-alpha, and HLA-A,B,C, and do not produce any of
CD31, CD34, CD45, CD117, CD141, or HLA-DR,DP,DQ, as detected by
flow cytometry.
[0162] It is preferred that postpartum-derived cells exhibit
expression, which relative to a human cell that is a fibroblast, a
mesenchymal stem cell, or an ileac crest bone marrow cell, is
increased for at least one of at least one, two, or three of
interleukin 8; reticulon 1; chemokine (C--X--C motif) ligand 1
(melanoma growth stimulating activity, alpha); chemokine (C--X--C
motif) ligand 6 (granulocyte chemotactic protein 2); chemokine
(C--X--C motif) ligand 3; and tumor necrosis factor, alpha-induced
protein 3; or at least one, two, or three of C-type lectin
superfamily member A2, Wilms tumor 1, aldehyde dehydrogenase 1
family member A2, renin, oxidized low density lipoprotein receptor
1, protein kinase C zeta, clone IMAGE:4179671, hypothetical protein
DKFZp564F013, downregulated in ovarian cancer 1, and clone
DKFZp547K1113. More preferred are those cells which exhibit
elevated relative expression of four or five, and still more
preferred are cell capable of increased relative expression of six,
seven, or eight of the foregoing genes of the respective gene sets.
Most preferably, the cells exhibit expression, which relative to a
human cell that is a fibroblast, a mesenchymal stem cell, or an
ileac crest bone marrow cell, is increased for a combination of
interleukin 8; reticulon 1; chemokine (C--X--C motif) ligand 1
(melanoma growth stimulating activity, alpha); chemokine (C--X--C
motif) ligand 6 (granulocyte chemotactic protein 2); chemokine
(C--X--C motif) ligand 3; tumor necrosis factor, alpha-induced
protein 3 or a combination of C-type lectin superfamily member A2,
Wilms tumor 1, aldehyde dehydrogenase 1 family member A2, renin,
oxidized low density lipoprotein receptor 1, protein kinase C zeta,
clone IMAGE:4179671, hypothetical protein DKFZp564F013,
downregulated in ovarian cancer 1, and clone DKFZp547K1113.
[0163] For some embodiments, preferred are cells, which relative to
a human cell that is a fibroblast, a mesenchymal stem cell, or an
ileac crest bone marrow cell, have reduced expression for at least
one of the genes corresponding to: short stature homeobox 2; heat
shock 27 kDa protein 2; chemokine (C--X--C motif) ligand 12
(stromal cell-derived factor 1); elastin; cDNA DKFZp586M2022 (from
clone DKFZp586M2022); mesenchyme homeobox 2; sine oculis homeobox
homolog 1; crystallin, alpha B; dishevelled associated activator of
morphogenesis 2; DKFZP586B2420 protein; similar to neuralin 1;
tetranectin; src homology three (SH3) and cysteine rich domain;
B-cell translocation gene 1, anti-proliferative; cholesterol
25-hydroxylase; runt-related transcription factor 3; hypothetical
protein FLJ23191; interleukin 11 receptor, alpha; procollagen
C-endopeptidase enhancer; frizzled homolog 7; hypothetical gene
BC008967; collagen, type VIII, alpha 1; tenascin C; iroquois
homeobox protein 5; hephaestin; integrin, beta 8; synaptic vesicle
glycoprotein 2; cDNA FLJ12280 fis, clone MAMMA1001744; cytokine
receptor-like factor 1; potassium intermediate/small conductance
calcium-activated channel, subfamily N, member 4; integrin, alpha
7; DKFZP586L151 protein; transcriptional co-activator with
PDZ-binding motif (TAZ); sine oculis homeobox homolog 2; KIAA1034
protein; early growth response 3; distal-less homeobox 5;
hypothetical protein FLJ20373; aldo-keto reductase family 1, member
C3 (3-alpha hydroxysteroid dehydrogenase, type II); biglycan;
fibronectin 1; proenkephalin; integrin, beta-like 1 (with EGF-like
repeat domains); cDNA clone EUROIMAGE 1968422; EphA3; KIAA0367
protein; natriuretic peptide receptor C/guanylate cyclase C
(atrionatriuretic peptide receptor C); hypothetical protein
FLJ14054; cDNA DKFZp564B222 (from clone DKFZp564B222);
vesicle-associated membrane protein 5; EGF-containing fibulin-like
extracellular matrix protein 1; BCL2/adenovirus E1B 19 kDa
interacting protein 3-like; AE binding protein 1; cytochrome c
oxidase subunit VIIa polypeptide 1 (muscle); neuroblastoma,
suppression of tumorigenicity 1; and insulin-like growth factor
binding protein 2, 36 kDa. More preferred are cells that have,
relative to human fibroblasts, mesenchymal stem cells, or ileac
crest bone marrow cells, reduced expression of at least 5, 10, 15
or 20 genes corresponding to those listed above. Presently more
preferred are cell with reduced expression of at least 25, 30, or
35 of the genes corresponding to the listed sequences. Also more
preferred are those postpartum-derived cells having expression that
is reduced, relative to that of a human fibroblast, a mesenchymal
stem cell, or an ileac crest bone marrow cell, of genes
corresponding to 35 or more, 40 or more, or even all of the
sequences listed.
[0164] Secretion of certain growth factors and other cellular
proteins can make cells of the invention particularly useful.
Preferred postpartum-derived cells secrete at least one, two, three
or four of MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF,
TPO, MIP1a, RANTES, and TIMP1. Cells which secrete five, six, seven
or eight of the listed proteins are also preferred. Cells which can
secrete at least nine, ten, eleven or more of the factors are more
preferred, as are cells which can secrete twelve or more, or even
all thirteen of the proteins in the foregoing list.
[0165] While secretion of such factors is useful, PPDCs can also be
characterized by their lack of secretion of factors into the
medium. Postpartum-derived cells that lack secretion of at least
one, two, three or four of TGF-beta2, ANG2, PDGFbb, MIP1b, I309,
MDC, and VEGF, as detected by ELISA, are presently preferred for
use. Cells that are characterized in their lack secretion of five
or six of the foregoing proteins are more preferred. Cells which
lack secretion of all seven of the factors listed above are also
preferred.
[0166] Examples of placenta-derived cells of the invention were
deposited with the American Type Culture Collection (ATCC,
Manassas, Va.) and assigned ATCC Accession Numbers as follows: (1)
strain designation PLA 071003 (P8) was deposited Jun. 15, 2004 and
assigned Accession No. PTA-6074; (2) strain designation PLA 071003
(P11) was deposited Jun. 15, 2004 and assigned Accession No.
PTA-6075; and (3) strain designation PLA 071003 (P16) was deposited
Jun. 16, 2004 and assigned Accession No. PTA-6079.
[0167] Examples of umbilical cord-derived cells of the invention
were deposited with the American Type Culture Collection (ATCC,
Manassas, Va.) on Jun. 10, 2004, and assigned ATCC Accession
Numbers as follows: (1) strain designation UMB 022803 (P7) was
assigned Accession No. PTA-6067; and (2) strain designation UMB
022803 (P17) was assigned Accession No. PTA-6068.
[0168] PPDCs can be isolated. The invention also provides
compositions of PPDCs, including populations of PPDCs. In some
embodiments, the cell population is heterogeneous. A heterogeneous
cell population of the invention may comprise at least about 5%,
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% PPDCs of the
invention. In some embodiments, the heterogeneous cell populations
of the invention may further comprise bone marrow cells, stem
cells, chondroblasts, chondrocytes, and/or progenitor cells. In
some embodiments, the heterogeneous cell populations of the
invention may further comprise bone marrow cells, stem cells,
osteoblasts, osteocytes, osteoclasts, bone lining cells, and/or
progenitor cells. In some embodiments, the population is
substantially homogeneous, i.e., comprises substantially only PPDCs
(preferably at least about 96%, 97%, 98%, 99% or more PPDCs). The
homogeneous cell population of the invention may comprise umbilical
cord- or placenta-derived cells. Homogeneous populations of
umbilical cord-derived cells may be free of cells of maternal
lineage. Homogeneous populations of placenta-derived cells may be
of neonatal or maternal lineage. Homogeneity of a cell population
may be achieved by any method known in the art, for example, by
cell sorting (e.g., flow cytometry), bead separation, or by clonal
expansion.
[0169] Methods of the invention further include methods for
producing a population of postpartum-derived cells by expanding a
cell of the invention in culture. The postpartum-derived cells of
the invention preferably expand in the presence of from about 5% to
about 20% oxygen. The postpartum-derived cells of the invention
preferably are expanded in culture medium such as but not limited
to Dulbecco's modified Eagle's medium (DMEM), mesenchymal stem cell
growth medium, advanced DMEM (Gibco), DMEM/MCDB201 (Sigma),
RPMI1640, CELL-GRO FREE, advanced DMEM (Gibco), DMEM/MCDB201
(Sigma), Ham's F10 medium, Ham's F12 medium, DMEM/F12, Iscove's
modified Dulbecco's medium, or Eagle's basal medium. The culture
medium preferably contains low or high glucose, about 2%-15% (v/v)
serum, betamercaptoethanol, and an antibiotic agent. The culture
medium may contain at least one of fibroblast growth factor,
platelet-derived growth factor, vascular endothelial growth factor,
and epidermal growth factor. The cells of the invention may be
grown on an uncoated or coated surface. Surfaces for growth of the
cells may be coated for example with gelatin, collagen (e.g.,
native or denatured), fibronectin, laminin, ornithine, vitronectin,
or extracellular membrane protein (e.g., MATRIGEL). In some
embodiments, a population of postpartum-derived cells is mixed with
another population of cells.
Culture of PPDCs in a Chondrogenic Medium
[0170] PPDCs may be induced to differentiate into a chondrogenic
lineage by subjecting them to differentiation-inducing cell culture
conditions. PPDCs may be cultured in a chondrogenic medium
comprising specific exogenous chondrogenic growth factors (e.g., in
culture), such as, for example, one or more of GDF-5 or
transforming growth factor beta3 (TGF-beta3), with or without
ascorbate.
[0171] Preferred chondrogenic medium is supplemented with an
antibiotic agent, amino acids including proline and glutamine,
sodium pyruvate, dexamethasone, ascorbic acid, and
insulin/tranferrin/selenium. Chondrogenic medium is preferably
supplemented with sodium hydroxide and/or collagen. Most
preferably, chondrogenic culture medium is supplemented with
collagen. The cells may be cultured at high or low density. Cells
are preferably cultured in the absence of serum.
Culture of PPDCs in an Osteogenic Medium
[0172] PPDCs may be induced to differentiate into an osteogenic
lineage by subjecting them to differentiation-inducing cell culture
conditions. In some embodiments, PPDCs are cultured in osteogenic
medium such as, but not limited to, media (e.g., DMEM-low glucose)
containing about 10.sup.-7 molar and about 10.sup.-9 molar
dexamethasone in combination with about 10 micromolar to about 50
micromolar ascorbate phosphate salt (e.g., ascorbate-2-phosphate)
and between about 10 nanomolar and about 10 millimolar
beta-glycerophosphate. The medium preferably includes serum (e.g.,
bovine serum, horse serum). Osteogenic medium also may comprise one
or more antibiotic/antimycotic agents. The osteogenic medium is
preferably supplemented with transforming growth factor-beta (e.g.,
TGF-beta1) and/or bone morphogenic protein (e.g., BMP-2, BMP-4, or
a combination thereof; most preferably BMP-4)
Assessment of Differentiation
[0173] PPDCs may be induced to differentiate to an ectodermal,
endodermal, or mesodermal lineage. Methods to characterize
differentiated cells that develop from the PPDCs of the invention,
include, but are not limited to, histological, morphological,
biochemical and immunohistochemical methods, or using cell surface
markers, or genetically or molecularly, or by identifying factors
secreted by the differentiated cell, and by the inductive qualities
of the differentiated PPDCs.
[0174] Chondrogenic differentiation may be assessed, for example,
by Safranin-O staining for glycosaminoglycan expression by the
cells or hematoxylin/eosin staining or by detection of a
chondrogenic lienage marker (e.g., sulfated glycosaminoglycans and
proteoglycans, keratin, chondroitin, Type II collagen) in the
culture or more preferably in the cells themselves.
[0175] PPDCs may be analyzed for an osteogenic phenotype by any
method known in the art, e.g., von Kossa staining or by detection
of osteogenic markers such as osteocalcin, bone sialoprotein,
alkaline phosphatase, osteonectin, osteopontin, type I collagen,
bone morphogenic proteins, and/or core binding factor a1 in the
culture or more preferably in the cells themselves.
Methods of Using PPDCs or Components or Products Thereof
[0176] Genetic Engineering of PPDCs
[0177] The cells of the invention can be engineered using any of a
variety of vectors including, but not limited to, integrating viral
vectors, e.g., retrovirus vector or adeno-associated viral vectors;
non-integrating replicating vectors, e.g., papilloma virus vectors,
SV40 vectors, adenoviral vectors; or replication-defective viral
vectors. Other methods of introducing DNA into cells include the
use of liposomes, electroporation, a particle gun, or by direct DNA
injection.
[0178] Hosts cells are preferably transformed or transfected with
DNA controlled by or in operative association with, one or more
appropriate expression control elements such as promoter or
enhancer sequences, transcription terminators, polyadenylation
sites, among others, and a selectable marker.
[0179] Following the introduction of the foreign DNA, engineered
cells may be allowed to grow in enriched media and then switched to
selective media. The selectable marker in the foreign DNA confers
resistance to the selection and allows cells to stably integrate
the foreign DNA as, for example, on a plasmid, into their
chromosomes and grow to form foci which, in turn, can be cloned and
expanded into cell lines.
[0180] This method can be advantageously used to engineer cell
lines which express the gene product.
[0181] Any promoter may be used to drive the expression of the
inserted gene. For example, viral promoters include, but are not
limited to, the CMV promoter/enhancer, SV 40, papillomavirus,
Epstein-Barr virus or elastin gene promoter. Preferably, the
control elements used to control expression of the gene of interest
should allow for the regulated expression of the gene so that the
product is synthesized only when needed in vivo. If transient
expression is desired, constitutive promoters are preferably used
in a non-integrating and/or replication-defective vector.
Alternatively, inducible promoters could be used to drive the
expression of the inserted gene when necessary.
[0182] Inducible promoters include, but are not limited to, those
associated with metallothionein and heat shock proteins.
[0183] The cells of the invention may be genetically engineered to
"knock out" or "knock down" expression of factors that promote
inflammation or rejection at the implant site. Negative modulatory
techniques for the reduction of target gene expression levels or
target gene product activity levels are discussed below. "Negative
modulation," as used herein, refers to a reduction in the level
and/or activity of target gene product relative to the level and/or
activity of the target gene product in the absence of the
modulatory treatment. The expression of a gene native to a
chondrocyte or osteocyte can be reduced or knocked out using a
number of techniques including, for example, inhibition of
expression by inactivating the gene completely (commonly termed
"knockout") using the homologous recombination technique. Usually,
an exon encoding an important region of the protein (or an exon 5'
to that region) is interrupted by a positive selectable marker,
e.g., neo, preventing the production of normal mRNA from the target
gene and resulting in inactivation of the gene. A gene may also be
inactivated by creating a deletion in part of a gene, or by
deleting the entire gene. By using a construct with two regions of
homology to the target gene that are far apart in the genome, the
sequences intervening the two regions can be deleted (Mombaerts et
al., 1991, Proc. Nat. Acad. Sci. U.S.A. 88:3084).
[0184] Antisense, small interfering RNA, DNAzymes and ribozyme
molecules which inhibit expression of the target gene can also be
used in accordance with the invention to reduce the level of target
gene activity. For example, antisense RNA molecules which inhibit
the expression of major histocompatibility gene complexes (HLA)
have been shown to be most versatile with respect to immune
responses. Still further, triple helix molecules can be utilized in
reducing the level of target gene activity.
[0185] These techniques are described in detail by L. G. Davis et
al. (eds), 1994, BASIC METHODS IN MOLECULAR BIOLOGY, 2nd ed.,
Appleton & Lange, Norwalk, Conn., which is incorporated herein
by reference.
[0186] IL-1 is a potent stimulator of cartilage resorption and of
the production of inflammatory mediators by chondrocytes (Campbell
et al., 1991, J. Immun. 147: 1238). Using any of the foregoing
techniques, the expression of IL-1 can be knocked out or knocked
down in the cells of the invention to reduce the risk of resorption
of implanted cartilage or the production of inflammatory mediators
by the cells of the invention. Likewise, the expression of MHC
class II molecules can be knocked out or knocked down in order to
reduce the risk of rejection of the implanted tissue.
[0187] Once the cells of the invention have been genetically
engineered, they may be directly implanted into the patient to
allow for the treatment of a bone or cartilage condition, for
example, rheumatoid or joint disease, or to produce an
anti-inflammatory gene product such as, for example, peptides or
polypeptides corresponding to the idiotype of neutralizing
antibodies for GM-CSF, TNF, IL-1, IL-2, or other inflammatory
cytokines.
[0188] Alternatively, the genetically engineered cells may be used
to produce new tissue in vitro, which is then implanted in the
subject, as described herein.
[0189] Secretion of Trophic Factors
[0190] The secretion of growth factors by PPDCs may provide trophic
support for a second cell type in vitro or in vivo. PPDCs may
secrete, for example, at least one of monocyte chemotactic protein
1 (MCP-1), interleukin-6 (IL6), interleukin 8 (IL-8), GCP-2,
hepatocyte growth factor (HGF), keratinocyte growth factor (KGF),
fibroblast growth factor (FGF), heparin binding epidermal growth
factor (HB-EGF), brain-derived neurotrophic factor (BDNF),
thrombopoietin (TPO), macrophage inflammatory protein 1 alpha
(MIP1a), RANTES, and tissue inhibitor of matrix metalloproteinase 1
(TIMP1), which can be augmented by a variety of techniques,
including ex vivo cultivation of the cells in chemically defined
medium.
[0191] In some aspects of the invention, a population of PPDCs
supports the survival, proliferation, growth, maintenance,
maturation, differentiation, or increased activity of cells
including stem cells, such as embryonic stem cells, bone marrow
cells, chondrocytes, chondroblasts, and mixtures thereof. In some
aspects of the invention, a population of PPDCs supports cells
including stem cells, such as embryonic stem cells, bone marrow
cells, osteoblasts, osteocytes, osteoclasts, bone lining cells, and
mixtures thereof. In some aspects of the invention, a population of
PPDCs supports cells including stem cells, such as embryonic stem
cells, bone marrow cells, chondrocytes, chondroblasts, osteoblasts,
osteocytes, osteoclasts, bone lining cells, and mixtures thereof.
In other embodiments, the population is substantially homogeneous,
i.e., comprises substantially only PPDCs (preferably at least about
96%, 97%, 98%, 99% or more PPDCs).
[0192] PPDCs have the ability to support survival, growth, and
differentiation of other cell types in co-culture. In some
embodiments, PPDCs are co-cultured in vitro to provide trophic
support to other cells, including but not limited to stem cells,
osteocytes, osteoblasts, osteoclasts, bone lining cells,
chondrocytes, chondroblasts, and/or bone marrow cells, or
combinations thereof. For co-culture, it may be desirable for the
PPDCs and the desired other cells to be co-cultured under
conditions in which the two cell types are in contact. This can be
achieved, for example, by seeding the cells as a heterogeneous
population of cells in culture medium or onto a suitable culture
substrate. Alternatively, the PPDCs can first be grown to
confluence and employed as a substrate for the second desired cell
type in culture. In this latter embodiment, the cells may further
be physically separated, e.g., by a membrane or similar device,
such that the other cell type may be removed and used separately
following the co-culture period. In other embodiments, the desired
other cells are cultured in contact with the conditioned medium,
extracellular matrix, and/or cell lysate of the PPDCs. Use of PPDCs
in co-culture to promote expansion and differentiation of other
cell types may find applicability in research and in
clinical/therapeutic areas. For instance, PPDC co-culture may be
utilized to facilitate growth and differentiation of cells of a
given phenotype in culture, for example, chondrocytes or
osteocytes, for basic research purposes or for use in drug
screening assays, for example. PPDC co-culture may also be utilized
for ex vivo expansion of cells of an osteogenic or chondrogenic
phenotype for later administration for therapeutic purposes. For
example, cells may be harvested from an individual, expanded ex
vivo in co-culture with PPDCs, then returned to that individual
(autologous transfer) or another individual (syngeneic or
allogeneic transfer). In these embodiments, it will be appreciated
that, following ex vivo expansion, the mixed population of cells
comprising the PPDCs could be administered to a patient in need of
treatment, for example, of a bone or cartilage condition as
described herein. Alternatively, in situations where autologous
transfer is appropriate or desirable, the co-cultured cell
populations may be physically separated in culture, enabling
removal of the autologous cells for administration to the
patient.
[0193] Conditioned Medium of PPDCs
[0194] Another embodiment of the invention features use of PPDCs
for production of conditioned medium, either from undifferentiated
PPDCs or from PPDCs incubated under conditions that stimulate
differentiation into a chondrogenic or osteogenic lineage. Such
conditioned media are contemplated for use in in vitro or ex vivo
culture of cells, for example, stem or progenitor cells, including
but not limited to bone marrow cells, osteoblasts, osteocytes,
osteoclasts, bone lining cells, chondroblasts, and chondrocytes, or
in vivo to support transplanted cells comprising homogeneous or
heterogeneous populations of PPDCs and/or stem cells, osteocytes,
osteoblasts, osteoclasts, bone lining cells, chondrocytes,
chondroblasts, and bone marrow cells, for example.
Therapeutic Applications of PPDCs
[0195] PPDCs of the invention may be used to treat patients
requiring the repair or replacement of cartilage or bone tissue
resulting from disease or trauma or failure of the tissue to
develop normally, or to provide a cosmetic function, such as to
augment facial or other features of the body. Treatment may entail
the use of the cells of the invention to produce new cartilage
tissue or bone tissue. For example, the undifferentiated or
chondrogenic differentiation-induced cells of the invention may be
used to treat a cartilage condition, for example, rheumatoid
arthritis or osteoarthritis or a traumatic or surgical injury to
cartilage. As another example, the undifferentiated or osteogenic
differentiation-induced cells of the invention may be used to treat
bone conditions, including metabolic and non-metabolic bone
diseases. Examples of bone conditions include meniscal tears,
spinal fusion, spinal disc removal, spinal reconstruction, bone
fractures, bone/spinal deformation, osteosarcoma, myeloma, bone
dysplasia, scoliosis, osteoporosis, periodontal disease, dental
bone loss, osteomalacia, rickets, fibrous osteitis, renal bone
dystrophy, and Paget's disease of bone.
[0196] The cells of the invention may be administered alone or as
admixtures with other cells. Cells that may be administered in
conjunction with PPDCs include, but are not limited to, other
multipotent or pluripotent cells or chondrocytes, chondroblasts,
osteocytes, osteoblasts, osteoclasts, bone lining cells, stem
cells, or bone marrow cells. The cells of different types may be
admixed with the PPDCs immediately or shortly prior to
administration, or they may be co-cultured together for a period of
time prior to administration.
[0197] The PPDCs may be administered with other beneficial drugs or
biological molecules (growth factors, trophic factors). When PPDCs
are administered with other agents, they may be administered
together in a single pharmaceutical composition, or in separate
pharmaceutical compositions, simultaneously or sequentially with
the other agents (either before or after administration of the
other agents). Bioactive factors which may be co-administered
include anti-apoptotic agents (e.g., EPO, EPO mimetibody, TPO,
IGF-I and IGF-II, HGF, caspase inhibitors); anti-inflammatory
agents (e.g., p38 MAPK inhibitors, TGF-beta inhibitors, statins,
IL-6 and IL-1 inhibitors, PEMIROLAST, TRANILAST, REMICADE,
SIROLIMUS, and NSAIDs (non-steroidal anti-inflammatory drugs; e.g.,
TEPOXALIN, TOLMETIN, SUPROFEN); immunosupressive/immunomodulatory
agents (e.g., calcineurin inhibitors, such as cyclosporine,
tacrolimus; mTOR inhibitors (e.g., SIROLIMUS, EVEROLIMUS);
anti-proliferatives (e.g., azathioprine, mycophenolate mofetil);
corticosteroids (e.g., prednisolone, hydrocortisone); antibodies
such as monoclonal anti-IL-2Ralpha receptor antibodies (e.g.,
basiliximab, daclizumab), polyclonal anti-T-cell antibodies (e.g.,
anti-thymocyte globulin (ATG); anti-lymphocyte globulin (ALG);
monoclonal anti-T cell antibody OKT3)); anti-thrombogenic agents
(e.g., heparin, heparin derivatives, urokinase, PPack
(dextrophenylalanine proline arginine chloromethylketone),
antithrombin compounds, platelet receptor antagonists,
anti-thrombin antibodies, anti-platelet receptor antibodies,
aspirin, dipyridamole, protamine, hirudin, prostaglandin
inhibitors, and platelet inhibitors); and anti-oxidants (e.g.,
probucol, vitamin A, ascorbic acid, tocopherol, coenzyme Q-10,
glutathione, L-cysteine, N-acetylcysteine) as well as local
anesthetics. As another example, the cells may be co-administered
with scar inhibitory factor as described in U.S. Pat. No.
5,827,735, incorporated herein by reference.
[0198] In one embodiment, PPDCs are administered as
undifferentiated cells, i.e., as cultured in Growth Medium.
Alternatively, PPDCs may be administered following exposure in
culture to conditions that stimulate differentiation toward a
desired phenotype, for example, a chondrogenic or osteogenic
phenotype.
[0199] The cells of the invention may be surgically implanted,
injected, delivered (e.g., by way of a catheter or syringe), or
otherwise administered directly or indirectly to the site in need
of repair or augmentation. The cells may be administered by way of
a matrix (e.g., a three-dimensional scaffold). The cells may be
administered with conventional pharmaceutically acceptable
carriers. Routes of administration of the cells of the invention or
compositions or components (e.g., ECM, cell lysate, conditioned
medium) thereof include intramuscular, ophthalmic, parenteral
(including intravenous), intraarterial, subcutaneous, oral, and
nasal administration. Particular routes of parenteral
administration include, but are not limited to, intramuscular,
subcutaneous, intraperitoneal, intracerebral, intraventricular,
intracerebroventricular, intrathecal, intracisternal, intraspinal
and/or peri-spinal routes of administration.
[0200] When cells are administered in semi-solid or solid devices,
surgical implantation into a precise location in the body is
typically a suitable means of administration. Liquid or fluid
pharmaceutical compositions, however, may be administered to a more
general location (e.g., throughout a diffusely affected area, for
example), from which they migrate to a particular location, e.g.,
by responding to chemical signals.
[0201] Other embodiments encompass methods of treatment by
administering pharmaceutical compositions comprising PPDC cellular
components (e.g., cell lysates or components thereof) or products
(e.g., extracellular matrix, trophic and other biological factors
produced naturally by PPDCs or through genetic modification,
conditioned medium from PPDC culture). Again, these methods may
further comprise administering other active agents, such as
anti-apoptotic agents (e.g., EPO, EPO mimetibody, TPO, IGF-I and
IGF-II, HGF, caspase inhibitors); anti-inflammatory agents (e.g.,
p38 MAPK inhibitors, TGF-beta inhibitors, statins, IL-6 and IL-1
inhibitors, PEMIROLAST, TRANILAST, REMICADE, SIROLIMUS, and NSAIDs
(non-steroidal anti-inflammatory drugs; e.g., TEPOXALIN, TOLMETIN,
SUPROFEN); immunosupressive/immunomodulatory agents (e.g.,
calcineurin inhibitors, such as cyclosporine, tacrolimus; mTOR
inhibitors (e.g., SIROLIMUS, EVEROLIMUS); anti-proliferatives
(e.g., azathioprine, mycophenolate mofetil); corticosteroids (e.g.,
prednisolone, hydrocortisone); antibodies such as monoclonal
anti-IL-2Ralpha receptor antibodies (e.g., basiliximab,
daclizumab), polyclonal anti-T-cell antibodies (e.g.,
anti-thymocyte globulin (ATG); anti-lymphocyte globulin (ALG);
monoclonal anti-T cell antibody OKT3)); anti-thrombogenic agents
(e.g., heparin, heparin derivatives, urokinase, PPack
(dextrophenylalanine proline arginine chloromethylketone),
antithrombin compounds, platelet receptor antagonists,
anti-thrombin antibodies, anti-platelet receptor antibodies,
aspirin, dipyridamole, protamine, hirudin, prostaglandin
inhibitors, and platelet inhibitors); and anti-oxidants (e.g.,
probucol, vitamin A, ascorbic acid, tocopherol, coenzyme Q-10,
glutathione, L-cysteine, N-acetylcysteine), local anesthetics, and
scar inhibitory factor as described in U.S. Pat. No. 5,827,735,
incorporated herein by reference.
[0202] Dosage forms and regimes for administering PPDCs or any of
the other pharmaceutical compositions described herein are
developed in accordance with good medical practice, taking into
account the condition of the individual patient, e.g., nature and
extent of the condition being treated, age, sex, body weight and
general medical condition, and other factors known to medical
practitioners. Thus, the effective amount of a pharmaceutical
composition to be administered to a patient is determined by these
considerations as known in the art.
[0203] In some embodiments of the invention, it may not be
necessary or desirable to immunosuppress a patient prior to
initiation of cell therapy with PPDCs. In addition, PPDCs have been
shown not to stimulate allogeneic PBMCs in a mixed lymphocyte
reaction. Accordingly, transplantation with allogeneic, or even
xenogeneic, PPDCs may be tolerated in some instances.
[0204] However, in other instances it may be desirable or
appropriate to pharmacologically immunosuppress a patient prior to
initiating cell therapy. This may be accomplished through the use
of systemic or local immunosuppressive agents, or it may be
accomplished by delivering the cells in an encapsulated device.
PPDCs may be encapsulated in a capsule that is permeable to
nutrients and oxygen required by the cell and therapeutic factors
the cell is yet impermeable to immune humoral factors and cells.
Preferably the encapsulant is hypoallergenic, is easily and stably
situated in a target tissue, and provides added protection to the
implanted structure. These and other means for reducing or
eliminating an immune response to the transplanted cells are known
in the art. As an alternative, PPDCs may be genetically modified to
reduce their immunogenicity.
[0205] Survival of transplanted PPDCs in a living patient can be
determined through the use of a variety of scanning techniques,
e.g., computerized axial tomography (CAT or CT) scan, magnetic
resonance imaging (MRI) or positron emission tomography (PET)
scans. Determination of transplant survival can also be done post
mortem by removing the target tissue, and examining it visually or
through a microscope. Alternatively, cells can be treated with
stains that are specific for cells of a specific lineage.
Transplanted cells can also be identified by prior incorporation of
tracer dyes such as rhodamine- or fluorescein-labeled microspheres,
fast blue, bisbenzamide, ferric microparticles, or genetically
introduced reporter gene products, such as beta-galactosidase or
beta-glucuronidase.
[0206] Functional integration of transplanted PPDCs into a subject
can be assessed by examining restoration of the function that was
damaged or diseased, for example, restoration of joint or bone
function, or augmentation of function.
[0207] Compositions and Pharmaceutical Compositions
[0208] Compositions of PPDCs and related products (e.g.,
extracellular matrix, lysate, soluble cell fraction, conditioned
medium), including for example pharmaceutical compositions, are
included within the scope of the invention. Compositions of the
invention may include one or more bioactive factors, for example
but not limited to a growth factor, a differentiation-inducing
factor, a cell survival factor such as caspase inhibitor, an
anti-inflammatory agent such as p38 kinase inhibitor, or an
angiogenic factor such as VEGF or bFGF. Some examples of bioactive
factors include PDGF-bb, EGF, bFGF, IGF-1, and LIF. In some
embodiments, undifferentiated or differentiation-induced PDPCs are
cultured in contact with the bioactive factor. In some embodiments,
undifferentiated PPDCs remain undifferentiated upon contact with
the bioactive factor. In other embodiments, the bioactive factor
induces differentiation of the PPDCs.
[0209] Pharmaceutical compositions of the invention may comprise
homogeneous or heterogeneous populations of PPDCs, extracellular
matrix or cell lysate thereof, or conditioned medium thereof in a
pharmaceutically acceptable carrier. Pharmaceutically acceptable
carriers for the cells of the invention include organic or
inorganic carrier substances suitable which do not deleteriously
react with the cells of the invention or compositions or components
thereof. To the extent they are biocompatible, suitable
pharmaceutically acceptable carriers include water, salt solution
(such as Ringer's solution), alcohols, oils, gelatins, and
carbohydrates, such as lactose, amylose, or starch, fatty acid
esters, hydroxymethylcellulose, and polyvinyl pyrolidine. Such
preparations can be sterilized, and if desired, mixed with
auxiliary agents such as lubricants, preservatives, stabilizers,
wetting agents, emulsifiers, salts for influencing osmotic
pressure, buffers, and coloring. Pharmaceutical carriers suitable
for use in the present invention are known in the art and are
described, for example, in Pharmaceutical Sciences (17.sup.th Ed.,
Mack Pub. Co., Easton, Pa.) and WO 96/05309, each of which are
incorporated by reference herein.
[0210] The dosage (e.g., the number of cells to be administered)
and frequency of administration of the pharmaceutical compositions
of the invention will depend upon a number of factors, including
but not limited to, the nature of the condition to be treated, the
extent of the symptoms of the condition, characteristics of the
patient (e.g., age, size, gender, health).
[0211] For example but not by way of limitation, PPDCs,
extracellular matrix or cell lysates thereof, conditioned medium,
compositions, and matrices produced according to the invention can
be used to repair or replace damaged or destroyed cartilage tissue,
to augment existing cartilage tissue, to introduce new or altered
tissue, to modify artificial prostheses, or to join biological
tissues or structures. For example, some embodiments of the
invention would include (i) hip prostheses coated with replacement
cartilage tissue constructs grown in three-dimensional cultures;
(ii) knee reconstruction with cartilage tissue constructs; (iii)
prostheses of other joints requiring reconstruction and/or
replacement of articular cartilage; and (iv) cosmetic
reconstruction with cartilage tissue constructs.
[0212] For example, the evaluation of internal derangements of
articular cartilage in for example, the knee, hip, elbow, ankle and
the glenohumeral joint, may be performed by arthroscopic
techniques. In some embodiments, the injured or deteriorated
portion of cartilage tissue is removed, for example, by
arthroscopic surgery, followed by cartilage grafting. Cartilage
tissue constructs may also be employed in reconstructive surgery
for different types of joints. Detailed procedures have been
described in Resnick, D., and Niwayama, G., (eds), 1988, Diagnosis
of Bone and Joint Disorders, 2d ed., W.B. Sanders Co., which is
incorporated herein by reference.
[0213] Repair or replacement of damaged cartilage may be enhanced
by fixation of the implanted cells and/or cartilage tissue at the
site of repair. Various methods can be used to fix the new cells
and/or cartilage tissue in place, including: patches derived from
biocompatible tissues, which can be placed over the site;
bioabsorbable sutures or other fasteners, e.g., pins, staples,
tacks, screws and anchors; non-absorbable fixation devices, e.g.,
sutures, pins, screws and anchors; adhesives.
[0214] As another example but not by way of limitation, PPDCs,
extracellular matrix or cell lysates thereof, conditioned medium,
and the bone tissue produced according to the invention can be used
to repair or replace damaged or destroyed bone tissue, to augment
existing bone tissue, to introduce new or altered tissue, or to
modify artificial prostheses. The cells of the invention may be
administered alone, in a pharmaceutically acceptable carrier, or
seeded on or in a matrix as described herein.
[0215] Use of PPDCs for Transplantation
[0216] The treatment methods of the subject invention involve the
implantation of PPDCs or trans-differentiated cells into
individuals in need thereof. The cells of the present invention may
be allogeneic or autologous and may be delivered to the site of
therapeutic need or "home" to the site.
[0217] The cells of the present invention may be differentiated in
vitro prior to implantation in a patient. In vitro differentiation
allows for controlled application of bioactive factors.
[0218] The cells of the present invention may be induced to
differentiate in situ or may be introduced in vivo to provide
trophic support to endogenous cells. The appropriate cell
implantation dosage in humans can be determined from existing
information relating to either the activity of the cells or the
density of cells for bone or cartilage replacement. This
information is also useful in calculating an appropriate dosage of
implanted material. Additionally, the patient can be monitored to
determine if additional implantation can be made or implanted
material reduced accordingly.
[0219] To enhance the differentiation, survival or activity of
implanted cells, additional bioactive factors may be added
including growth factors, anti-apoptotic agents (e.g., EPO, EPO
mimetibody, TPO, IGF-I and IGF-II, HGF, caspase inhibitors);
anti-inflammatory agents (e.g., p38 MAPK inhibitors, TGF-beta
inhibitors, statins, IL-6 and IL-1 inhibitors, PEMIROLAST,
TRANILAST, REMICADE, SIROLIMUS, and NSAIDs (non-steroidal
anti-inflammatory drugs; e.g., TEPOXALIN, TOLMETIN, SUPROFEN);
immunosupressive/immunomodulatory agents (e.g., calcineurin
inhibitors, such as cyclosporine, tacrolimus; mTOR inhibitors
(e.g., SIROLIMUS, EVEROLIMUS); anti-proliferatives (e.g.,
azathioprine, mycophenolate mofetil); corticosteroids (e.g.,
prednisolone, hydrocortisone); antibodies such as monoclonal
anti-IL-2Ralpha receptor antibodies (e.g., basiliximab,
daclizumab), polyclonal anti-T-cell antibodies (e.g.,
anti-thymocyte globulin (ATG); anti-lymphocyte globulin (ALG);
monoclonal anti-T cell antibody OKT3)); anti-thrombogenic agents
(e.g., heparin, heparin derivatives, urokinase, PPack
(dextrophenylalanine proline arginine chloromethylketone),
antithrombin compounds, platelet receptor antagonists,
anti-thrombin antibodies, anti-platelet receptor antibodies,
aspirin, dipyridamole, protamine, hirudin, prostaglandin
inhibitors, and platelet inhibitors); and anti-oxidants (e.g.,
probucol, vitamin A, ascorbic acid, tocopherol, coenzyme Q-10,
glutathione, L-cysteine, N-acetylcysteine), and local anesthetics.
To enhance vascularization and survival of transplanted bone
tissue, angiogenic factors such as VEGF, PDGF or bFGF can be added
either alone or in combination with endothelial cells or their
precursors including CD34+, CD34+/CD117+ cells.
[0220] Alternatively, PPDCs to be transplanted may be genetically
engineered to express such growth factors, antioxidants,
antiapoptotic agents, anti-inflammatory agents, or angiogenic
factors.
[0221] PPDCs can be used to treat diseases or conditions of bone or
cartilage or to augment or replace bone or cartilage. The disease
or conditions to be treated include but are not limited to
osteoarthritis, osteoporosis, rheumatoid arthritis, chondrosis
deformans, dental and oral cavity disease (e.g., tooth fracture and
defects), joint replacement, congenital abnormalities, bone
fracture, and tumors (benign and malignant).
[0222] One or more other components may be added to transplanted
cells, including selected extracellular matrix components, such as
one or more types of collagen known in the art, and/or growth
factors, platelet-rich plasma, and drugs. Alternatively, the cells
of the invention may be genetically engineered to express and
produce for growth factors. Details on genetic engineering of the
cells of the invention are provided infra. Bioactive factors which
may be usefully incorporated into the cell formulation include
anti-apoptotic agents (e.g., EPO, EPO mimetibody, TPO, IGF-I and
IGF-II, HGF, caspase inhibitors); anti-inflammatory agents (e.g.,
p38 MAPK inhibitors, TGF-beta inhibitors, statins, IL-6 and IL-1
inhibitors, PEMIROLAST, TRANILAST, REMICADE, SIROLIMUS, and NSAIDs
(non-steroidal anti-inflammatory drugs; e.g., TEPOXALIN, TOLMETIN,
SUPROFEN); immunosupressive/immunomodulatory agents (e.g.,
calcineurin inhibitors, such as cyclosporine, tacrolimus; mTOR
inhibitors (e.g., SIROLIMUS, EVEROLIMUS); anti-proliferatives
(e.g., azathioprine, mycophenolate mofetil); corticosteroids (e.g.,
prednisolone, hydrocortisone); antibodies such as monoclonal
anti-IL-2Ralpha receptor antibodies (e.g., basiliximab,
daclizumab), polyclonal anti-T-cell antibodies (e.g.,
anti-thymocyte globulin (ATG); anti-lymphocyte globulin (ALG);
monoclonal anti-T cell antibody OKT3)); anti-thrombogenic agents
(e.g., heparin, heparin derivatives, urokinase, PPack
(dextrophenylalanine proline arginine chloromethylketone),
antithrombin compounds, platelet receptor antagonists,
anti-thrombin antibodies, anti-platelet receptor antibodies,
aspirin, dipyridamole, protamine, hirudin, prostaglandin
inhibitors, and platelet inhibitors); and anti-oxidants (e.g.,
probucol, vitamin A, ascorbic acid, tocopherol, coenzyme Q-10,
glutathione, L-cysteine, N-acetylcysteine), and local anesthetics.
For example, the cells may be co-administered with scar inhibitory
factor as described in U.S. Pat. No. 5,827,735, incorporated herein
by reference.
[0223] Formulation of PPDCs for Transplantation
[0224] In a non-limiting embodiment, a formulation comprising the
cells of the invention is prepared for administration directly to
the site where the production of new cartilage or bone tissue is
desired. For example, and not by way of limitation, the cells of
the invention may be suspended in a hydrogel solution for
injection. Examples of suitable hydrogels for use in the invention
include self-assembling peptides, such as RAD16. Alternatively, the
hydrogel solution containing the cells may be allowed to harden,
for instance in a mold, to form a matrix having cells dispersed
therein prior to implantation. Or, once the matrix has hardened,
the cell formations may be cultured so that the cells are
mitotically expanded prior to implantation. The hydrogel is an
organic polymer (natural or synthetic) which is cross-linked via
covalent, ionic, or hydrogen bonds to create a three-dimensional
open-lattice structure which entraps water molecules to form a gel.
Examples of materials which can be used to form a hydrogel 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
support for the PPDCs of the invention is biodegradable.
[0225] In some embodiments of the invention, the formulation
comprises an in situ polymerizable gel, as described, for example,
in 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).
[0226] 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
with 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.
[0227] Examples of polymers with basic side groups that can be
reacted with anions are poly(vinyl amines), poly(vinyl pyridine),
poly(vinyl imidazole), and some imino substituted polyphosphazenes.
The ammonium or quaternary salt of the polymers can also be formed
from the backbone nitrogens or pendant imino groups. Examples of
basic side groups are amino and imino groups.
[0228] Alginate can be ionically cross-linked with divalent
cations, in water, at room temperature, to form a hydrogel matrix.
Due to these mild conditions, alginate has been the most commonly
used polymer for hybridoma cell encapsulation, as described, for
example, in U.S. Pat. No. 4,352,883 to Lim. In the Lim process, an
aqueous solution containing the biological materials to be
encapsulated is suspended in a solution of a water soluble polymer,
the suspension is formed into droplets which are configured into
discrete microcapsules by contact with multivalent cations, then
the surface of the microcapsules is crosslinked with polyamino
acids to form a semipermeable membrane around the encapsulated
materials.
[0229] Polyphosphazenes are polymers with backbones consisting of
nitrogen and phosphorous separated by alternating single and double
bonds. Each phosphorous atom is covalently bonded to two side
chains.
[0230] The polyphosphazenes suitable for cross-linking have a
majority of side chain groups which are acidic and capable of
forming salt bridges with di- or trivalent cations. Examples of
preferred acidic side groups are carboxylic acid groups and
sulfonic acid groups. Hydrolytically stable polyphosphazenes are
formed of monomers having carboxylic acid side groups that are
crosslinked by divalent or trivalent cations such as Ca.sup.2+ or
Al.sup.3+. Polymers can be synthesized that degrade by hydrolysis
by incorporating monomers having imidazole, amino acid ester, or
glycerol side groups. For example, a polyanionic
poly[bis(carboxylatophenoxy)]phosphazene (PCPP) can be synthesized,
which is cross-linked with dissolved multivalent cations in aqueous
media at room temperature or below to form hydrogel matrices.
[0231] Biodegradable polyphosphazenes have at least two differing
types of side chains, acidic side groups capable of forming salt
bridges with multivalent cations, and side groups that hydrolyze
under in vivo conditions, e.g., imidazole groups, amino acid
esters, glycerol and glucosyl.
[0232] Hydrolysis of the side chain results in erosion of the
polymer. Examples of hydrolyzing side chains are unsubstituted and
substituted imidizoles and amino acid esters in which the group is
bonded to the phosphorous atom through an amino linkage
(polyphosphazene polymers in which both R groups are attached in
this manner are known as polyaminophosphazenes). For
polyimidazolephosphazenes, some of the "R" groups on the
polyphosphazene backbone are imidazole rings, attached to
phosphorous in the backbone through a ring nitrogen atom. Other "R"
groups can be organic residues that do not participate in
hydrolysis, such as methyl phenoxy groups or other groups shown in
the scientific paper of Allcock, et al., Macromolecule 10:824
(1977). Methods of synthesis of the hydrogel materials, as well as
methods for preparing such hydrogels, are known in the art.
[0233] Other components may also be included in the formulation,
including but not limited to any of the following: (1) buffers to
provide appropriate pH and isotonicity; (2) lubricants; (3) viscous
materials to retain the cells at or near the site of
administration, including, for example, alginates, agars and plant
gums; and (4) other cell types that may produce a desired effect at
the site of administration, such as, for example, enhancement or
modification of the formation of tissue or its physicochemical
characteristics, or as support for the viability of the cells, or
inhibition of inflammation or rejection. The cells may be covered
by an appropriate wound covering to prevent cells from leaving the
site. Such wound coverings are known as those of skill in the
art.
[0234] Formulation of a Cartilage or Bone Tissue Patch
[0235] Culture or co-cultures of PPDCs in a pre-shaped well enables
the manufacture of a tissue patch of pre-determined thickness and
volume. The volume of the resulting tissue patch is dependent upon
the volume of the well and upon the number of PPDCs in the well.
Tissue of optimal pre-determined volume may be prepared by routine
experimentation by altering either or both of the aforementioned
parameters.
[0236] The cell contacting surface of the well may be coated with a
molecule that discourages adhesion of PPDCs to the cell contacting
surface. Preferred coating reagents include silicon based reagents
i.e., dichlorodimethylsilane or polytetrafluoroethylene based
reagents, i.e., TEFLON. Procedures for coating materials with
silicon based reagents, specifically dichlorodimethylsilane, are
well known in the art. See for example, Sambrook et al. (1989)
"Molecular Cloning A Laboratory Manual", Cold Spring Harbor
Laboratory Press, the disclosure of which is incorporated by
reference herein. It is appreciated that other biocompatible
reagents that prevent the attachment of cells to the surface of the
well may be useful in the practice of the instant invention.
[0237] Alternatively, the well may be cast from a pliable or
moldable biocompatible material that does not permit attachment of
cells per se. Preferred materials that prevent such cell attachment
include, but are not limited to, agarose, glass, untreated cell
culture plastic and polytetrafluoroethylene, i.e., TEFLON.
Untreated cell culture plastics, i.e., plastics that have not been
treated with or made from materials that have an electrostatic
charge are commercially available, and may be purchased, for
example, from Falcon Labware, Becton-Dickinson, Lincoln Park, N.J.
The aforementioned materials, however, are not meant to be
limiting. It is appreciated that any other pliable or moldable
biocompatible material that inherently discourages the attachment
of PPDCs may be useful in the practice of the instant
invention.
[0238] The size and shape of the well may be determined by the size
and shape of the tissue defect to be repaired. For example, it is
contemplated that the well may have a cross-sectional surface area
of 25 cm.sup.2. This is the average cross-sectional surface area of
an adult, human femoral chondyle. Accordingly, it is anticipated
that a single piece of cartilage may be prepared in accordance with
the invention in order to resurface the entire femoral chondyle.
The depth of the well is preferably greater than about 0.3 cm and
preferably about 0.6 cm in depth. The thickness of natural
articular cartilage in an adult articulating joint is usually about
0.3 cm. Accordingly, the depth of the well should be large enough
to permit a cartilage patch of about 0.3 cm to form. The well
should be deep enough to contain culture medium overlaying the
tissue patch.
[0239] It is contemplated that a tissue patch prepared in
accordance with the invention may be "trimmed" to a pre-selected
size and shape by a surgeon performing surgical repair of the
damaged tissue. Trimming may be performed with the use of a sharp
cutting implement, i.e., a scalpel, a pair of scissors or an
arthroscopic device fitted with a cutting edge, using procedures
well known in the art.
[0240] The pre-shaped well may be cast in a block of agarose gel
under aseptic conditions. Agarose is an economical, biocompatible,
pliable and moldable material that can be used to cast pre-shaped
wells, quickly and easily. As mentioned above, the dimensions of
the well may dependent upon the size of the resulting tissue plug
that is desired.
[0241] A pre-shaped well may be prepared by pouring a hot solution
of molten LT agarose (BioRad, Richmond, Calif.) into a tissue
culture dish containing a cylinder, the cylinder having dimensions
that mirror the shape of the well to be formed. The size and shape
of the well may be chosen by the artisan and may be dependent upon
the shape of the tissue defect to be repaired. Once the agarose has
cooled and solidified around the cylinder, the cylinder is
carefully removed with forceps. The surface of the tissue culture
dish that is exposed by the removal of the cylinder is covered with
molten agarose. This seals the bottom of the well and provides a
cell adhesive surface at the base of the well. When the newly added
molten LT agarose cools and solidifies, the resulting pre-shaped
well is suitable for culturing, and inducing the differentiation of
PPDCs. It is appreciated, however, that alternative methods may be
used to prepare a pre-shaped well useful in the practice of the
invention.
[0242] PPDCs in suspension may be seeded into and cultured in the
pre-shaped well. The PPDCs may be induced to differentiate to a
chondrogenic or osteogenic phenotype in culture in the well or may
have been induced to differentiate prior to seeding in the well.
The cells may be diluted by the addition of culture medium to a
cell density of about 1.times.10.sup.5 to 1.times.10.sup.9 PPDCs
per milliliter.
[0243] The cells may form a cohesive plug of cells. The cohesive
plug of cells may be removed from the well and surgically implanted
into the tissue defect. It is anticipated that undifferentiated
PPDCs may differentiate in situ thereby to form tissue in vivo.
[0244] Cartilage and bone defects may be identified inferentially
by using computer aided tomography (CAT scanning); X-ray
examination, magnetic resonance imaging (MRI), analysis of synovial
fluid or serum markers or by any other procedures known in the art.
Defects in mammals also are readily identifiable visually during
arthroscopic examination or during open surgery of the joint.
Treatment of the defects can be effected during an arthroscopic or
open surgical procedure using the methods and compositions
disclosed herein.
[0245] Accordingly, once the defect has been identified, the defect
may be treated by the following steps of (1) surgically implanting
at the pre-determined site a tissue patch prepared by the
methodologies described herein, and (2) permitting the tissue patch
to integrate into pre-determined site.
[0246] The tissue patch optimally has a size and shape such that
when the patch is implanted into the defect, the edges of the
implanted tissue contact directly the edges of the defect. In
addition, the tissue patch may be fixed in place during the
surgical procedure. This can be effected by surgically fixing the
patch into the defect with biodegradable sutures and/or by applying
a bioadhesive to the region interfacing the patch and the
defect.
[0247] In some instances, damaged tissue maybe surgically excised
prior the to implantation of the patch of tissue.
[0248] Transplantation of PPDCs Using Scaffolds
[0249] The cells of the invention or co-cultures thereof may be
seeded onto or into a three-dimensional scaffold and implanted in
vivo, where the seeded cells will proliferate on the framework and
form a replacement cartilage or bone tissue in vivo in cooperation
with the cells of the patient.
[0250] In some aspects of the invention, the matrix comprises
decellularized tissue, such as extracellular matrix, cell lysates
(e.g., soluble cell fractions), or combinations thereof, of the
PPDCs. In some embodiments, the matrix is biodegradable. In some
aspects of the invention, the matrix comprises natural or synthetic
polymers. Matrices of the invention include biocompatible
scaffolds, lattices, self-assembling structures and the like,
whether biodegradable or not, liquid or solid. Such matrices are
known in the arts of cell-based therapy, surgical repair, tissue
engineering, and wound healing. Preferably the matrices are
pretreated (e.g., seeded, inoculated, contacted with) with the
cells, extracellular matrix, conditioned medium, cell lysate, or
combination thereof, of the invention. More preferably the matrices
are populated with cells in close association to the matrix or its
spaces. In some aspects of the invention, the cells adhere to the
matrix. In some embodiments, the cells are contained within or
bridge interstitial spaces of the matrix. Most preferred are those
seeded matrices wherein the cells are in close association with the
matrix and which, when used therapeutically, induce or support
ingrowth of the patient's cells and/or proper angiogenesis. The
seeded matrices can be introduced into a patient's body in any way
known in the art, including but not limited to implantation,
injection, surgical attachment, transplantation with other tissue,
injection, and the like. The matrices of the invention may be
configured to the shape and/or size of a tissue or organ in
vivo.
[0251] For example, but not by way of limitation, the scaffold may
be designed such that the scaffold structure: (1) supports the
seeded cells without subsequent degradation; (2) supports the cells
from the time of seeding until the tissue transplant is remodeled
by the host tissue; (2) allows the seeded cells to attach,
proliferate, and develop into a tissue structure having sufficient
mechanical integrity to support itself in vitro, at which point,
the scaffold is degraded. A review of scaffold design is provided
by Hutmacher, J. Biomat. Sci. Polymer Edn., 12(1):107-124
(2001).
[0252] Scaffolds of the invention can be administered in
combination with any one or more growth factors, cells, for example
stem cells, bone marrow cells, chondrocytes, chondroblasts,
osteocytes, osteoblasts, osteoclasts, bone lining cells, or their
precursors, drugs or other components described above that
stimulate tissue formation or otherwise enhance or improve the
practice of the invention. The PPDCs to be seeded onto the
scaffolds may be genetically engineered to express growth factors
or drugs.
[0253] The cells of the invention can be used to produce new tissue
in vitro, which can then be implanted, transplanted or otherwise
inserted into a site requiring tissue repair, replacement or
augmentation in a patient.
[0254] In a non-limiting embodiment, the cells of the invention are
used to produce a three-dimensional tissue construct in vitro,
which is then implanted in vivo. As an example of the production of
three-dimensional tissue constructs, see U.S. Pat. No. 4,963,489,
which is incorporated herein by reference. For example, the cells
of the invention may be inoculated or "seeded" onto a
three-dimensional framework or scaffold, and proliferated or grown
in vitro to form a living tissue that can be implanted in vivo.
[0255] The cells of the invention can be grown freely in a culture
vessel to sub-confluency or confluency, lifted from the culture and
inoculated onto a three-dimensional framework. Inoculation of the
three-dimensional framework with a high concentration of cells,
e.g., approximately 10.sup.6 to 5.times.10.sup.7 cells per
milliliter, will result in the establishment of the
three-dimensional support in relatively shorter periods of
time.
[0256] Examples of scaffolds which may be used in the present
invention include nonwoven mats, porous foams, or self assembling
peptides. Nonwoven mats may, for example, be formed using fibers
comprised of a synthetic absorbable copolymer of glycolic and
lactic acids (PGA/PLA), sold under the tradename VICRYL (Ethicon,
Inc., Somerville, N.J.). Foams, composed of, for example,
poly(epsilon-caprolactone)/poly(glycolic acid) (PCL/PGA) copolymer,
formed by processes such as freeze-drying, or lyophilized, as
discussed in U.S. Pat. No. 6,355,699, are also possible scaffolds.
Hydrogels such as self-assembling peptides (e.g., RAD16) may also
be used. These materials are frequently used as supports for growth
of tissue.
[0257] The three-dimensional framework may be made of ceramic
materials 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 (University of
Florida, Gainesville, Fla.), and mixtures thereof. There are a
number of suitable porous biocompatible ceramic materials currently
available on the commercial market such as SURGIBON (Unilab
Surgibone, Inc., Canada), ENDOBON (Merck Biomaterial France,
France), CEROS (Mathys, A. G., Bettlach, Switzerland), and
INTERPORE (Interpore, Irvine, Calif., United States), and
mineralized collagen bone grafting products such as HEALOS
(Orquest, Inc., Mountain View, Calif.) and VITOSS, RHAKOSS, and
CORTOSS (Orthovita, Malvern, Pa.). The framework may be a mixture,
blend or composite of natural and/or synthetic materials. In some
embodiments, the scaffold is in the form of a cage. In a preferred
embodiment, the scaffold is coated with collagen.
[0258] According to a preferred embodiment, the framework is a
felt, which can be composed of a multifilament yarn made from a
bioabsorbable material, e.g., PGA, PLA, PCL copolymers or blends,
or hyaluronic acid. The yarn is made into a felt using standard
textile processing techniques consisting of crimping, cutting,
carding and needling.
[0259] In another preferred embodiment the cells of the invention
are seeded onto foam scaffolds that may be composite structures. In
addition, the three-dimensional framework may be molded into a
useful shape, such as that of the external portion of the ear, a
bone, joint or other specific structure in the body to be repaired,
replaced or augmented.
[0260] In another preferred embodiment, the cells of the invention
are seeded onto a framework comprising a prosthetic device for
implantation into a patient, as described in U.S. Pat. No.
6,200,606, incorporated herein by reference. As described therein,
a variety of clinically useful prosthetic devices have been
developed for use in bone and cartilage grafting procedures. (see
e.g. Bone Grafts and Bone Substitutions. Ed. M. B. Habal & A.
H. Reddi, W.B. Saunders Co., 1992). For example, effective knee and
hip replacement devices have been and continue to be widely used in
the clinical environment. Many of these devices are fabricated
using a variety of inorganic materials having low immunogenic
activity, which safely function in the body. Examples of synthetic
materials which have been tried and proven include titanium alloys,
calcium phosphate, ceramic hydroxyapatite, and a variety of
stainless steel and cobalt-chrome alloys. These materials provide
structural support and can form a scaffolding into which host
vascularization and cell migration can occur. The present invention
provides a source of cells which may be used to "seed" such
prosthetic devices. In the preferred embodiment PPDCs are first
mixed with a carrier material before application to a device.
Suitable carriers well known to those skilled in the art include,
but are not limited to, gelatin, fibrin, collagen, starch,
polysaccharides, saccharides, proteoglycans, synthetic polymers,
calcium phosphate, or ceramics.
[0261] The framework may be treated prior to inoculation of the
cells of the invention in order to enhance cell attachment. For
example, prior to inoculation with the cells of the invention,
nylon matrices could be treated with 0.1 molar acetic acid and
incubated in polylysine, PBS, and/or collagen to coat the nylon.
Polystyrene could be similarly treated using sulfuric acid.
[0262] In addition, the external surfaces of the three-dimensional
framework may be modified to improve the attachment or growth of
cells and differentiation of tissue, such as by plasma coating the
framework 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), a
cellular matrix, and/or other materials such as, but not limited
to, gelatin, alginates, agar, agarose, and plant gums, among
others.
[0263] In some embodiments, the scaffold is comprised of 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 (The
Polymer Technology Group, Inc., Berkeley, Calif.). These materials
can be further treated to render the scaffold non-thrombogenic.
Such treatments include anti-thrombotic agents such as heparin, and
treatments which alter the surface charge of the material such as
plasma coating.
[0264] In some embodiments, the surface of the scaffold is
textured. For example, in some aspects of the invention, the
scaffold is provided with a groove and ridge pattern. The grooves
are preferably less than about 500 microns, more preferably less
than about 100 microns, and most preferably between about 10
nanometers and 10 microns. Such "microgrooves" allow the cells to
align and/or migrate guided by the surface grooves. See, e.g.,
Odontology. 2001; 89(1):2-11. The textured scaffold may be used,
for example, as a dental implant.
[0265] In some embodiments, it is important to re-create in culture
the cellular microenvironment found in vivo, such that the extent
to which the cells of the invention are grown prior to implantation
in vivo or use in vitro may vary. In addition, growth factors,
chondrogenic differentiation inducing agents, osteogenic inducing
agents, and angiogenic factors may be added to the culture medium
prior to, during, or subsequent to inoculation of the cells to
trigger differentiation and tissue formation by the PPDCs or
co-cultures thereof.
[0266] The three-dimensional framework may be modified so that the
growth of cells and the production of tissue thereon is enhanced,
or so that the risk of rejection of the implant is reduced. Thus,
one or more biologically active compounds, including, but not
limited to, anti-inflammatories, immunosuppressants or growth
factors, may be added to the framework.
[0267] Therapeutic Uses for Extracellular Matrix or Cell Lysate
Derived from PPDCs
[0268] As an alternative to implanting the cells of the invention,
or living tissue produced therefrom, a subject in need of tissue
repair, replacement, or augmentation may benefit from the
administration of a component or product of PPDCs, such as the
extracellular matrix (ECM) or cell lysate produced by those
cells.
[0269] In some embodiments, after the cells of the invention have
been cultured in vitro, such as, for example, by using a
three-dimensional scaffold system described herein, such that a
desired amount of ECM has been secreted onto the framework. Once
ECM is secreted onto the framework, the cells may be removed. The
ECM may be processed for further use, for example, as an injectable
preparation.
[0270] In some embodiments, the cells are killed and cellular
debris (e.g., cellular membranes) is removed from the framework.
This process may be carried out in a number of different ways. For
example, the living tissue can be flash-frozen in liquid nitrogen
without a cryopreservative, or the tissue can be immersed in
sterile distilled water so that the cells burst in response to
osmotic pressure. Once the cells have been killed, the cellular
membranes may be disrupted and cellular debris removed by treatment
with a mild detergent rinse, such as EDTA, CHAPS or a zwitterionic
detergent. An advantage to using a mild detergent rinse is that it
solubilizes membrane-bound proteins, which are often highly
antigenic.
[0271] Alternatively, the tissue can be enzymatically digested
and/or extracted with reagents that break down cellular membranes.
Example of such enzymes include, but are not limited to,
hyaluronidase, dispase, proteases, and nucleases (for example,
deoxyribonuclease and ribonuclease). Examples of detergents include
non-ionic detergents such as, for example, alkylaryl polyether
alcohol (TRITON.RTM. X-100), octylphenoxy polyethoxy-ethanol (Rohm
and Haas Philadelphia, Pa.), BRIJ-35, a polyethoxyethanol lauryl
ether (Atlas Chemical Co., San Diego, Calif.), polysorbate 20
(TWEEN 20.RTM.), a polyethoxyethanol sorbitan monolaureate (Rohm
and Haas), polyethylene lauryl ether (Rohm and Haas); and ionic
detergents such as, for example, sodium dodecyl sulphate, sulfated
higher aliphatic alcohols, sulfonated alkanes and sulfonated
alkylarenes containing 7 to 22 carbon atoms in a branched or
unbranched chain.
[0272] Scaffolds comprising the ECm may be used therapeutically as
described above. Alternatively, ECM may be collected from the
scaffold. The collection of the ECM can be accomplished in a
variety of ways, depending, for example, on whether the scaffold is
biodegradable or non-biodegradable. For example, if the framework
is non-biodegradable, the ECM can be removed by subjecting the
framework to sonication, high pressure water jets, mechanical
scraping, or mild treatment with detergents or enzymes, or any
combination of the above.
[0273] If the framework is biodegradable, the ECM can be collected,
for example, by allowing the framework to degrade or dissolve in
solution. Alternatively, if the biodegradable framework is composed
of a material that can itself be injected along with the ECM, the
framework and the ECM can be processed in toto for subsequent
injection. Alternatively, the ECM can be removed from the
biodegradable framework by any of the methods described above for
collection of ECM from a non-biodegradable framework. All
collection processes are preferably designed so as not to denature
the ECM or cell lysate produced by the cells of the invention.
[0274] Once the ECM has been collected, it may be processed
further. The ECM can be homogenized to fine particles using
techniques well known in the art such as, for example, by
sonication, so that they can pass through a surgical needle. ECM
components can be crosslinked, if desired, by gamma irradiation.
Preferably, the ECM can be irradiated between 0.25 to 2 mega rads
to sterilize and crosslink the ECM. Chemical crosslinking using
agents that are toxic, such as glutaraldehyde, is possible but not
generally preferred.
[0275] Cell lysates prepared from the populations of the
postpartum-derived cells also have many utilities. In one
embodiment, whole cell lysates are prepared, e.g., by disrupting
cells without subsequent separation of cell fractions. In another
embodiment, a cell membrane fraction is separated from a soluble
fraction of the cells by routine methods known in the art, e.g.,
centrifugation, filtration, or similar methods. Use of soluble cell
fractions in vivo allows the beneficial intracellular milieu to be
used in a patient without triggering rejection or an adverse
response. Methods of lysing cells are well-known in the art and
include various means of mechanical disruption, enzymatic
disruption, or chemical disruption, or combinations thereof. Such
cell lysates may be prepared from cells directly in their growth
medium and thus containing secreted growth factors and the like, or
may be prepared from cells washed free of medium in, for example,
PBS or other solution. Washed cells may be resuspended at
concentrations greater than the original population density if
preferred. Cell lysates prepared from populations of
postpartum-derived cells may be used as is, further concentrated,
by for example, ultrafiltration or lyophilization, or even dried,
partially purified, combined with pharmaceutically acceptable
carriers or diluents as are known in the art, or combined with
other compounds such as biologicals, for example pharmaceutically
useful protein compositions. Cell lysates may be used in vitro or
in vivo, alone or for example, with cells. The cell lysates, if
introduced in vivo, may be introduced locally at a site of
treatment, or remotely to provide, for example needed cellular
growth factors to a patient.
[0276] The amounts and/or ratios of proteins may be adjusted by
mixing the ECM or cell lysate produced by the cells of the
invention with ECM or cell lysate of one or more other cell types.
In addition, biologically active substances such as proteins,
growth factors and/or drugs, can be incorporated into the ECM or
cell lysate preparation. Exemplary biologically active substances
include anti-inflammatory agents and growth factors which promote
healing and tissue repair. Cells may be co-administered with the
ECM or cell lysates of the invention. ECM or cell lysate of PPDCs
may be formulated for administration as described above for
PPDCs.
[0277] The above described process for preparing injectable ECM or
cell lysate is preferably carried out under sterile conditions
using sterile materials. The processed ECM or cell lysate in a
pharmaceutically acceptable carrier can be injected intradermally
or subcutaneously to treat bone or cartilage conditions, for
example, by augmenting tissue or repairing or correcting congenital
anomalies, acquired defects or cosmetic defects.
[0278] Use of PPDCs for In Vitro Screening of Drug Efficacy or
Toxicity
[0279] The cells and tissues of the invention may be used in vitro
to screen a wide variety of compounds for effectiveness and
cytotoxicity of pharmaceutical agents, growth/regulatory factors,
anti-inflammatory agents. To this end, the cells of the invention,
or tissue cultures described above, are maintained in vitro and
exposed to the compound to be tested. The activity of a cytotoxic
compound can be measured by its ability to damage or kill cells in
culture. This may readily be assessed by vital staining techniques.
The effect of growth/regulatory factors may be assessed by
analyzing the number of living cells in vitro, e.g., by total cell
counts, and differential cell counts. This may be accomplished
using standard cytological and/or histological techniques,
including the use of immunocytochemical techniques employing
antibodies that define type-specific cellular antigens. The effect
of various drugs on the cells of the invention either in suspension
culture or in the three-dimensional system described above may be
assessed.
[0280] The cells and tissues of the invention may be used as model
systems for the study of physiological or pathological conditions.
For example, joints that are immobilized suffer relatively quickly
in a number of respects. The metabolic activity of chondrocytes
appears affected as loss of proteoglycans and an increase in water
content are soon observed. The normal white, glistening appearance
of the cartilage changes to a dull, bluish color, and the cartilage
thickness is reduced. However, the amount of this change that is
due to nutritional deficiency versus the amount due to upset in the
stress-dependent metabolic homeostasis is not yet clear. The cells
and tissues of the invention may be used to determine the
nutritional requirements of cartilage under different physical
conditions, e.g., intermittent pressurization, and by pumping
action of nutrient medium into and out of the cartilage construct.
This may be especially useful in studying underlying causes for
age-related or injury-related decrease in tensile strength of, for
example, articular cartilage, e.g., in the knee, that predispose
the weakened cartilage to traumatic damage.
[0281] The cells and tissues of the invention may also be used to
study the mechanism of action of cytokines, growth factors and
inflammatory mediators, e.g., IL-1, TNF and prostaglandins. In
addition, cytotoxic and/or pharmaceutical agents can be screened
for those that are most efficacious for a particular patient, such
as those that reduce or prevent resorption of cartilage or bone
otherwise enhance the balanced growth thereof. Agents that prove to
be efficacious in vitro could then be used to treat the patient
therapeutically.
[0282] Use of PPDCs to Produce Biological Molecules
[0283] In a further embodiment, the cells of the invention can be
cultured in vitro to produce biological products in high yield. For
example, such cells, which either naturally produce a particular
biological product of interest (e.g., a growth factor, regulatory
factor, or peptide hormone), or have been genetically engineered to
produce a biological product, could be clonally expanded using, for
example, the three-dimensional culture system described above. If
the cells excrete the biological product into the nutrient medium,
the product can be readily isolated from the spent or conditioned
medium using standard separation techniques, e.g., such as
differential protein precipitation, ion-exchange chromatography,
gel filtration chromatography, electrophoresis, and high
performance liquid chromatography. A "bioreactor" may be used to
take advantage of the flow method for feeding, for example, a
three-dimensional culture in vitro.
[0284] Essentially, as fresh media is passed through the
three-dimensional culture, the biological product is washed out of
the culture and may then be isolated from the outflow, as
above.
[0285] Alternatively, a biological product of interest may remain
within the cell and, thus, its collection may require that the
cells be lysed. The biological product may then be purified using
any one or more of the above-listed techniques.
[0286] Kits
[0287] The PPDCs and components and products thereof can
conveniently be employed as part of a kit, for example, for culture
or implantation. Accordingly, the invention provides a kit
including the PPDCs and additional components, such as a matrix
(e.g., a scaffold), hydrating agents (e.g.,
physiologically-compatible saline solutions, prepared cell culture
media), cell culture substrates (e.g., culture dishes, plates,
vials, etc.), cell culture media (whether in liquid or powdered
form), antibiotic compounds, hormones, and the like. While the kit
can include any such components, preferably it includes all
ingredients necessary for its intended use. If desired, the kit
also can include cells (typically cryopreserved), which can be
seeded into the lattice as described herein.
[0288] In another aspect, the invention provides kits that utilize
the PPDCs, PPDC populations, components and products of PPDCs in
various methods for augmentation, regeneration, and repair as
described above. In some embodiments, the kits may include one or
more cell populations, including at least PPDCs and a
pharmaceutically acceptable carrier (liquid, semi-solid or solid).
The kits also optionally may include a means of administering the
cells, for example by injection. The kits further may include
instructions for use of the cells. Kits prepared for field hospital
use, such as for military use, may include full-procedure supplies
including tissue scaffolds, surgical sutures, and the like, where
the cells are to be used in conjunction with repair of acute
injuries. Kits for assays and in vitro methods as described herein
may contain one or more of (1) PPDCs or components or products of
PPDCs, (2) reagents for practicing the in vitro method, (3) other
cells or cell populations, as appropriate, and (4) instructions for
conducting the in vitro method.
Cryopreservation and Banking PPDCs
[0289] PDCs of the invention can be cryopreserved and maintained or
stored in a "cell bank". Cryopreservation of cells of the invention
may be carried out according to known methods. For example, but not
by way of limitation, cells may be suspended in a "freeze medium"
such as, for example, culture medium further comprising 0 to 95
percent FBS and 0 to 10 percent dimethylsulfoxide (DMSO), with or
without 5 to 10 percent glycerol, at a density, for example, of
about 0.5 to 10.times.10.sup.6 cells per milliliter. The
cryopreservation medium may comprise cryopreservation agents
including but not limited to methylcellulose. The cells are
dispensed into glass or plastic ampoules that are then sealed and
transferred to the freezing chamber of a controlled rate freezer.
The optimal rate of freezing may be determined empirically. A
programmable rate freezer for example, can give a change in
temperature of -1 to -10.degree. C. per minute. The preferred
cryopreservation temperature is about -80.degree. C. to about
-180.degree. C., more preferably is about -90.degree. C. to about
-160.degree. C., and most preferably is about -125 to about
-140.degree. C. Cryopreserved cells preferably are 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 can be stored for a period of years.
[0290] The cryopreserved cells of the invention constitute a bank
of cells, portions of which can be "withdrawn" by thawing and then
used as needed. Thawing should generally be carried out rapidly,
for example, by transferring an ampoule from liquid nitrogen to a
37.degree. C. water bath. The thawed contents of the ampoule should
be immediately transferred under sterile conditions to a culture
vessel containing an appropriate medium such as DMEM conditioned
with 10 percent FBS.
[0291] In yet another aspect, the invention also provides for
banking of tissues, cells, cellular components and cell populations
of the invention. As discussed above, the cells are readily
cryopreserved. The invention therefore provides methods of
cryopreserving the cells in a bank, wherein the cells are stored
frozen and associated with a complete characterization of the cells
based on immunological, biochemical and genetic properties of the
cells. The cells so frozen can be used for autologous, syngeneic,
or allogeneic therapy, depending on the requirements of the
procedure and the needs of the patient. Preferably, the information
on each cryopreserved sample is stored in a computer, which is
searchable based on the requirements of the surgeon, procedure and
patient with suitable matches being made based on the
characterization of the cells or populations. Preferably, the cells
of the invention are grown and expanded to the desired quantity of
cells and therapeutic cell compositions are prepared either
separately or as co-cultures, in the presence or absence of a
matrix or support. While for some applications it may be preferable
to use cells freshly prepared, the remainder can be cryopreserved
and banked by freezing the cells and entering the information in
the computer to associate the computer entry with the samples. Even
where it is not necessary to match a source or donor with a
recipient of such cells, for immunological purposes, the bank
system makes it easy to match, for example, desirable biochemical
or genetic properties of the banked cells to the therapeutic needs.
Upon matching of the desired properties with a banked sample, the
sample is retrieved, and readied for therapeutic use. Cell lysates
or components prepared as described herein may also be preserved
(e.g., cryopreserved, lyophilized) and banked in accordance with
the present invention.
[0292] The following examples describe several aspects of
embodiments of the invention in greater detail. These examples are
provided to further illustrate, not to limit, aspects of the
invention described herein.
EXAMPLES
Example 1
Derivation of Cells from Postpartum Tissues
[0293] The objective of this study was to derive populations of
cells from placental and umbilical cord tissues. Postpartum
umbilical cord and placenta were obtained upon birth of either a
full term or pre-term pregnancy. Cells were harvested from 5
separate donors of umbilical cord and placental tissue. Different
methods of cell isolation were tested for their ability to yield
cells with: 1) the potential to differentiate into cells with
different phenotypes, or 2) the potential to provide critical
trophic factors useful for other cells and tissues.
[0294] Methods & Materials
[0295] Umbilical cord cell derivation. Umbilical cords were
obtained from National Disease Research Interchange (NDRI,
Philadelphia, Pa.). The tissues were obtained following normal
deliveries. The cell isolation protocol was performed aseptically
in a laminar flow hood. To remove blood and debris, the umbilical
cord was washed in phosphate buffered saline (PBS; Invitrogen,
Carlsbad, Calif.) in the presence of antimycotic and antibiotic
(100 Units/milliliter penicillin, 100 micrograms/milliliter
streptomycin, 0.25 micrograms/milliliter amphotericin B (Invitrogen
Carlsbad, Calif.)). The tissues were then mechanically dissociated
in 150 cm.sup.2 tissue culture plates in the presence of 50
milliliters of medium (DMEM-Low glucose or DMEM-High glucose;
Invitrogen) until the tissue was minced into a fine pulp. The
chopped tissues were transferred to 50 milliliter conical tubes
(approximately 5 grams of tissue per tube). The tissue was then
digested in either DMEM-Low glucose medium or DMEM-High glucose
medium, each containing antimycotic and antibiotic (100
Units/milliliter penicillin, 100 micrograms/milliliter
streptomycin, 0.25 micrograms/milliliter amphotericin B
(Invitrogen)) and digestion enzymes. In some experiments, an enzyme
mixture of collagenase and dispase was used ("C:D;" collagenase
(Sigma, St Louis, Mo.), 500 Units/milliliter; and dispase
(Invitrogen), 50 Units/milliliter in DMEM-Low glucose medium). In
other experiments a mixture of collagenase, dispase and
hyaluronidase ("C:D:H") was used (collagenase, 500
Units/milliliter; dispase, 50 Units/milliliter; and hyaluronidase
(Sigma), 5 Units/milliliter, in DMEM-Low glucose). The conical
tubes containing the tissue, medium and digestion enzymes were
incubated at 37.degree. C. in an orbital shaker (Environ, Brooklyn,
N.Y.) at 225 rpm for 2 hrs.
[0296] After digestion, the tissues were centrifuged at 150.times.g
for 5 minutes, and the supernatant was aspirated. The pellet was
resuspended in 20 milliliters of Growth medium (DMEM-Low glucose
(Invitrogen), 15 percent (v/v) fetal bovine serum (FBS; defined
bovine serum; Lot#AND18475; Hyclone, Logan, Utah), 0.001% (v/v)
2-mercaptoethanol (Sigma), 100 Units/milliliter of penicillin, 100
microgram/milliliter streptomycin, 0.25 microgram/milliliter
amphotericin B (Invitrogen, Carlsbad, Calif.). The cell suspension
was filtered through a 70-micrometer nylon cell strainer (BD
Biosciences). An additional 5 milliliter rinse comprising Growth
medium was passed through the strainer. The cell suspension was
then passed through a 40-micrometer nylon cell strainer (BD
Biosciences) and chased with a rinse of an additional 5 milliliters
of Growth medium.
[0297] The filtrate was resuspended in Growth medium (total volume
50 milliliters) and centrifuged at 150.times.g for 5 minutes. The
supernatant was aspirated, and the cells were resuspended in 50
milliliters of fresh Growth medium. This process was repeated twice
more.
[0298] Upon the final centrifugation supernatant was aspirated and
the cell pellet was resuspended in 5 milliliters of fresh Growth
medium. The number of viable cells was determined using Trypan Blue
staining. Cells were then cultured under standard conditions.
[0299] The cells isolated from umbilical cord cells were seeded at
5,000 cells/cm.sup.2 onto gelatin-coated T-75 cm.sup.2 flasks
(Corning Inc., Corning, N.Y.) in Growth medium (DMEM-Low glucose
(Invitrogen), 15 percent (v/v) defined bovine serum (Hyclone,
Logan, Utah; Lot#AND18475), 0.001 percent (v/v) 2-mercaptoethanol
(Sigma), 100 Units/milliliter penicillin, 100 micrograms/milliliter
streptomycin, 0.25 micrograms/milliliter amphotericin B
(Invitrogen)). After about 2-4 days, spent medium was aspirated
from the flasks. Cells were washed with PBS three times to remove
debris and blood-derived cells. Cells were then replenished with
Growth medium and allowed to grow to confluence (about 10 days from
passage 0 to passage 1). On subsequent passages (from passage 1 to
2, etc.), cells reached sub-confluence (75-85 percent confluence)
in 4-5 days. For these subsequent passages, cells were seeded at
5000 cells/cm.sup.2. Cells were grown in a humidified incubator
with 5 percent carbon dioxide and 20 percent oxygen at 37.degree.
C.
[0300] Placental Cell Isolation. Placental tissue was obtained from
NDRI (Philadelphia, Pa.). The tissues were from a pregnancy and
were obtained at the time of a normal surgical delivery. Placental
cells were isolated as described for umbilical cord cell
isolation.
[0301] The following example applies to the isolation of separate
populations of maternal-derived and neonatal-derived cells from
placental tissue.
[0302] The cell isolation protocol was performed aseptically in a
laminar flow hood. The placental tissue was washed in phosphate
buffered saline (PBS; Invitrogen, Carlsbad, Calif.) in the presence
of antimycotic and antibiotic (100 U/milliliter penicillin, 100
microgram/milliliter streptomycin, 0.25 microgram/milliliter
amphotericin B; Invitrogen) to remove blood and debris. The
placental tissue was then dissected into three sections: top-line
(neonatal side or aspect), mid-line (mixed cell isolation neonatal
and maternal or villous region), and bottom line (maternal side or
aspect).
[0303] The separated sections were individually washed several
times in PBS with antibiotic/antimycotic to further remove blood
and debris. Each section was then mechanically dissociated in 150
cm.sup.2 tissue culture plates in the presence of 50 milliliters of
DMEM-Low glucose (Invitrogen) to a fine pulp. The pulp was
transferred to 50 milliliter conical tubes. Each tube contained
approximately 5 grams of tissue. The tissue was digested in either
DMEM-Low glucose or DMEM-High glucose medium containing antimycotic
and antibiotic (100 Units/milliliter penicillin, 100
micrograms/milliliter streptomycin, 0.25 micrograms/milliliter
amphotericin B (Invitrogen)) and digestion enzymes. In some
experiments an enzyme mixture of collagenase and dispase ("C:D")
was used containing collagenase (Sigma, St Louis, Mo.) at 500
Units/milliliter and dispase (Invitrogen) at 50 Units/milliliterin
DMEM-Low glucose medium. In other experiments a mixture of
collagenase, dispase, and hyaluronidase (C:D:H) was used
(collagenase, 500 Units/milliliter; dispase, 50 Units/milliliter;
and hyaluronidase (Sigma), 5 Units/milliliter in DMEM-Low glucose).
The conical tubes containing the tissue, medium, and digestion
enzymes were incubated for 2 h at 37.degree. C. in an orbital
shaker (Environ, Brooklyn, N.Y.) at 225 rpm.
[0304] After digestion, the tissues were centrifuged at 150.times.g
for 5 minutes, the resultant supernatant was aspirated off. The
pellet was resuspended in 20 milliliter of Growth medium (DMEM-Low
glucose (Invitrogen), 15% (v/v) fetal bovine serum (FBS; defined
bovine serum; Lot#AND18475; Hyclone, Logan, Utah), 0.001% (v/v)
2-mercaptoethanol (Sigma, St. Louis, Mo.), antibiotic/antimycotic
(100 U/milliliter penicillin, 100 microgram/milliliter
streptomycin, 0.25 microgram/milliliter amphotericin B;
Invitrogen)). The cell suspension was filtered through a 70
micrometer nylon cell strainer (BD Biosciences), chased by a rinse
with an additional 5 milliliters of Growth medium. The total cell
suspension was passed through a 40 micrometer nylon cell strainer
(BD Biosciences) followed with an additional 5 milliliters of
Growth medium as a rinse.
[0305] The filtrate was resuspended in Growth medium (total volume
50 milliliters) and centrifuged at 150.times.g for 5 minutes. The
supernatant was aspirated and the cell pellet was resuspended in 50
milliliters of fresh Growth medium. This process was repeated twice
more. After the final centrifugation, supernatant was aspirated and
the cell pellet was resuspended in 5 milliliters of fresh Growth
medium. A cell count was determined using the Trypan Blue Exclusion
test. Cells were then cultured at standard conditions.
[0306] LIBERASE (Boehringer Mannheim Corp., Indianapolis, Ind.)
Cell Isolation. Cells were isolated from umbilical cord in DMEM-Low
glucose medium with LIBERASE (Boehringer Mannheim Corp.,
Indianapolis, Ind.) (2.5 milligrams per milliliter, Blendzyme 3;
Roche Applied Sciences, Indianapolis, Ind.) and hyaluronidase (5
Units/milliliter, Sigma). Digestion of the tissue and isolation of
the cells was as described for other protease digestions above
using a LIBERASE (Boehringer Mannheim Corp., Indianapolis,
Ind.)/hyaluronidase mixture in place of the C:D or C:D:H enzyme
mixture. Tissue digestion with LIBERASE (Boehringer Mannheim Corp.,
Indianapolis, Ind.) resulted in the isolation of cell populations
from postpartum tissues that expanded readily.
[0307] Cell isolation using other enzyme combinations. Procedures
were compared for isolating cells from the umbilical cord using
differing enzyme combinations. Enzymes compared for digestion
included: i) collagenase; ii) dispase; iii) hyaluronidase; iv)
collagenase:dispase mixture (C;D); v) collagenase:hyaluronidase
mixture (C:H); vi) dispase:hyaluronidase mixture (D:H); and vii)
collagenase:dispase:hyaluronidase mixture (C:D:H). Differences in
cell isolation utilizing these different enzyme digestion
conditions were observed (Table 1-1).
[0308] Isolation of cells from residual blood in the cords.
Attempts were made to isolate pools of cells from umbilical cord by
different approaches. In one instance umbilical cord was sliced and
washed with Growth medium to dislodge the blood clots and
gelatinous material. The mixture of blood, gelatinous material, and
Growth medium was collected and centrifuged at 150.times.g. The
pellet was resuspended and seeded onto gelatin-coated flasks in
Growth medium. From these experiments a cell population was
isolated that readily expanded.
[0309] Isolation of cells from Cord Blood. Cells have also been
isolated from cord blood samples attained from NDRI. The isolation
protocol used here was that of International Patent Application
WO02/29971 by Ho et al. Samples (50 milliliter and 10.5
milliliters, respectively) of umbilical cord blood (NDRI,
Philadelphia Pa.) were mixed with lysis buffer (filter-sterilized
155 millimolar ammonium chloride, 10 millimolar potassium
bicarbonate, 0.1 millimolar EDTA buffered to pH 7.2 (all components
from Sigma, St. Louis, Mo.)). Cells were lysed at a ratio of 1:20
cord blood to lysis buffer. The resulting cell suspension was
vortexed for 5 seconds, and incubated for 2 minutes at ambient
temperature. The soluble cell fraction was centrifuged (10 minutes
at 200.times.g). The cell pellet was resuspended in complete
minimal essential medium (Gibco, Carlsbad Calif.) containing 10
percent fetal bovine serum (Hyclone, Logan Utah), 4 millimolar
glutamine (Mediatech Herndon, Va.), 100 Units penicillin per 100
milliliters and 100 micrograms streptomycin per 100 milliliters
(Gibco, Carlsbad, Calif.). The resuspended cells were centrifuged
(10 minutes at 200.times.g), the supernatant was aspirated, and the
cell pellet was washed in complete medium. Cells were seeded
directly into either T75 flasks (Corning, N.Y.), T75 laminin-coated
flasks, or T175 fibronectin-coated flasks (both Becton Dickinson,
Bedford, Mass.).
[0310] Isolation of postpartum-derived cells using different enzyme
combinations and growth conditions. To determine whether cell
populations can be isolated under different conditions and expanded
under a variety of conditions immediately after isolation, cells
were digested in Growth medium with or without 0.001 percent (v/v)
2-mercaptoethanol (Sigma, St. Louis, Mo.), using the enzyme
combination of C:D:H, according to the procedures provided above.
Placenta-derived cells so isolated were seeded under a variety of
conditions. All cells were grown in the presence of
penicillin/streptomycin. (Table 1-2).
[0311] Isolation of postpartum-derived cells using different enzyme
combinations and growth conditions. In all conditions, cells
attached and expanded well between passage 0 and 1 (Table 1-2).
Cells in conditions 5 to 8 and 13 to 16 were demonstrated to
proliferate well up to 4 passages after seeding at which point they
were cryopreserved. All cells were banked.
[0312] Results
[0313] Cell isolation using different enzyme combinations. The
combination of C:D:H provided the best cell yield following
isolation and generated cells which expanded for many more
generations in culture than the other conditions (Table 1-1). An
expandable cell population was not attained using collagenase or
hyaluronidase alone. No attempt was made to determine if this
result is specific to the collagen that was tested.
[0314] Isolation of postpartum-derived cells using different enzyme
combinations and growth conditions. Cells attached and expanded
well between passage 0 and 1 under all conditions tested for enzyme
digestion and growth (Table 1-2). Cells in experimental conditions
5-8 and 13-16 proliferated well up to 4 passages after seeding, at
which point they were cryopreserved. All cells were banked.
[0315] Isolation of cells from residual blood in the cords.
Nucleated cells attached and grew rapidly. These cells were
analyzed by flow cytometry and were similar to cells obtained by
enzyme digestion.
[0316] Isolation of cells from Cord Blood. The preparations
contained red blood cells and platelets. No nucleated cells
attached and divided during the first 3 weeks. The medium was
changed 3 weeks after seeding and no cells were observed to attach
and grow.
[0317] Summary. Populations of cells can be isolated from umbilical
cord and placental tissue most efficiently using the enzyme
combination collagenase (a matrix metalloprotease), dispase
(neutral protease), and hyaluronidase (a mucolytic enzyme which
breaks down hyaluronic acid). LIBERASE (Boehringer Mannheim Corp.,
Indianapolis, Ind.), which is a Blendzyme, may also be used. In the
present study Blendzyme 3 which is collagenase (4 Wunsch units/g)
and thermolysin (1714 casein Units/g) was also used together with
hyaluronidase to isolate cells. These cells expand readily over
many passages when cultured in Growth medium on gelatin-coated
plastic.
[0318] Postpartum-derived cells were isolated from residual blood
in the cords but not from cord blood. The presence of cells in
blood clots washed from the tissue that adhere and grow under the
conditions used may be due to cells being released during the
dissection process. TABLE-US-00001 TABLE 1-1 Isolation of cells
from umbilical cord tissue using varying enzyme combinations Enzyme
Digest Cells Isolated Cell Expansion Collagenase X X Dispase
+(>10 h) + Hyaluronidase X X Collagenase:Dispase ++(<3 h) ++
Collagenase:Hyaluronidase ++(<3 h) + Dispase:Hyaluronidase
+(>10 h) + Collagenase:Dispase:Hyaluronidase +++(<3 h) +++
Key: + = good, ++ = very good, +++ = excellent, X = no success
[0319] TABLE-US-00002 TABLE 1-2 Isolation and culture expansion of
postpartum-derived cells under varying conditions: Condition Medium
15% FBS BME Gelatin 20% O2 Growth Factors 1 DMEM-Lg Y Y Y Y N 2
DMEM-Lg Y Y Y N (5%) N 3 DMEM-Lg Y Y N Y N 4 DMEM-Lg Y Y N N (5%) N
5 DMEM-Lg N (2%) Y N (Laminin) Y EGF/FGF (20 ng/mL) 6 DMEM-Lg N
(2%) Y N (Laminin) N (5%) EGF/FGF (20 ng/mL) 7 DMEM-Lg N (2%) Y N Y
PDGF/VEGF (Fibronectin) 8 DMEM-Lg N (2%) Y N N (5%) PDGF/VEGF
(Fibronectin) 9 DMEM-Lg Y N Y Y N 10 DMEM-Lg Y N Y N (5%) N 11
DMEM-Lg Y N N Y N 12 DMEM-Lg Y N N N (5%) N 13 DMEM-Lg N (2%) N N
(Laminin) Y EGF/FGF (20 ng/mL) 14 DMEM-Lg N (2%) N N (Laminin) N
(5%) EGF/FGF (20 ng/mL) 15 DMEM-Lg N (2%) N N Y PDGF/VEGF
(Fibronectin) 16 DMEM-Lg N (2%) N N N (5%) PDGF/VEGF
(Fibronectin)
REFERENCE
[0320] 1. HO, Tony, W.; KOPEN, Gene, C.; RIGHTER, William, F.;
RUTKOWSKI, J., Lynn; HERRING, W., Joseph; RAGAGLIA, Vanessa;
WAGNER, Joseph WO2003025149 A2 CELL POPULATIONS WHICH CO-EXPRESS
CD49C AND CD90, NEURONYX, INC. Application No. US0229971 US, Filed
20020920, A2 Published 20030327, A3 Published 20031218.
Example 2
Evaluation of Growth Media for Postpartum-Derived Cells
[0321] Several cell culture media were evaluated for their ability
to support the growth of placenta-derived cells. The growth of
placenta-derived cells in normal (20%) and low (5%) oxygen was
assessed after 3 days using the MTS calorimetric assay.
[0322] Methods & Materials
[0323] Placenta-derived cells at passage 8 (P8) were seeded at
1.times.10.sup.3 cells/well in 96 well plates in Growth medium
(DMEM-low glucose (Gibco, Carlsbad Calif.), 15% (v/v) fetal bovine
serum (Cat. #SH30070.03; Hyclone, Logan, Utah), 0.001% (v/v)
betamercaptoethanol (Sigma, St. Louis, Mo.), 50 Units/milliliter
penicillin, 50 micrograms/milliliter streptomycin (Gibco)). After 8
hours, the medium was changed as described in Table 2-1, and cells
were incubated in normal (20%, v/v) or low (5%, v/v) oxygen at
37.degree. C., 5% CO.sub.2 for 48 hours. MTS was added to the
culture medium (CELLTITER 96 AQueous One Solution Cell
Proliferation Assay, Promega, Madison, Wis.) for 3 hours and the
absorbance measured at 490 nanometers (Molecular Devices, Sunnyvale
Calif.). TABLE-US-00003 TABLE 2-1 Culture medium Added fetal bovine
serum % Culture Medium Supplier (v/v) DMEM-low glucose Gibco
Carlsbad CA 0, 2, 10 DMEM-high glucose Gibco 0, 2, 10 RPMI 1640
Mediatech, Inc. 0, 2, 10 Herndon, VA Cell gro-free (Serum-free,
Mediatech, Inc. -- Protein-free) Ham's F10 Mediatech, Inc. 0, 2, 10
MSCGM (complete with Cambrex, Walkersville, 0, 2, 10 serum) MD
Complete-serum free Mediatech, Inc. -- w/albumin Growth medium NA
-- Ham's F12 Mediatech, Inc. 0, 2, 10 Iscove's Mediatech, Inc. 0,
2, 10 Basal Medium Eagle's Mediatech, Inc. 0, 2, 10 DMEM/F12 (1:1)
Mediatech, Inc. 0, 2, 10
[0324] Results
[0325] Standard curves for the MTS assay established a linear
correlation between an increase in absorbance and an increase in
cell number. The absorbance values obtained were converted into
estimated cell numbers and the change (%) relative to the initial
seeding was calculated.
[0326] The Effect of Serum. The addition of serum to media at
normal oxygen conditions resulted in a reproducible dose-dependent
increase in absorbance and thus the viable cell number. The
addition of serum to complete MSCGM resulted in a dose-dependent
decrease in absorbance. In the media without added serum, cells
grew in Cellgro, Ham's F10, and DMEM.
[0327] The Effect of Oxygen. Reduced oxygen appeared to increase
the growth rate of cells in Growth Medium, Ham's F10, and
MSCGM.
[0328] In decreasing order of growth, the media resulting in the
best growth of the cells were Growth
medium>MSCGM>Iscove's+10% FBS=DMEM-HG+10% FBS=Ham's F12+10%
FBS=RPMI 1640+10% FBS.
[0329] Summary. Postpartum-derived cells may be grown in a variety
of culture media in normal or low oxygen. Short-term growth of
placenta-derived cells was determined in 12 basal media with 0, 2,
and 10% (v/v) serum in 5% or 20% O.sub.2. In general
placenta-derived cells did not grow in serum-free conditions with
the exceptions of Ham's F10 and Cellgro-free, which are also
protein-free. Growth in these serum-free media was approximately
25-33% of the maximal growth observed with Growth medium containing
15% serum. This study demonstrates that placenta-derived cells may
be grown in serum-free conditions and that Growth medium is one of
several media (10% serum in Iscove's, RPMI or Ham's F12 media) that
can be used to grow placenta-derived cells.
[0330] The most promising serum-free media was CELLGRO-FREE, a
serum and protein-free medium without hormones or growth factors,
which is designed for the growth of mammalian cells in vitro
(Mediatech product information).
[0331] Complete-serum free medium also developed for serum-free
culture was not as effective in supporting growth of the
placenta-derived cells. Complete-serum free was developed by
Mediatech, based on a 50/50 mix of DMEM/F12 with smaller
percentages of RPMI 1640 and McCoy's 5A. This medium also contains
selected trace elements and high molecular weight carbohydrates,
extra vitamins, a non-animal protein source, and a small amount of
BSA (1 gram/liter). It does not contain any insulin, transferrin,
cholesterol, or growth or attachment factors. It is bicarbonate
buffered for use with 5% CO.sub.2. Originally designed for
hybridomas and suspension cell lines, it may be suitable for some
anchorage dependent cell lines.
Example 3
Growth of Postpartum-Derived Cells in Medium Containing
D-Valine
[0332] It has been reported that medium containing D-valine instead
of the normal L-valine isoform can be used to selectively inhibit
the growth of fibroblast-like cells in culture (Hongpaisan, 2000;
Sordillo et al., 1988). The growth of postpartum-derived cells in
medium containing D-valine in the absence of L-valine was
evaluated.
[0333] Methods & Materials
[0334] Placenta-derived cells (P3), fibroblasts (P9), and umbilical
cord-derived cells (P5) were seeded at 5.times.10.sup.3
cells/cm.sup.2 in gelatin-coated T75 flasks (Corning, Corning,
N.Y.). After 24 hours the medium was removed and the cells were
washed with phosphate buffered saline (PBS) (Gibco, Carlsbad,
Calif.) to remove residual medium. The medium was replaced with a
Modified Growth medium (DMEM with D-valine (special order Gibco),
15% (v/v) dialyzed fetal bovine serum (Hyclone, Logan, Utah),
0.001% (v/v) betamercaptoethanol (Sigma), 50 Units/milliliter
penicillin, 50 microgram/milliliter streptomycin (Gibco)).
[0335] Results
[0336] Placenta-derived, umbilical cord-derived, and fibroblast
cells seeded in the D-valine-containing medium did not proliferate,
unlike cells seeded in Growth medium containing dialyzed serum.
Fibroblasts changed morphologically, increasing in size and
changing shape. All of the cells died and eventually detached from
the flask surface after 4 weeks.
[0337] Summary. Postpartum-derived cells require L-valine for cell
growth and for long-term viability. L-valine is preferably not
removed from the growth medium for postpartum-derived cells.
REFERENCES
[0338] Hongpaisan J. (2000) Inhibition of proliferation of
contaminating fibroblasts by D-valine in cultures of smooth muscle
cells from human myometrium. Cell Biol Int. 24:1-7. [0339] Sordillo
L M, Oliver S P, Akers R M. (1988) Culture of bovine mammary
epithelial cells in D-valine modified medium: selective removal of
contaminating fibroblasts. Cell Biol Int Rep. 12:355-64.
Example 4
Cryopreservation Media for Postpartum-Derived Cells
[0340] The objective of this study was to determine a suitable
cryopreservation medium for the cryopreservation of
postpartum-derived cells.
[0341] Methods & Materials
[0342] Placenta-derived cells grown in Growth medium (DMEM-low
glucose (Gibco, Carlsbad Calif.), 15% (v/v) fetal bovine serum
(Cat. #SH30070.03, Hyclone, Logan, Utah), 0.001% (v/v)
betamercaptoethanol (Sigma, St. Louis, Mo.), 50 Units/milliliter
penicillin, 50 microgram/milliliter streptomycin (Gibco)), in a
gelatin-coated T75 flask were washed with phosphate buffered saline
(PBS; Gibco) and trypsinized using 1 milliliter Trypsin/EDTA
(Gibco). The trypsinization was stopped by adding 10 milliliter
Growth medium. The cells were centrifuged at 150.times.g,
supernatant removed, and the cell pellet was resuspended in 1
milliliter Growth medium. An aliquot of cell suspension, 60
microliter, was removed and added to 60 microliter
.quadrature.trypan blue (Sigma). The viable cell number was
estimated using a hemocytometer. The cell suspension was divided
into four equal aliquots each containing 88.times.10.sup.4 cells
each. The cell suspension was centrifuged and resuspended in 1
milliliter of each media below and transferred into Cryovials
(Nalgene).
[0343] 1.) Growth medium+10% (v/v) DMSO (Hybrimax, Sigma, St.
Louis, Mo.)
[0344] 2.) Cell Freezing medium w/DMSO, w/methylcellulose,
serum-free (C6295, Sigma, St. Louis, Mo.)
[0345] 3.) Cell Freezing medium serum-free (C2639, Sigma, St.
Louis, Mo.)
[0346] 4.) Cell Freezing Medium w/glycerol (C6039, Sigma, St.
Louis, Mo.)
[0347] The cells were cooled at approximately 1.degree. C./min
overnight in a -80.degree. C. freezer using a "Mr Frosty" freezing
container according to the manufacturer's instructions (Nalgene,
Rochester, N.Y.). Vials of cells were transferred into liquid
nitrogen for 2 days before thawing rapidly in a 37.degree. C. water
bath. The cells were added to 10 milliliter Growth medium and
centrifuged before the cell number and viability was estimated as
before. Cells were seeded onto gelatin-coated flasks at 5,000
cells/cm.sup.2 to determine whether the cells would attach and
proliferate.
[0348] Results
[0349] The initial viability of the cells to be cryopreserved was
assessed by trypan blue staining to be 100%.
[0350] There was a commensurate reduction in cell number with
viability for C6295 due to cells lysis. The viable cells
cryopreserved in all four solutions attached, divided, and produced
a confluent monolayer within 3 days. There was no discernable
difference in estimated growth rate.
[0351] Summary. The cryopreservation of cells is one procedure
available for preparation of a cell bank or a cell product. Four
cryopreservation mixtures were compared for their ability to
protect human placenta-derived cells from freezing damage.
Dulbecco's modified Eagle's medium (DMEM) and 10% (v/v)
dimethylsulfoxide (DMSO) is the preferred medium of those compared
for cryopreservation of placenta-derived cells.
Example 5
Growth Characteristics of Postpartum-Derived Cells
[0352] The cell expansion potential of postpartum-derived cells was
compared to other populations of isolated stem cells. The art of
cell expansion to senescence is referred to as Hayflick's limit
(Hayflick L. The longevity of cultured human cells. J. Am. Geriatr.
Soc. 22(1):1-12, 1974; Hayflick L. The strategy of senescence.
Gerontologist 14(1):37-45), 1974). Postpartum-derived cells are
highly suited for therapeutic use because they can be readily
expanded to sufficient cell numbers.
[0353] Materials and Methods
[0354] Gelatin-coating flasks. Tissue culture plastic flasks were
coated by adding 20 milliliter 2% (w/v) porcine gelatin (Type B:
225 Bloom; Sigma, St Louis, Mo.) to a T75 flask (Corning, Corning,
N.Y.) for 20 minutes at room temperature. After removing the
gelatin solution, 10 milliliter phosphate-buffered saline (PBS)
(Invitrogen, Carlsbad, Calif.) were added and then aspirated.
[0355] Comparison of expansion potential of postpartum-derived
cells to other cell populations. For comparison of growth expansion
potential, the following cell populations were utilized: i)
Mesenchymal stem cells (MSC; Cambrex, Walkersville, Md.); ii)
Adipose-derived cells (U.S. Pat. No. 6,555,374 B1; U.S. Patent
Application Publication No. US2004/0058412); iii) Normal dermal
skin fibroblasts (cc-2509 lot # 9F0844; Cambrex, Walkersville,
Md.); iv) Umbilical cord-derived cells; and v) Placenta-derived
cells. Cells were initially seeded at 5,000 cells/cm.sup.2 on
gelatin-coated T75 flasks in DMEM-Low glucose growth medium
((Invitrogen, Carlsbad, Calif.), with 15% (v/v) defined bovine
serum (Hyclone, Logan, Utah; Lot#AND18475), 0.001% (v/v)
2-mercaptoethanol (Sigma, St. Louis, Mo.), 100 Units/milliliter
penicillin, 100 micrograms/milliliter streptomycin, 0.25
micrograms/milliliter amphotericin B; Invitrogen, Carlsbad,
Calif.). For subsequent passages, cell cultures were treated as
follows. After trypsinization, viable cells were counted after
Trypan Blue staining. Cell suspension (50 microliters) was combined
with Trypan Blue (50 microliters, Sigma, St. Louis Mo.). Viable
cell numbers were estimated using a hemocytometer.
[0356] Following counting, cells were seeded at 5,000
cells/cm.sup.2 onto gelatin-coated T 75 flasks in 25 milliliter of
fresh Growth medium. Cells were grown under standard atmosphere
with 5% carbon dioxide at 37.degree. C. The growth medium was
changed twice per week. When cells reached about 85 percent
confluence, they were passaged; this process was repeated until the
cells reached senescence.
[0357] At each passage, cells were trypsinized and counted. The
viable cell yield, population doubling [ln(cell final/cell
initial)/ln 2] and doubling time (time in culture (h)/population
doubling) were calculated. For the purposes of determining optimal
cell expansion, the total cell yield per passage was determined by
multiplying the total yield for the previous passage by the
expansion factor for each passage (i.e., expansion factor=cell
final/cell initial).
[0358] Expansion potential of cell banks at low density. The
expansion potential of cells banked at passage 10 was also tested.
A different set of conditions was used. Normal dermal skin
fibroblasts (cc-2509 lot # 9F0844; Cambrex, Walkersville, Md.),
umbilical cord-derived cells, and placenta-derived cells were
tested. These cell populations had been banked at passage 10
previously, having been seeded at 5,000 cells/cm.sup.2 and grown to
confluence at each passage to that point. The effect of cell
density on the cell populations following cell thaw at passage 10
was determined. Cells were thawed under standard conditons, counted
using Trypan Blue staining. Thawed cells were then seeded at 1,000
cells/cm.sup.2 in Growth medium (DMEM-Low glucose (Invitrogen,
Carlsbad, Calif.) with 15 percent (v/v) defined bovine serum
(Hyclone, Logan, Utah; Lot#AND18475), 0.001 percent
2-mercaptoethanol (Sigma, St. Louis, Mo.), antibiotic/antimycotic
(100 Units/milliliter penicillin, 100 micrograms/milliliter
streptomycin, 0.25 micrograms/milliliter amphotericin B
(Invitrogen, Carlsbad, Calif.)). Cells were grown under standard
atmospheric conditions at 37.degree. C. Growth medium was changed
twice a week and cells were passaged as they reached about 85%
confluence. Cells were subsequently passaged until senescence,
i.e., until they could not be expanded any further. Cells were
trypsinized and counted at each passage. The cell yield, population
doubling (ln(cell final/cell initial)/ln 2) and doubling time (time
in culture (h)/population doubling) were calculated. The total cell
yield per passage was determined by multiplying total yield for the
previous passage by the expansion factor for each passage (i.e.,
expansion factor=cell final/cell initial).
[0359] Expansion of postpartum-derived cells at low density from
initial cell seeding. The expansion potential of freshly isolated
postpartum-derived cell cultures under low cell seeding conditions
was tested in another experiment. Umbilical cord- and
placenta-derived cells were isolated as described herein. Cells
were seeded at 1000 cells/cm.sup.2 and passaged as described above
until senescence. Cells were grown under standard atmospheric
conditions at 37.degree. C. Growth medium was changed twice per
week. Cells were passaged as they reached about 85% confluence. At
each passage, cells were trypsinized and counted by Trypan Blue
staining. The cell yield, population doubling (ln(cell final/cell
initial)/ln 2), and doubling time (time in culture (h)/population
doubling) were calculated for each passage. The total cell yield
per passage was determined by multiplying the total yield for the
previous passage by the expansion factor for each passage (i.e.,
expansion factor=cell final/cell initial). Cells were grown on
gelatin- and non-gelatin-coated flasks.
[0360] Expansion of Clonal Neonatal or Maternal Placenta-derived
Cells. Cloning may be used in order to expand a population of
neonatal or maternal cells successfully from placental tissue.
Following isolation of three different cell populations from the
placenta (neonatal aspect, maternal aspect, and villous region),
these cell populations are expanded under standard growth
conditions and then karyotyped to reveal the identity of the
isolated cell populations. By isolating the cells from a mother who
delivers a boy, it is possible to distinguish between the male and
female chromosomes by performing metaphase spreads. These
experiments can be used to demonstrate that top-line cells are
karyotype positive for neonatal phenotype, mid-line cells are
karyotype positive for both neonatal and maternal phenotypes, and
bottom-line cells are karyotype positive for maternal cells.
[0361] Expansion of cells in low oxygen culture conditions. It has
been demonstrated that low O.sub.2 cell culture conditions can
improve cell expansion in certain circumstances (Csete, Marie;
Doyle, John; Wold, Barbara J.; McKay, Ron; Studer, Lorenz. Low
oxygen culturing of central nervous system progenitor cells.
US20040005704). In order to determine if cell expansion of
postpartum-derived cells could be improved by altering cell culture
conditions, cultures of umbilical cord-derived cells were grown in
low oxygen conditions. Cells were seeded at 5,000 cells/cm.sup.2 in
Growth medium on gelatin-coated flasks. Cells were initially
cultured under standard atmospheric conditions through passage 5,
at which point they were transferred to low oxygen (5% O.sub.2)
culture conditions.
[0362] Evaluation of other growth conditions. In other experiments,
postpartum-derived cells were expanded on non-coated,
collagen-coated, fibronectin-coated, laminin-coated, and
extracellular membrane protein (e.g., MATRIGEL (BD Discovery
Labware, Bedford, Mass.))-coated plates. Cultures have been
demonstrated to expand well on these different matrices.
[0363] Results
[0364] Comparison of expansion potential of postpartum-derived
cells vs. other stem cell and non-stem cell populations. Both
umbilical cord-derived and placenta-derived cells expanded for
greater than 40 passages generating cell yields of >1E17 cells
in 60 days. In contrast, MSCs and fibroblasts senesced after <25
days and <60 days, respectively. Although both adipose-derived
and omental cells expanded for almost 60 days, they generated total
cell yields of 4.5E12 and 4.24E13 respectively. Thus, when seeded
at 5,000 cells/cm.sup.2 under the experimental conditions utilized,
postpartum-derived cells expanded much better than the other cell
types grown under the same conditions (Table 5-1).
[0365] Expansion of potential of cell banks at low density.
Umbilical cord-derived, placenta-derived, and fibroblast cells
expanded for greater than 10 passages generating cell yields of
>1E11 cells in 60 days (Table 5-2). After 60 days under these
conditions, the fibroblasts became senescent, whereas the umbilical
cord-derived and placenta-derived cell populations senesced after
80 days, completing >50 and >40 population doublings,
respectively.
[0366] Expansion of postpartum-derived cells at low density from
initial cell seeding. Postpartum-derived cells were seeded at low
density (1,000 cells/cm.sup.2) on gelatin-coated and uncoated
plates or flasks. Growth potential of these cells under these
conditions was good. The cells expanded readily in a log phase
growth. The rate of cell expansion was similar to that observed
when postpartum-derived cells were seeded at 5,000 cells/cm.sup.2
on gelatin-coated flasks in Growth medium. No differences were
observed in cell expansion potential between culturing on either
uncoated flasks or gelatin-coated flasks. However, cells appeared
phenotypically much smaller on gelatin-coated flasks, and more,
larger cell phenotypes were observed on uncoated flasks.
[0367] Expansion of Clonal Neonatal or Maternal Placenta-Derived
Cells. A clonal neonatal or maternal cell population can be
expanded from placenta-derived cells isolated from the neonatal
aspect or the maternal aspect, respectively, of the placenta. Cells
are serially diluted and then seeded onto gelatin-coated plates in
Growth medium for expansion at 1 cell/well in 96-well gelatin
coated plates. From this initial cloning, expansive clones are
identified, trypsinized, and reseeded in 12-well gelatin-coated
plates in Growth medium and then subsequently passaged into T25
gelatin-coated flasks at 5,000 cells/cm.sup.2 in Growth medium.
Subcloning is performed to ensure that a clonal population of cells
has been identified. For subcloning experiments, cells are
trypsinized and reseeded at 0.5 cells/well. The subclones that grow
well are expanded in gelatin-coated T25 flasks at 5,000 cells
cm.sup.2/flask. Cells are passaged at 5,000 cells cm.sup.2/T75
flask. The growth characteristics of a clone may be plotted to
demonstrate cell expansion. Karyotyping analysis can confirm that
the clone is either neonatal or maternal.
[0368] Expansion of cells in low oxygen culture conditions.
Postpartum-derived cells expanded well under the reduced oxygen
conditions. Culturing under low oxygen conditions does not appear
to have a significant effect on cell expansion for
postpartum-derived cells. Standard atmospheric conditions have
already proven successful for growing sufficient numbers of cells,
and low oxygen culture is not required for the growth of
postpartum-derived cells.
[0369] Summary. Commercially viable cell products must be able to
be produced in sufficient quantities to provide therapeutic
treatment to patients in need of the treatment. Postpartum-derived
cells can be expanded in culture for such purposes. Comparisons
were made of the growth of postpartum-derived cells in culture to
that of other cell populations including mesenchymal stem cells.
The data demonstrated that postpartum-derived cell lines as
developed herein can expand for greater than 40 doublings to
provide sufficient cell numbers, for example, for pre-clinical
banks. Furthermore, these postpartum-derived cell populations can
be expanded well at low or high density. This study has
demonstrated that mesenchymal stem cells, in contrast, cannot be
expanded to obtain large quantities of cells.
[0370] The current cell expansion conditions of growing isolated
postpartum-derived cells at densities of about 5,000 cells/cm.sup.2
in Growth medium on gelatin-coated or uncoated flasks, under
standard atmospheric oxygen, are sufficient to generate large
numbers of cells at passage 11. Furthermore, the data suggests that
the cells can be readily expanded using lower density culture
conditions (e.g. 1,000 cells/cm.sup.2). Postpartum-derived cell
expansion in low oxygen conditions also facilitates cell expansion,
although no incremental improvement in cell expansion potential has
yet been observed when utilizing these conditions for growth.
Presently, culturing postpartum-derived cells under standard
atmospheric conditions is preferred for generating large pools of
cells. However, when the culture conditions are altered,
postpartum-derived cell expansion can likewise be altered. This
strategy may be used to enhance the proliferative and
differentiative capacity of these cell populations.
[0371] Under the conditions utilized, while the expansion potential
of MSC and adipose-derived cells is limited, postpartum-derived
cells expand readily to large numbers.
REFERENCES
[0372] 1) Hayflick L. The longevity of cultured human cells. J Am
Geriatr Soc. 1974 January; 22(1): 1-12. [0373] 2) Hayflick L. The
strategy of senescence. Gerontologist. 1974 February; 14(1):37-45.
[0374] 3) Patent US20040058412 [0375] 4) Patent US20040048372
[0376] 6) Csete, Marie; (Ann Arbor, Mich.); Doyle, John; (South
Pasadena, Calif.); Wold, Barbara J.; (San Marino, Calif.); McKay,
Ron; (Bethesda, Md.); Studer, Lorenz; (New York, N.Y.). Low oxygen
culturing of central nervous system progenitor cells.
US20040005704. TABLE-US-00004 TABLE 5-1 Growth characteristics for
different cell populations grown to senescence Total Population
Total Cell Cell Type Senescence Doublings Yield MSC 24 days 8
4.72E7 Adipose- 57 days 24 4.5E12 derived cells (Artecel, U.S. Pat.
No. 6,555,374) Fibroblasts 53 days 26 2.82E13 Umbilical cord- 65
days 42 6.15E17 derived cells Placenta- 80 days 46 2.49E19 derived
cells
[0377] TABLE-US-00005 TABLE 5-2 Growth characteristics for
different cell populations using low density growth expansion from
passage 10 to senescence Total Population Total Cell Cell Type
Senescence Doublings Yield Fibroblast (P10) 80 days 43.68 2.59E11
Umbilical cord- 80 days 53.6 1.25E14 derived cells (P10)
Placenta-derived 60 days 32.96 6.09E12 cells (P10)
Example 6
Karyotype Analysis of PPDCs
[0378] Cell lines used in cell therapy are preferably homogeneous
and free from any contaminating cell type. Human cells used in cell
therapy should have a normal chromosome number (46) and structure.
To identify postpartum-derived placental and umbilical cord cell
lines that are homogeneous and free from cells of non-postpartum
tissue origin, karyotypes of cell samples were analyzed.
[0379] Materials and Methods
[0380] PPDCs from postpartum tissue of a male neonate were cultured
in Growth medium (DMEM-low glucose (Gibco Carlsbad, Calif.), 15%
(v/v) fetal bovine serum (FBS) (Hyclone, Logan, Utah), 0.001% (v/v)
betamercaptoethanol (Sigma, St. Louis, Mo.), and 50
Units/milliliter penicillin, 50 micrograms/milliliter streptomycin
(Gibco, Carlsbad, Calif.)). Postpartum tissue from a male neonate
(X,Y) was selected to allow distinction between neonatal-derived
cells and maternal-derived cells (X,X). Cells were seeded at 5,000
cells per square centimeter in Growth medium in a T25 flask
(Corning, Corning, N.Y.) and expanded to about 80% confluence. A
T25 flask containing cells was filled to the neck with Growth
medium. Samples were delivered to a clinical cytogenetics lab by
courier (estimated lab to lab transport time is one hour).
Chromosome analysis was performed by the Center for Human &
Molecular Genetics at the New Jersey Medical School, Newark, N.J.
Cells were analyzed during metaphase when the chromosomes are best
visualized. Of twenty cells in metaphase counted, five were
analyzed for normal homogeneous karyotype number (two). A cell
sample was characterized as homogeneous if two karyotypes were
observed. A cell sample was characterized as heterogeneous if more
than two karyotypes were observed. Additional metaphase cells were
counted and analyzed when a heterogeneous karyotype number (four)
was identified.
[0381] Results
[0382] All cell samples sent for chromosome analysis were
interpreted by the cytogenetics laboratory staff as exhibiting a
normal appearance. Three of the sixteen cell lines analyzed
exhibited a heterogeneous phenotype (XX and XY) indicating the
presence of cells derived from both neonatal and maternal origins
(Table 6-1). Cells derived from tissue Placenta-N were isolated
from the neonatal aspect of placenta. At passage zero, this cell
line appeared homogeneous XY. However, at passage nine, the cell
line was heterogeneous (XX/XY), indicating a previously undetected
presence of cells of maternal origin. TABLE-US-00006 TABLE 6-1
Karyotype results of PPDCs. Metaphase cells Metaphase cells Number
of Tissue passage counted analyzed karyotypes ISCN Karyotype
Placenta 22 20 5 2 46, XX Umbilical 23 20 5 2 46, XX Umbilical 6 20
5 2 46, XY Placenta 2 20 5 2 46, XX Umbilical 3 20 5 2 46, XX
Placenta-N 0 20 5 2 46, XY Placenta-V 0 20 5 2 46, XY Placenta-M 0
21 5 4 46, XY[18]/46, XX[3] Placenta-M 4 20 5 2 46, XX Placenta-N 9
25 5 4 46, XY[5]/46, XX[20] Placenta-N 1 20 5 2 46, XY C1
Placenta-N 1 20 6 4 46, XY[2]/46, C3 XX[18] Placenta-N 1 20 5 2 46,
XY C4 Placenta-N 1 20 5 2 46, XY C15 Placenta-N 1 20 5 2 46, XY C20
Placenta-N 1 20 5 2 46, XY C22 Key: N--Neonatal side; V--villous
region; M--maternal side; C--clone
[0383] Summary. Chromosome analysis identified placenta- and
umbilical cord-derived PPDCs whose karyotypes appear normal as
interpreted by a clinical cytogenetic laboratory. Karyotype
analysis also identified cell lines free from maternal cells, as
determined by homogeneous karyotype.
Example 7
Evaluation of Human Postpartum-Derived Cell Surface Markers by Flow
Cytometry
[0384] Characterization of cell surface proteins or "markers" by
flow cytometry can be used to determine a cell line's identity. The
consistency of expression can be determined from multiple donors,
and in cells exposed to different processing and culturing
conditions. Postpartum-derived cell lines isolated from the
placenta and umbilical cord were characterized by flow cytometry,
thereby providing a profile for the identification of the cells of
the invention.
[0385] Materials and Methods
[0386] Media. Cells were cultured in DMEM-low glucose Growth medium
(Gibco Carlsbad, Calif.), with 15% (v/v) fetal bovine serum (FBS);
(Hyclone, Logan, Utah), 0.001% (v/v) betamercaptoethanol (Sigma,
St. Louis, Mo.), and 50 Units/milliliter penicillin, 50
micrograms/milliliter streptomycin (Gibco, Carlsbad, Calif.).
[0387] Culture Vessels. Cells were cultured in plasma-treated T75,
T150, and T225 tissue culture flasks (Corning, Corning, N.Y.) until
confluent. The growth surfaces of the flasks were coated with
gelatin by incubating 2% (w/v) gelatin (Sigma, St. Louis, Mo.) for
20 minutes at room temperature.
[0388] Antibody Staining. Adherent cells in flasks were washed in
phosphate buffered saline (PBS); (Gibco, Carlsbad, Calif.) and
detached with Trypsin/EDTA (Gibco, Carlsbad, Calif.). Cells were
harvested, centrifuged, and resuspended in 3% (v/v) FBS in PBS at a
cell concentration of 1.times.10.sup.7 per milliliter. In
accordance with the manufacturer's specifications, antibody to the
cell surface marker of interest (Table 7-1) was added to one
hundred microliters of cell suspension and the mixture was
incubated in the dark for 30 minutes at 4.degree. C. After
incubation, cells were washed with PBS and centrifuged to remove
unbound antibody. Cells were resuspended in 500 microliter PBS and
analyzed by flow cytometry.
[0389] Flow Cytometry Analysis. Flow cytometry analysis was
performed with a FACScalibur instrument (Becton Dickinson, San
Jose, Calif.).
[0390] Antibodies to Cell Surface Markers. The following antibodies
to cell surface markers were used. TABLE-US-00007 TABLE 7-1
Antibodies to Cell Surface markers Antibody Manufacture Catalog
Number CD10 BD Pharmingen (San Diego, CA) 555375 CD13 BD Pharmingen
(San Diego, CA) 555394 CD31 BD Pharmingen (San Diego, CA) 555446
CD34 BD Pharmingen (San Diego, CA) 555821 CD44 BD Pharmingen (San
Diego, CA) 555478 CD45RA BD Pharmingen (San Diego, CA) 555489 CD73
BD Pharmingen (San Diego, CA) 550257 CD90 BD Pharmingen (San Diego,
CA) 555596 CD117 BD Biosciences (San Jose, CA) 340529 CD141 BD
Pharmingen (San Diego, CA) 559781 PDGFr-alpha BD Pharmingen (San
Diego, CA) 556002 HLA-A, B, C BD Pharmingen (San Diego, CA) 555553
HLA-DR, DP, BD Pharmingen (San Diego, CA) 555558 DQ IgG-FITC Sigma
(St. Louis, MO) F-6522 IgG-PE Sigma (St. Louis, MO) P-4685
[0391] Placenta- and Umbilical Cord-Derived Cell Comparison.
Placenta-derived cells were compared to umbilical cord-derived
cells at passage 8.
[0392] Passage to Passage Comparison. Placenta- and umbilical cord
cells were analyzed at passages 8, 15, and 20.
[0393] Donor to Donor Comparison. To compare differences among
donors, placenta-derived cells from different donors were compared
to each other, and umbilical cord-derived cells from different
donors were compared to each other.
[0394] Surface Coating Comparison. Placenta-derived cells cultured
on gelatin-coated flasks were compared to placenta-derived cells
cultured on uncoated flasks. Umbilical cord-derived cells cultured
on gelatin-coated flasks were compared to umbilical cord-derived
cells cultured on uncoated flasks.
[0395] Digestion Enzyme Comparison. Four treatments used for
isolation and preparation of cells were compared. Cells derived
from postpartum tissue by treatment with 1) collagenase; 2)
collagenase/dispase; 3) collagenase/hyaluronidase; and 4)
collagenase/hyaluronidase/dispase were compared.
[0396] Placental Layer Comparison. Cells isolated from the maternal
aspect of placental tissue were compared to cells isolated from the
villous region of placental tissue and cells isolated from the
neonatal fetal aspect of placenta.
[0397] Results
[0398] Placenta-derived cells were compared to Umbilical
cord-derived cells. Placenta- and umbilical cord-derived cells
analyzed by flow cytometry showed positive for production of CD10,
CD13, CD44, CD73, CD 90, PDGFr-alpha and HLA-A, B, C, indicated by
the increased values of fluorescence relative to the IgG control.
These cells were negative for detectable expression of CD31, CD34,
CD45, CD117, CD141, and HLA-DR, DP, DQ, indicated by fluorescence
values comparable to the IgG control. Variations in fluorescence
values of positive curves was accounted. The mean (i.e., CD13) and
range (i.e., CD90) of the positive curves showed some variation,
but the curves appeared normal, confirming a homogeneous
population. Both curves individually exhibited values greater than
the IgG control.
[0399] Passage to Passage Comparison of Placenta-derived cells.
Placenta-derived cells at passages 8, 15, and 20 analyzed by flow
cytometry all were positive for production of CD10, CD13, CD44,
CD73, CD 90, PDGFr-alpha and HLA-A, B, C, as reflected in the
increased value of fluorescence relative to the IgG control. The
cells were negative for production of CD31, CD34, CD45, CD117,
CD141, and HLA-DR, DP, DQ having fluorescence values consistent
with the IgG control.
[0400] Passage to Passage Comparison of Umbilical cord-derived
cells. Umbilical cord-derived cells at passage 8, 15, and 20
analyzed by flow cytometry all expressed CD10, CD13, CD44, CD73, CD
90, PDGFr-alpha and HLA-A, B, C, indicated by increased
fluorescence relative to the IgG control. These cells were negative
for CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ, indicated
by fluorescence values consistent with the IgG control.
[0401] Donor to Donor Comparison of Placenta-derived cells.
Placenta-derived cells isolated from separate donors analyzed by
flow cytometry each expressed CD10, CD13, CD44, CD73, CD 90,
PDGFr-alpha and HLA-A, B, C, with increased values of fluorescence
relative to the IgG control. The cells were negative for production
of CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ as indicated
by fluorescence value consistent with the IgG control.
[0402] Donor to Donor Comparison of Umbilical cord-derived cells.
Umbilical cord-derived cells isolated from separate donors analyzed
by flow cytometry each showed positive for production of CD10,
CD13, CD44, CD73, CD 90, PDGFr-alpha and HLA-A, B, C, reflected in
the increased values of fluorescence relative to the IgG control.
These cells were negative for production of CD31, CD34, CD45,
CD117, CD141, and HLA-DR, DP, DQ with fluorescence values
consistent with the IgG control.
[0403] The Effect of Surface Coating with Gelatin on
Placenta-derived Cells. Placenta-derived cells expanded on either
gelatin-coated or uncoated flasks analyzed by flow cytometry all
expressed of CD10, CD13, CD44, CD73, CD 90, PDGFr-alpha and HLA-A,
B, C, reflected in the increased values of fluorescence relative to
the IgG control. These cells were negative for production of CD31,
CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ indicated by
fluorescence values consistent with the IgG control.
[0404] The Effect of Surface Coating with Gelatin on Umbilical
cord-derived Cells. Umbilical cord-derived cells expanded on
gelatin and uncoated flasks analyzed by flow cytometry all were
positive for production of CD10, CD13, CD44, CD73, CD 90,
PDGFr-alpha and HLA-A, B, C, with increased values of fluorescence
relative to the IgG control. These cells were negative for
production of CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ,
with fluorescence values consistent with the IgG control.
[0405] Evaluation of Effect of Enzyme Digestion Procedure Used for
Preparation and Isolation of the Cells on the Cell Surface Marker
Profile. Placenta-derived cells isolated using various digestion
enzymes analyzed by flow cytometry all expressed CD10, CD13, CD44,
CD73, CD 90, PDGFr-alpha and HLA-A, B, C, as indicated by the
increased values of fluorescence relative to the IgG control. These
cells were negative for production of CD31, CD34, CD45, CD117,
CD141, and HLA-DR, DP, DQ as indicated by fluorescence values
consistent with the IgG control.
[0406] Placental Layer Comparison. Cells derived from the maternal,
villous, and neonatal layers of the placenta, respectively,
analyzed by flow cytometry showed positive for production of CD10,
CD13, CD44, CD73, CD90, PDGFr-alpha and HLA-A, B, C, as indicated
by the increased value of fluorescence relative to the IgG control.
These cells were negative for production of CD31, CD34, CD45,
CD117, CD141, and HLA-DR, DP, DQ as indicated by fluorescence
values consistent with the IgG control.
[0407] Summary. Analysis of placenta- and umbilical cord-derived
postpartum cells by flow cytometry has established of an identity
of these cell lines. Placenta- and umbilical cord-derived
postpartum cells are positive for CD10, CD13, CD44, CD73, CD90,
PDGFr-alpha, HLA-A,B,C and negative for CD31, CD34, CD45, CD117,
CD141 and HLA-DR, DP, DQ. This identity was consistent between
variations in variables including the donor, passage, culture
vessel surface coating, digestion enzymes, and placental layer.
Some variation in individual fluorescence value histogram curve
means and ranges were observed, but all positive curves under all
conditions tested were normal and expressed fluorescence values
greater than the IgG control, thus confirming that the cells
comprise a homogeneous population which has positive expression of
the markers.
Example 8
Analysis of Postpartum Tissue-Derived Cells by Affymetrix
GeneChip.RTM. Arrays
[0408] Affymetrix GeneChip.RTM. arrays were used to compare gene
expression profiles of umbilical cord- and placenta-derived cells
with fibroblasts, human mesenchymal stem cells, and another cell
line derived from human bone marrow. This analysis provided a
characterization of the postpartum-derived cells and identified
unique molecular markers for these cells.
[0409] Materials and Methods
[0410] Isolation and Culture of Cells
[0411] Postpartum tissue-derived cells. Human umbilical cords and
placenta were obtained from National Disease Research Interchange
(NDRI, Philadelphia, Pa.) from normal full term deliveries with
patient consent. The tissues were received and cells were isolated
as described in Example 1. Cells were cultured in Growth medium
(Dulbecco's Modified Essential Media (DMEM-low glucose; Invitrogen,
Carlsbad, Calif.) with 15% (v/v) fetal bovine serum (Hyclone, Logan
Utah), 100 Units/milliliter penicillin, 100 micrograms/milliliter
streptomycin (Invitrogen, Carlsbad, Calif.), and 0.001% (v/v)
2-mercaptoethanol (Sigma, St. Louis Mo.)) on gelatin-coated tissue
culture plastic flasks. The cultures were incubated at 37.degree.
C. in standard atmosphere.
[0412] Fibroblasts. Human dermal fibroblasts were purchased from
Cambrex Incorporated (Walkersville, Md.; Lot number 9F0844) and
were obtained from ATCC CRL-1501 (CCD39SK). Both lines were
cultured in DMEM/F12 medium (Invitrogen, Carlsbad, Calif.) with 10%
(v/v) fetal bovine serum (Hyclone) and 100 Units/milliliter
penicillin, 100 micrograms/milliliter streptomycin (Invitrogen).
The cells were grown on standard tissue-treated plastic.
[0413] Human Mesenchymal Stem Cells (hMSC). hMSCs were purchased
from Cambrex Incorporated (Walkersville, Md.; Lot numbers 2F1655,
2F1656 and 2F1657) and cultured according to the manufacturer's
specifications in MSCGM Media (Cambrex). The cells were grown on
standard tissue cultured plastic at 37.degree. C. with 5%
CO.sub.2.
[0414] Human Ileac Crest Bone Marrow Cells (ICBM). Human ileac
crest bone marrow was received from NDRI with patient consent. The
marrow was processed according to the method outlined by Ho, et al.
(WO03/025149). The marrow was mixed with lysis buffer (155
micromolar NH.sub.4Cl, 10 micromolar KHCO.sub.3, and 0.1 micromolar
EDTA, pH 7.2) at a ratio of 1 part bone marrow to 20 parts lysis
buffer. The cell suspension was vortexed, incubated for 2 minutes
at ambient temperature, and centrifuged for 10 minutes at
500.times.g. The supernatant was discarded and the cell pellet was
resuspended in Minimal Essential Medium-alpha (Invitrogen)
supplemented with 10% (v/v) fetal bovine serum and 4 micromolar
glutamine. The cells were centrifuged again and the cell pellet was
resuspended in fresh medium. The viable mononuclear cells were
counted using trypan-blue exclusion (Sigma, St. Louis, Mo.). The
mononuclear cells were seeded in tissue-cultured plastic flasks at
5.times.10.sup.4 cells/cm.sup.2. The cells were incubated at
37.degree. C. with 5% CO.sub.2 at either standard atmospheric
O.sub.2 or at 5% O.sub.2. Cells were cultured for 5 days without a
media change. Media and non-adherent cells were removed after 5
days of culture. The adherent cells were maintained in culture.
[0415] Isolation of mRNA and Gene Chip Analysis. Actively growing
cultures of cells were removed from the flasks with a cell scraper
in cold phosphate buffered saline (PBS). The cells were centrifuged
for 5 minutes at 300.times.g. The supernatant was removed and the
cells were resuspended in fresh PBS and centrifuged again. The
supernatant was removed and the cell pellet was immediately frozen
and stored at -80.degree. C. Cellular mRNA was extracted and
transcribed into cDNA. cDNA was then transcribed into cRNA and
biotin-labeled. The biotin-labeled cRNA was hybridized with
HG-U133A GENECHIP oligonucleotide array (Affymetrix, Santa Clara
Calif.). The hybridization and data collection was performed
according to the manufacturer's specifications. Analyses were
performed using "Significance Analysis of Microarrays" (SAM)
version 1.21 computer software (Stanford University,
www-stat.stanford.edu/.about.tibs/SAM; Tusher, V. G. et al., 2001,
Proc. Natl. Acad. Sci. USA 98: 5116-5121).
[0416] Results
[0417] Fourteen different populations of cells were analyzed in
this study. The cells along with passage information, culture
substrate, and culture media are listed in Table 8-1.
TABLE-US-00008 TABLE 8-1 Cells analyzed by the microarray study.
The cells lines are listed by their identification code along with
passage at the time of analysis, cell growth substrate, and growth
media. Cell Population Passage Substrate Media Umbilical (022803) 2
Gelatin DMEM, 15% FBS, BME Umbilical (042103) 3 Gelatin DMEM, 15%
FBS, BME Umbilical (071003) 4 Gelatin DMEM, 15% FBS, BME Placenta
(042203) 12 Gelatin DMEM, 15% FBS, BME Placenta (042903) 4 Gelatin
DMEM, 15% FBS, BME Placenta (071003) 3 Gelatin DMEM, 15% FBS, BME
ICBM (070203) (5% 3 Plastic MEM 10% FBS O.sub.2) ICBM (062703) (std
O.sub.2) 5 Plastic MEM 10% FBS ICBM (062703)(5% 5 Plastic MEM 10%
FBS O.sub.2) hMSC (Lot 2F1655) 3 Plastic MSCGM hMSC (Lot 2F1656) 3
Plastic MSCGM hMSC (Lot 2F1657) 3 Plastic MSCGM hFibroblast
(9F0844) 9 Plastic DMEM-F12, 10% FBS hFibroblast (ATCC 4 Plastic
DMEM-F12, 10% FBS CRL-1501)
[0418] The data were evaluated by a Principle Component Analysis,
analyzing the 290 genes that were differentially expressed in the
cells. This analysis allows for a relative comparison for the
similarities between the populations. Table 8-2 shows the Euclidean
distances that were calculated for the comparison of the cell
pairs. The Euclidean distances were based on the comparison of the
cells based on the 290 genes that were differentially expressed
among the cell types. The Euclidean distance is inversely
proportional to similarity between the expression of the 290 genes.
TABLE-US-00009 TABLE 8-2 The Euclidean Distances for the Cell
Pairs. The Euclidean distance was calculated for the cell types
using the 290 genes that were differentially expressed between the
cell types. Similarity between the cells is inversely proportional
to the Euclidean distance. Cell Pair Euclidean Distance ICBM-hMSC
24.71 Placenta-umbilical 25.52 ICBM-Fibroblast 36.44 ICBM-placenta
37.09 Fibroblast-MSC 39.63 ICBM-Umbilical 40.15 Fibroblast- 41.59
Umbilical MSC-Placenta 42.84 MSC-Umbilical 46.86 ICBM-placenta
48.41
[0419] Tables 8-3, 8-4, and 8-5 show the expression of genes
increased in placenta-derived cells (Table 8-3), increased in
umbilical cord-derived cells (Table 8-4), and reduced in umbilical
cord- and placenta-derived cells (Table 8-5). The column entitled
"Probe Set ID" refers to the manufacturer's identification code for
the sets of several oligonucleotide probes located on a particular
site on the chip, which hybridize to the named gene (column "Gene
Name"), comprising a sequence that can be found within the NCBI
(GenBank) database at the specified accession number (column "NCBI
Accession Number"). TABLE-US-00010 TABLE 8-3 Genes shown to have
specifically increased expression in the placenta-derived cells as
compared to the other cell lines assayed. Genes Increased in
Placenta-Derived Cells Probe Set ID Gene Name NCBI Accession Number
209732_at C-type (calcium dependent, carbohydrate- AF070642
recognition domain) lectin, superfamily member 2
(activation-induced) 206067_s_at Wilms tumor 1 NM_024426
207016_s_at aldehyde dehydrogenase 1 family, member A2 AB015228
206367_at renin NM_000537 210004_at oxidised low density
lipoprotein (lectin-like) receptor 1 AF035776 214993_at Homo
sapiens, clone IMAGE: 4179671, mRNA, AF070642 partial cds 202178_at
protein kinase C, zeta NM_002744 209780_at hypothetical protein
DKFZp564F013 AL136883 204135_at downregulated in ovarian cancer 1
NM_014890 213542_at Homo sapiens mRNA; cDNA DKFZp547K1113 AI246730
(from clone DKFZp547K1113)
[0420] TABLE-US-00011 TABLE 8-4 Genes shown to have specifically
increased expression in umbilical cord-derived cells as compared to
the other cell lines assayed. Genes Increased in Umbilical
Cord-Derived Cells Probe Set ID Gene Name NCBI Accession Number
202859_x_at interleukin 8 NM_000584 211506_s_at interleukin 8
AF043337 210222_s_at reticulon 1 BC000314 204470_at chemokine
(C--X--C motif) ligand 1 (melanoma NM_001511 growth stimulating
activity 206336_at chemokine (C--X--C motif) ligand 6 (granulocyte
NM_002993 chemotactic protein 2) 207850_at chemokine (C--X--C
motif) ligand 3 NM_002090 203485_at reticulon 1 NM_021136
202644_s_at tumor necrosis factor, alpha-induced protein 3
NM_006290
[0421] TABLE-US-00012 TABLE 8-5 Genes that were shown to have
decreased expression in the umbilical cord- and placenta-derived
cells as compared to the other cell lines assayed. Genes Decreased
in Umbilical Cord- and Placenta-Derived Cells Probe Set ID Gene
name NCBI Accession Number 210135_s_at short stature homeobox 2
AF022654.1 205824_at heat shock 27 kDa protein 2 NM_001541.1
209687_at chemokine (C--X--C motif) ligand 12 (stromal cell-
U19495.1 derived factor 1) 203666_at chemokine (C--X--C motif)
ligand 12 (stromal cell- NM_000609.1 derived factor 1) 212670_at
elastin (supravalvular aortic stenosis, Williams- AA479278 Beuren
syndrome) 213381_at Homo sapiens mRNA; cDNA DKFZp586M2022 N91149
(from clone DKFZp586M2022) 206201_s_at mesenchyme homeobox 2
(growth arrest- NM_005924.1 specific homeobox) 205817_at sine
oculis homeobox homolog 1 (Drosophila) NM_005982.1 209283_at
crystallin, alpha B AF007162.1 212793_at dishevelled associated
activator of BF513244 morphogenesis 2 213488_at DKFZP586B2420
protein AL050143.1 209763_at similar to neuralin 1 AL049176
205200_at tetranectin (plasminogen binding protein) NM_003278.1
205743_at src homology three (SH3) and cysteine rich NM_003149.1
domain 200921_s_at B-cell translocation gene 1, anti-proliferative
NM_001731.1 206932_at cholesterol 25-hydroxylase NM_003956.1
204198_s_at runt-related transcription factor 3 AA541630 219747_at
hypothetical protein FLJ23191 NM_024574.1 204773_at interleukin 11
receptor, alpha NM_004512.1 202465_at procollagen C-endopeptidase
enhancer NM_002593.2 203706_s_at frizzled homolog 7 (Drosophila)
NM_003507.1 212736_at hypothetical gene BC008967 BE299456 214587_at
collagen, type VIII, alpha 1 BE877796 201645_at tenascin C
(hexabrachion) NM_002160.1 210239_at iroquois homeobox protein 5
U90304.1 203903_s_at hephaestin NM_014799.1 205816_at integrin,
beta 8 NM_002214.1 203069_at synaptic vesicle glycoprotein 2
NM_014849.1 213909_at Homo sapiens cDNA FLJ12280 fis, clone
AU147799 MAMMA1001744 206315_at cytokine receptor-like factor 1
NM_004750.1 204401_at potassium intermediate/small conductance
NM_002250.1 calcium-activated channel, subfamily N, member 4
216331_at integrin, alpha 7 AK022548.1 209663_s_at integrin, alpha
7 AF072132.1 213125_at DKFZP586L151 protein AW007573 202133_at
transcriptional co-activator with PDZ-binding AA081084 motif (TAZ)
206511_s_at sine oculis homeobox homolog 2 (Drosophila) NM_016932.1
213435_at KIAA1034 protein AB028957.1 206115_at early growth
response 3 NM_004430.1 213707_s_at distal-less homeobox 5
NM_005221.3 218181_s_at hypothetical protein FLJ20373 NM_017792.1
209160_at aldo-keto reductase family 1, member C3 (3- AB018580.1
alpha hydroxysteroid dehydrogenase, type II) 213905_x_at biglycan
AA845258 201261_x_at biglycan BC002416.1 202132_at transcriptional
co-activator with PDZ-binding AA081084 motif (TAZ) 214701_s_at
fibronectin 1 AJ276395.1 213791_at proenkephalin NM_006211.1
205422_s_at integrin, beta-like 1 (with EGF-like repeat NM_004791.1
domains) 214927_at Homo sapiens mRNA full length insert cDNA
AL359052.1 clone EUROIMAGE 1968422 206070_s_at EphA3 AF213459.1
212805_at KIAA0367 protein AB002365.1 219789_at natriuretic peptide
receptor C/guanylate cyclase AI628360 C (atrionatriuretic peptide
receptor C) 219054_at hypothetical protein FLJ14054 NM_024563.1
213429_at Homo sapiens mRNA; cDNA DKFZp564B222 AW025579 (from clone
DKFZp564B222) 204929_s_at vesicle-associated membrane protein 5
NM_006634.1 (myobrevin) 201843_s_at EGF-containing fibulin-like
extracellular matrix NM_004105.2 protein 1 221478_at
BCL2/adenovirus E1B 19 kDa interacting protein AL132665.1 3-like
201792_at AE binding protein 1 NM_001129.2 204570_at cytochrome c
oxidase subunit VIIa polypeptide 1 NM_001864.1 (muscle) 201621_at
neuroblastoma, suppression of tumorigenicity 1 NM_005380.1
202718_at insulin-like growth factor binding protein 2, NM_000597.1
36 kDa
[0422] Tables 8-6, 8-7, and 8-8 show the expression of genes
increased in human fibroblasts (Table 8-6), ICBM cells (Table 8-7),
and MSCs (Table 8-8). TABLE-US-00013 TABLE 8-6 Genes that were
shown to have increased expression in fibroblasts as compared to
the other cell lines assayed. Genes increased in fibroblasts dual
specificity phosphatase 2 KIAA0527 protein Homo sapiens cDNA:
FLJ23224 fis, clone ADSU02206 dynein, cytoplasmic, intermediate
polypeptide 1 ankyrin 3, node of Ranvier (ankyrin G) inhibin, beta
A (activin A, activin AB alpha polypeptide) ectonucleotide
pyrophosphatase/phosphodiesterase 4 (putative function) KIAA1053
protein microtubule-associated protein 1A zinc finger protein 41
HSPC019 protein Homo sapiens cDNA: FLJ23564 fis, clone LNG10773
Homo sapiens mRNA; cDNA DKFZp564A072 (from clone DKFZp564A072) LIM
protein (similar to rat protein kinase C-binding enigma) inhibitor
of kappa light polypeptide gene enhancer in B-cells, kinase
complex-associated protein hypothetical protein FLJ22004 Human
(clone CTG-A4) mRNA sequence ESTs, Moderately similar to cytokine
receptor-like factor 2; cytokine receptor CRL2 precursor [Homo
sapiens] transforming growth factor, beta 2 hypothetical protein
MGC29643 antigen identified by monoclonal antibody MRC OX-2
[0423] TABLE-US-00014 TABLE 8-7 Genes that were shown to have
increased expression in the ICBM-derived cells as compared to the
other cell lines assayed. Genes Increased In ICBM Cells cardiac
ankyrin repeat protein MHC class I region ORF integrin, alpha 10
hypothetical protein FLJ22362 UDP-N-acetyl-alpha-D-galactosamine:
polypeptide N- acetylgalactosaminyltransferase 3 (GalNAc-T3)
interferon-induced protein 44 SRY (sex determining region Y)-box 9
(campomelic dysplasia, autosomal sex-reversal) keratin associated
protein 1--1 hippocalcin-like 1 jagged 1 (Alagille syndrome)
proteoglycan 1, secretory granule
[0424] TABLE-US-00015 TABLE 8-8 Genes that were shown to have
increased expression in the MSC cells as compared to the other cell
lines assayed. Genes Increased In MSC Cells interleukin 26
maltase-glucoamylase (alpha-glucosidase) nuclear receptor subfamily
4, group A, member 2 v-fos FBJ murine osteosarcoma viral oncogene
homolog hypothetical protein DC42 nuclear receptor subfamily 4,
group A, member 2 FBJ murine osteosarcoma viral oncogene homolog B
WNT1 inducible signaling pathway protein 1 MCF.2 cell line derived
transforming sequence potassium channel, subfamily K, member 15
cartilage paired-class homeoprotein 1 Homo sapiens cDNA FLJ12232
fis, clone MAMMA1001206 Homo sapiens cDNA FLJ34668 fis, clone
LIVER2000775 jun B proto-oncogene B-cell CLL/lymphoma 6 (zinc
finger protein 51) zinc finger protein 36, C3H type, homolog
(mouse)
[0425] Summary. The GENECHIP analysis was performed to provide a
molecular characterization of the postpartum cells derived from
umbilical cord and placenta. This analysis included cells derived
from three different umbilical cords and three different placentas.
The study also included two different lines of dermal fibroblasts,
three lines of mesenchymal stem cells, and three lines of ileac
crest bone marrow cells. The mRNA that was expressed by these cells
was analyzed by AffyMetrix GENECHIP that contained oligonucleotide
probes for 22,000 genes.
[0426] Results showed that 290 genes are differentially expressed
in these five different cell types. These genes include ten genes
that are specifically increased in the placenta-derived cells and
seven genes specifically increased in the umbilical cord-derived
cells. Fifty-four genes were found to have specifically lower
expression levels in placenta and umbilical cord.
[0427] The expression of selected genes has been confirmed by PCR
in Example 9. These results demonstrate that the postpartum-derived
cells have a distinct gene expression profile, for example, as
compared to bone marrow-derived cells and fibroblasts.
REFERENCE
[0428] Lockhart et al., Expression monitoring by hybridization to
high-density oligonucleotide arrays. Nat. Biotechnol. 1996,
14(13):1675-1680.
Example 9
Cell Markers in Postpartum-Derived Cells
[0429] Similarities and differences in cells derived from the human
placenta and the human umbilical cord were assessed by comparing
their gene expression profiles with those of cells derived from
other sources (using an Affymetrix GENECHIP array). Six "signature"
genes were identified: oxidized LDL receptor 1, interleukin-8,
renin, reticulon, chemokine receptor ligand 3 (CXC ligand 3), and
granulocyte chemotactic protein 2 (GCP-2). These "signature" genes
were expressed at relatively high levels in postpartum-derived
cells.
[0430] The present studies were conducted to verify the microarray
data and to identify accordance/discordance between gene and
protein expression, as well as to establish a series of reliable
assays for detection of unique identifiers for placenta- and
umbilical cord-derived cells.
[0431] Methods & Materials
[0432] Cells. Placenta-derived cells (three isolates, including one
isolate predominately neonatal as identified by karyotyping
analysis), umbilical cord-derived cells (four isolates), and Normal
Human Dermal Fibroblasts (NHDF; neonatal and adult) were grown in
Growth medium (DMEM-low glucose (Gibco, Carlsbad, Calif.), 15%
(v/v) fetal bovine serum (Cat. #SH30070.03; Hyclone, Logan, Utah),
0.001% (v/v) beta-mercaptoethanol (Sigma, St. Louis, Mo.), 50
Units/milliliter penicillin, 50 micrograms/milliliter streptomycin
(Gibco, Carlsbad, Calif.) in a gelatin-coated T75 flask.
Mesenchymal Stem Cells (MSCs) were grown in Mesenchymal Stem Cell
Growth Medium Bullet kit (MSCGM; Cambrex, Walkerville, Md.).
[0433] For the IL-8 secretion experiment, cells were thawed from
liquid nitrogen and plated in gelatin-coated flasks at 5,000
cells/cm.sup.2, grown for 48 hours in Growth medium, and then grown
for an additional 8 hours in 10 milliliter of serum starvation
medium (DMEM-low glucose (Gibco, Carlsbad, Calif.), 50
Units/milliliter penicillin, 50 micrograms/milliliter streptomycin
(Gibco, Carlsbad, Calif.), and 0.1% (w/v) Bovine Serum Albumin
(BSA; Sigma, St. Louis, Mo.)). After this treatment, RNA was
extracted and the supernatants were centrifuged at 150.times.g for
5 minutes to remove cellular debris. Supernatants were then frozen
at -80.degree. C. for ELISA analysis.
[0434] Cell culture for ELISA assay. Postpartum cells derived from
placenta and umbilical cord, as well as human fibroblasts derived
from human neonatal foreskin, were cultured in Growth medium in
gelatin-coated T75 flasks. Cells were frozen at passage 11 in
liquid nitrogen. Cells were thawed and transferred to 15 milliliter
centrifuge tubes. After centrifugation at 150.times.g for 5
minutes, the supernatant was discarded. Cells were resuspended in 4
milliliter culture medium and counted. Cells were grown in a 75
cm.sup.2 flask containing 15 milliliter of Growth medium at 375,000
cell/flask for 24 hours. The medium was changed to a serum
starvation medium for 8 hours. Serum starvation medium was
collected at the end of incubation, centrifuged at 14,000.times.g
for 5 minutes, and stored at -20.degree. C.
[0435] To estimate the number of cells in each flask, 2 milliliter
of tyrpsin/EDTA (Gibco, Carlsbad, Calif.) was added to each flask.
After cells detached from the flask, trypsin activity was
neutralized with 8 milliliter of Growth medium. Cells were
transferred to a 15 milliliter centrifuge tube and centrifuged at
150.times.g for 5 minutes. Supernatant was removed, and 1
milliliter Growth medium was added to each tube to resuspend the
cells. Cell number was estimated using a hemocytometer.
[0436] ELISA assay. The amount of IL-8 secreted by the cells into
serum starvation medium was analyzed using ELISA assays (R&D
Systems, Minneapolis, Minn.). All assays were tested according to
the instructions provided by the manufacturer.
[0437] Total RNA isolation. RNA was extracted from confluent
postpartum-derived cells and fibroblasts or for IL-8 expression
from cells treated as described above. Cells were lysed with 350
microliter buffer RLT containing beta-mercaptoethanol (Sigma, St.
Louis, Mo.) according to the manufacturer's instructions (RNeasy
Mini Kit; Qiagen, Valencia, Calif.). RNA was extracted according to
the manufacturer's instructions (RNeasy Mini Kit; Qiagen, Valencia,
Calif.) and subjected to DNase treatment (2.7 U/sample) (Sigma St.
Louis, Mo.). RNA was eluted with 50 microliter DEPC-treated water
and stored at -80.degree. C. RNA was also extracted from human
placenta and umbilical cord. Tissue (30 milligram) was suspended in
700 microliter of buffer RLT containing beta-mercaptoethanol.
Samples were mechanically homogenized, and the RNA extraction
proceeded according to manufacturer's specification. RNA was
extracted with 50 microliter of DEPC-treated water and stored at
-80.degree. C.
[0438] Reverse transcription. RNA was reversed transcribed using
random hexamers with the TaqMan.RTM. reverse transcription reagents
(Applied Biosystems, Foster City, Calif.) at 25.degree. C. for 10
minutes, 37.degree. C. for 60 minutes, and 95.degree. C. for 10
minutes. Samples were stored at -20.degree. C.
[0439] Genes identified by cDNA microarray as uniquely regulated in
postpartum-derived cells (signature genes--including oxidized LDL
receptor, interleukin-8, renin, and reticulon), were further
investigated using real-time and conventional PCR.
[0440] Real-time PCR. PCR was performed on cDNA samples using
ASSAYS-ON-DEMAND gene expression products: oxidized LDL receptor
(Hs00234028); renin (Hs00166915); reticulon (Hs00382515); CXC
ligand 3 (Hs00171061); GCP-2 (Hs00605742); IL-8 (Hs00174103); and
GAPDH were mixed with cDNA and TaqMan Universal PCR master mix
according to the manufacturer's instructions (Applied Biosystems,
Foster City, Calif.) using a 7000 sequence detection system with
ABI Prism 7000 SDS software (Applied Biosystems, Foster City,
Calif.). Thermal cycle conditions were initially 50.degree. C. for
2 minutes and 95.degree. C. for 10 min, followed by 40 cycles of
95.degree. C. for 15 seconds and 60.degree. C. for 1 minute. PCR
data was analyzed according to manufacturer's specifications (User
Bulletin #2 from Applied Biosystems for ABI Prism 7700 Sequence
Detection System).
[0441] Conventional PCR. Conventional PCR was performed using an
ABI PRISM 7700 (Perkin Elmer Applied Biosystems, Boston, Mass.) to
confirm the results from real-time PCR. PCR was performed using 2
microliter of cDNA solution, 1.times.TAQ polymerase (tradename
AMPLITAQ GOLD) universal mix PCR reaction buffer (Applied
Biosystems, Foster City, Calif.), and initial denaturation at
94.degree. C. for 5 minutes. Amplification was optimized for each
primer set: for IL-8, CXC ligand 3, and reticulon (94.degree. C.
for 15 seconds, 55.degree. C. for 15 seconds and 72.degree. C. for
30 seconds for 30 cycles); for renin (94.degree. C. for 15 seconds,
53.degree. C. for 15 seconds and 72.degree. C. for 30 seconds for
38 cycles); for oxidized LDL receptor and GAPDH (94.degree. C. for
15 seconds, 55.degree. C. for 15 seconds and 72.degree. C. for 30
seconds for 33 cycles). Primers used for amplification are listed
in Table 1. Primer concentration in the final PCR reaction was 1
micromolar except for GAPDH which was 0.5 micromolar. GAPDH primers
were the same as real-time PCR, except that the manufacturer's
TaqMan probe was not added to the final PCR reaction. Samples were
run on 2% (w/v) agarose gel and stained with ethidium bromide
(Sigma, St. Louis, Mo.). Images were captured using a 667 Universal
Twinpack film (VWR International, South Plainfield, N.J.) using a
focal-length POLAROID camera (VWR International, South Plainfield,
N.J.). TABLE-US-00016 TABLE 9-1 Primers used Primer name Primers
Oxidized LDL S: 5'-GAGAAATCCAAAGAGCAAATGG-3' (SEQ ID NO:1) receptor
A: 5'-AGAATGGAAAACTGGAATAGG-3' (SEQ ID NO:2) Renin S:
5'-TCTTCGATGCTTCGGATTCC-3' (SEQ ID NO:3) A:
5'-GAATTCTCGGAATCTCTGTTG-3' (SEQ ID NO:4) Reticulon S: 5'-
TTACAAGCAGTGCAGAAAACC-3' (SEQ ID NO:5) A: 5'-
AGTAAACATTGAAACCACAGCC-3' (SEQ ID NO:6) Interleukin-8 S: 5'-
TCTGCAGCTCTGTGTGAAGG-3' (SEQ ID NO:7) A: 5'-CTTCAAAAACTTCTCCACAACC-
3' (SEQ ID NO:8) Chemokine (CXC) S: 5'- CCCACGCCACGCTCTCC-3' (SEQ
ID NO:9) ligand 3 A: 5'-TCCTGTCAGTTGGTGCTCC -3' (SEQ ID NO:10)
[0442] Immunofluorescence. Postpartum-derived cells were fixed with
cold 4% (w/v) paraformaldehyde (Sigma-Aldrich, St. Louis, Mo.) for
10 minutes at room temperature. One isolate each of umbilical cord-
and placenta-derived cells at passage 0 (P0) (directly after
isolation) and passage 11 (P11) (two isolates of Placenta-derived,
two isolates of Umbilical cord-derived cells) and fibroblasts (P11)
were used. Immunocytochemistry was performed using antibodies
directed against the following epitopes: vimentin (1:500, Sigma,
St. Louis, Mo.), desmin (1:150; Sigma--raised against rabbit; or
1:300; Chemicon, Temecula, Calif.--raised against mouse),
alpha-smooth muscle actin (SMA; 1:400; Sigma), cytokeratin 18
(CK18; 1:400; Sigma), von Willebrand Factor (vWF; 1:200; Sigma),
and CD34 (human CD34 Class III; 1:100; DAKOCytomation, Carpinteria,
Calif.). In addition, the following markers were tested on passage
11 postpartum-derived cells: anti-human GROalpha-PE (1:100; Becton
Dickinson, Franklin Lakes, N.J.), anti-human GCP-2 (1:100; Santa
Cruz Biotech, Santa Cruz, Calif.), anti-human oxidized LDL receptor
1 (ox-LDL R1; 1:100; Santa Cruz Biotech), and anti-human NOGA-A
(1:100; Santa Cruz, Biotech).
[0443] Cultures were washed with phosphate-buffered saline (PBS)
and exposed to a protein blocking solution containing PBS, 4% (v/v)
goat serum (Chemicon, Temecula, Calif.), and 0.3% (v/v) Triton
(Triton X-100; Sigma, St. Louis, Mo.) for 30 minutes to access
intracellular antigens. Where the epitope of interest was located
on the cell surface (CD34, ox-LDL R1), Triton X-100 was omitted in
all steps of the procedure in order to prevent epitope loss.
Furthermore, in instances where the primary antibody was raised
against goat (GCP-2, ox-LDL R1, NOGO-A), 3% (v/v) donkey serum was
used in place of goat serum throughout the process. Primary
antibodies, diluted in blocking solution, were then applied to the
cultures for a period of 1 hour at room temperature. The primary
antibody solutions were removed and the cultures were washed with
PBS prior to application of secondary antibody solutions (1 hour at
room temperature) containing block along with goat anti-mouse
IgG--Texas Red (1:250; Molecular Probes, Eugene, Oreg.) and/or goat
anti-rabbit IgG--Alexa 488 (1:250; Molecular Probes) or donkey
anti-goat IgG--FITC (1:150, Santa Cruz Biotech). Cultures were then
washed and 10 micromolar DAPI (Molecular Probes) applied for 10
minutes to visualize cell nuclei.
[0444] Following immunostaining, fluorescence was visualized using
an appropriate fluorescence filter on an Olympus inverted
epi-fluorescent microscope (Olympus, Melville, N.Y.). In all cases,
positive staining represented fluorescence signal above control
staining where the entire procedure outlined above was followed
with the exception of application of a primary antibody solution
(no 1.degree. control). Representative images were captured using a
digital color videocamera and ImagePro software (Media Cybernetics,
Carlsbad, Calif.). For triple-stained samples, each image was taken
using only one emission filter at a time. Layered montages were
then prepared using Adobe Photoshop software (Adobe, San Jose,
Calif.).
[0445] Preparation of cells for FACS analysis. Adherent cells in
flasks were washed in phosphate buffered saline (PBS) (Gibco,
Carlsbad, Calif.) and detached with Trypsin/EDTA (Gibco, Carlsbad,
Calif.). Cells were harvested, centrifuged, and re-suspended 3%
(v/v) FBS in PBS at a cell concentration of
1.times.10.sup.7/milliliter. One hundred microliter aliquots were
delivered to conical tubes. Cells stained for intracellular
antigens were permeabilized with Perm/Wash buffer (BD Pharmingen,
San Diego, Calif.). Antibody was added to aliquots as per
manufacturer's specifications, and the cells were incubated for in
the dark for 30 minutes at 4.degree. C. After incubation, cells
were washed with PBS and centrifuged to remove excess antibody.
Cells requiring a secondary antibody were resuspended in 100
microliter of 3% FBS. Secondary antibody was added as per
manufacturer's specification, and the cells were incubated in the
dark for 30 minutes at 4.degree. C. After incubation, cells were
washed with PBS and centrifuged to remove excess secondary
antibody. Washed cells were resuspended in 0.5 milliliter PBS and
analyzed by flow cytometry. The following antibodies were used:
oxidized LDL receptor 1 (sc-5813; Santa Cruz, Biotech), GROa
(555042; BD Pharmingen, Bedford, Mass.), Mouse IgG1 kappa, (P-4685
and M-5284; Sigma), and Donkey against Goat IgG (sc-3743; Santa
Cruz, Biotech.).
[0446] FACS analysis. Flow cytometry analysis was performed with
FACScalibur (Becton Dickinson San Jose, Calif.).
[0447] Results
[0448] Results of real-time PCR for selected "signature" genes
performed on cDNA from cells derived from human placentas, adult
and neonatal fibroblasts, and Mesenchymal Stem Cells (MSCs)
indicate that both oxidized LDL receptor and renin were expressed
at higher level in the placenta-derived cells as compared to other
cells. The data obtained from real-time PCR were analyzed by the
.DELTA..DELTA.CT method and expressed on a logarithmic scale.
Levels of reticulon and oxidized LDL receptor expression were
higher in umbilical cord-derived cells as compared to other cells.
No significant difference in the expression levels of CXC ligand 3
and GCP-2 were found between postpartum-derived cells and controls
(data not shown). CXC-ligand 3 was expressed at very low levels.
GCP-2 was expressed at levels comparable to human adult and
neonatal fibroblasts. The results of real-time PCR were confirmed
by conventional PCR. Sequencing of PCR products further validated
these observations. No significant difference in the expression
level of CXC ligand 3 was found between postpartum-derived cells
and controls using conventional PCR CXC ligand 3 primers listed in
Table 9-1.
[0449] The expression of the cytokine IL-8 in postpartum-derived
cells is elevated in both Growth medium-cultured and serum-starved
postpartum-derived cells. All real-time PCR data was validated with
conventional PCR and by sequencing PCR products.
[0450] When supernatants of cells grown in serum-free medium were
examined for the presence of IL-8, the highest amounts were
detected in media derived from umbilical cord-derived cells and
some isolates of placenta-derived cells (Table 9-2). No IL-8 was
detected in medium derived from human dermal fibroblasts.
TABLE-US-00017 TABLE 9-2 IL-8 protein expression measured by ELISA
Cell type IL-8 Human fibroblasts ND Placenta Isolate 1 ND UMBC
Isolate 1 2058.42 .+-. 144.67 Placenta Isolate 2 ND UMBC Isolate 2
2368.86 .+-. 22.73 Placenta Isolate3 (normal O.sub.2) 17.27 .+-.
8.63 Placenta Isolate 3 (low O.sub.2, W/O 264.92 .+-. 9.88 BME)
Results of the ELISA assay for interleukin-8 (IL-8) performed on
placenta-and umbilical cord-derived cells as well as human skin
fibroblasts. Values are presented here are picogram/million cells,
n = 2, sem. ND: Not Detected
[0451] Placenta-derived cells were also examined for the expression
of oxidized LDL receptor, GCP-2, and GROalpha by FACS analysis.
Cells tested positive for GCP-2. Oxidized LDL receptor and GRO were
not detected by this method.
[0452] Placenta-derived cells were also tested for the production
of selected proteins by immunocytochemical analysis. Immediately
after isolation (passage 0), cells derived from the human placenta
were fixed with 4% paraformaldehyde and exposed to antibodies for
six proteins: von Willebrand Factor, CD34, cytokeratin 18, desmin,
alpha-smooth muscle actin, and vimentin. Cells stained positive for
both alpha-smooth muscle actin and vimentin. This pattern was
preserved through passage 11. Only a few cells (<5%) at passage
0 stained positive for cytokeratin 18.
[0453] Cells derived from the human umbilical cord at passage 0
were probed for the production of selected proteins by
immunocytochemical analysis. Immediately after isolation (passage
0), cells were fixed with 4% paraformaldehyde and exposed to
antibodies for six proteins: von Willebrand Factor, CD34,
cytokeratin 18, desmin, alpha-smooth muscle actin, and vimentin.
Umbilical cord-derived cells were positive for alpha-smooth muscle
actin and vimentin, with the staining pattern consistent through
passage 11.
[0454] Placenta-derived cells at passage 11 were also investigated
by immunocytochemistry for the production of GROalpha and GCP-2.
Placenta-derived cells were GCP-2 positive, but GROalpha production
was not detected by this method.
[0455] The production of GROalpha, GCP-2, oxidized LDL receptor 1
and reticulon (NOGO-A) in umbilical cord-derived cells at passage
11 was investigated by immunocytochemistry. Umbilical cord-derived
cells were GCP-2 positive, but GRO alpha production was not
detected by this method. Furthermore, cells were NOGO-A
positive.
[0456] Summary. Accordance between gene expression levels measured
by microarray and PCR (both real-time and conventional) has been
established for four genes: oxidized LDL receptor 1, renin,
reticulon, and IL-8. The expression of these genes was
differentially regulated at the mRNA level in postpartum-derived
cells, with IL-8 also differentially regulated at the protein
level. The presence of oxidized LDL receptor was not detected at
the protein level by FACS analysis in cells derived from the
placenta. Differential expression of GCP-2 and CXC ligand 3 was not
confirmed at the mRNA level; however, GCP-2 was detected at the
protein level by FACS analysis in the placenta-derived cells.
Although this result does not support data originally obtained from
the microarray experiment, this may be due to a difference in the
sensitivity of the methodologies.
[0457] Immediately after isolation (passage 0), cells derived from
the human placenta stained positive for both alpha-smooth muscle
actin and vimentin. This pattern was also observed in cells at
passage 11. These results suggest that vimentin and alpha-smooth
muscle actin expression may be preserved in cells with passaging,
at least in the Growth medium used here.
[0458] Cells derived from the human umbilical cord at passage 0
were probed for the expression of alpha-smooth muscle actin and
vimentin. and were positive for both. The staining pattern was
preserved through passage 11.
[0459] In conclusion, the complete mRNA data at least partially
verifies the data obtained from the microarray experiments.
Example 10
Immunohistochemical Characterization of PPDC Phenotype
[0460] The phenotypes of cells found within human postpartum
tissues, namely umbilical cord and placenta, were analyzed by
immunohistochemistry.
[0461] Materials & Methods
[0462] Tissue Preparation. Human umbilical cord and placenta tissue
were harvested and immersion fixed in 4% (w/v) paraformaldehyde
overnight at 4.degree. C. Immunohistochemistry was performed using
antibodies directed against the following epitopes (see Table
10-1): vimentin (1:500; Sigma, St. Louis, Mo.), desmin (1:150,
raised against rabbit; Sigma; or 1:300, raised against mouse;
Chemicon, Temecula, Calif.), alpha-smooth muscle actin (SMA; 1:400;
Sigma), cytokeratin 18 (CK18; 1:400; Sigma), von Willebrand Factor
(vWF; 1:200; Sigma), and CD34 (human CD34 Class III; 1:100;
DAKOCytomation, Carpinteria, Calif.). In addition, the following
markers were tested: anti-human GROalpha-PE (1:100; Becton
Dickinson, Franklin Lakes, N.J.), anti-human GCP-2 (1:100; Santa
Cruz Biotech, Santa Cruz, Calif.), anti-human oxidized LDL receptor
1 (ox-LDL R1; 1:100; Santa Cruz Biotech), and anti-human NOGO-A
(1:100; Santa Cruz Biotech). Fixed specimens were trimmed with a
scalpel and placed within OCT embedding compound (Tissue-Tek OCT;
Sakura, Torrance, Calif.) on a dry ice bath containing ethanol.
Frozen blocks were then sectioned (10 micron thick) using a
standard cryostat (Leica Microsystems) and mounted onto glass
slides for staining. TABLE-US-00018 TABLE 10-1 Summary of Primary
Antibodies Used Antibody Concentration Vendor Vimentin 1:500 Sigma,
St. Louis, MO Desmin (rb) 1:150 Sigma Desmin (m) 1:300 Chemicon,
Temecula, CA alpha-smooth muscle 1:400 Sigma actin (SMA)
Cytokeratin 18 (CK18) 1:400 Sigma von Willebrand factor 1:200 Sigma
(vWF) CD34 III 1:100 DakoCytomation, Carpinteria, CA GROalpha-PE
1:100 BD, Franklin Lakes, NJ GCP-2 1:100 Santa Cruz Biotech Ox-LDL
R1 1:100 Santa Cruz Biotech NOGO-A 1:100 Santa Cruz Biotech
[0463] Immunohistochemistry. Immunohistochemistry was performed
similar to previous studies (e.g., Messina, et al. (2003) Exper.
Neurol. 184: 816-829). Tissue sections were washed with
phosphate-buffered saline (PBS) and exposed to a protein blocking
solution containing PBS, 4% (v/v) goat serum (Chemicon, Temecula,
Calif.), and 0.3% (v/v) Triton (Triton X-100; Sigma) for 1 hour to
access intracellular antigens. In instances where the epitope of
interest would be located on the cell surface (CD34, ox-LDL R1),
triton was omitted in all steps of the procedure in order to
prevent epitope loss. Furthermore, in instances where the primary
antibody was raised against goat (GCP-2, ox-LDL R1, NOGO-A), 3%
(v/v) donkey serum was used in place of goat serum throughout the
procedure. Primary antibodies, diluted in blocking solution, were
then applied to the sections for a period of 4 hours at room
temperature. Primary antibody solutions were removed, and cultures
washed with PBS prior to application of secondary antibody
solutions (1 hour at room temperature) containing block along with
goat anti-mouse IgG--Texas Red (1:250; Molecular Probes, Eugene,
Oreg.) and/or goat anti-rabbit IgG--Alexa 488 (1:250; Molecular
Probes) or donkey anti-goat IgG--FITC (1:150; Santa Cruz Biotech).
Cultures were washed, and 10 micromolar DAPI (Molecular Probes) was
applied for 10 minutes to visualize cell nuclei.
[0464] Following immunostaining, fluorescence was visualized using
the appropriate fluorescence filter on an Olympus inverted
epi-fluorescent microscope (Olympus, Melville, N.Y.). Positive
staining was represented by fluorescence signal above control
staining. Representative images were captured using a digital color
videocamera and ImagePro software (Media Cybernetics, Carlsbad,
Calif.). For triple-stained samples, each image was taken using
only one emission filter at a time. Layered montages were then
prepared using Adobe Photoshop software (Adobe, San Jose,
Calif.).
[0465] Results
[0466] Umbilical Cord Characterization. Vimentin, desmin, SMA,
CK18, vWF, and CD34 markers were expressed in a subset of the cells
found within umbilical cord (data not shown). In particular, vWF
and CD34 expression were restricted to blood vessels contained
within the cord. CD34+ cells were on the innermost layer (lumen
side). Vimentin expression was found throughout the matrix and
blood vessels of the cord. SMA was limited to the matrix and outer
walls of the artery & vein, but not contained with the vessels
themselves. CK18 and desmin were observed within the vessels only,
desmin being restricted to the middle and outer layers.
[0467] Placenta Characterization. Vimentin, desmin, SMA, CK18, vWF,
and CD34 were all observed within the placenta and regionally
specific.
[0468] GROalpha, GCP-2, ox-LDL R1, and NOGO-A Tissue Expression.
None of these markers were observed within umbilical cord or
placental tissue (data not shown).
[0469] Summary. Vimentin, desmin, alpha-smooth muscle actin,
cytokeratin 18, von Willebrand Factor, and CD 34 are expressed in
cells within human umbilical cord and placenta. Based on in vitro
characterization studies showing that only vimentin and
alpha-smooth muscle actin are expressed, the data suggests that the
current process of postpartum-derived cell isolation harvests a
subpopulation of cells or that the cells isolated change expression
of markers to express vimentin and alpha-smooth muscle actin.
Example 11
In Vitro Immunology of Postpartum-Derived Cells
[0470] Postpartum-derived cell lines were evaluated in vitro for
their immunological characteristics in an effort to predict the
immunological response, if any, these cells would elicit upon in
vivo transplantation. Postpartum-derived cell lines were assayed by
flow cytometry for the expression of HLA-DR, HLA-DP, HLA-DQ, CD80,
CD86, and B7-H2. These proteins are expressed by antigen-presenting
cells (APC) and are required for the direct stimulation of naive
CD4.sup.+ T cells (Abbas & Lichtman, CELLULAR AND MOLECULAR
IMMUNOLOGY, 5th Ed. (2003) Saunders, Philadelphia, p. 171). The
cell lines were also analyzed by flow cytometry for the expression
of HLA-G (Abbas & Lichtman, CELLULAR AND MOLECULAR IMMUNOLOGY,
5th Ed. (2003) Saunders, Philadelphia, p. 171), CD 178 (Coumans,
et. al., (1999) Journal of Immunological Methods 224, 185-196), and
PD-L2 (Abbas & Lichtman, CELLULAR AND MOLECULAR IMMUNOLOGY, 5th
Ed. (2003) Saunders, Philadelphia, p. 171; Brown, et. al. (2003)
The Journal of Immunology 170, 1257-1266). The expression of these
proteins by cells residing in placental tissues is thought to
mediate the immuno-privileged status of placental tissues in utero.
To predict the extent to which postpartum placenta- and umbilical
cord-derived cell lines elicit an immune response in vivo, the cell
lines were tested in a one-way mixed lymphocyte reaction (MLR).
[0471] Materials and Methods
[0472] Cell culture. Cells were cultured in Growth medium (DMEM-low
glucose (Gibco, Carlsbad, Calif.), 15% (v/v) fetal bovine serum
(FBS); (Hyclone, Logan, Utah), 0.001% (v/v) betamercaptoethanol
(Sigma, St. Louis, Mo.), 50 Units/milliliter penicillin, 50
micrograms/milliliter streptomycin (Gibco, Carlsbad, Calif.)) until
confluent in T75 flasks (Corning, Corning, N.Y.) coated with 2%
gelatin (Sigma, St. Louis, Mo.).
[0473] Antibody Staining. Cells were washed in phosphate buffered
saline (PBS) (Gibco, Carlsbad, Calif.) and detached with
Trypsin/EDTA (Gibco, Carlsbad, Calif.). Cells were harvested,
centrifuged, and re-suspended in 3% (v/v) FBS in PBS at a cell
concentration of 1.times.10.sup.7 per milliliter. Antibody (Table
11-1) was added to one hundred microliters of cell suspension as
per manufacturer's specifications and incubated in the dark for 30
minutes at 4.degree. C. After incubation, cells were washed with
PBS and centrifuged to remove unbound antibody. Cells were
re-suspended in five hundred microliters of PBS and analyzed by
flow cytometry using a FACSCalibur instrument (Becton Dickinson,
San Jose, Calif.). TABLE-US-00019 TABLE 11-1 Antibodies Antibody
Manufacturer Catalog Number HLA-DRDPDQ BD Pharmingen (San Diego,
555558 CA) CD80 BD Pharmingen (San Diego, 557227 CA) CD86 BD
Pharmingen (San Diego, 555665 CA) B7-H2 BD Pharmingen (San Diego,
552502 CA) HLA-G Abcam (Cambridgeshire, UK) ab 7904-100 CD 178
Santa Cruz (San Cruz, CA) sc-19681 PD-L2 BD Pharmingen (San Diego,
557846 CA) Mouse IgG2a Sigma (St. Louis, MO) F-6522 Mouse IgG1kappa
Sigma (St. Louis, MO) P-4685
[0474] Mixed Lymphocyte Reaction. Cryopreserved vials of passage 10
umbilical cord-derived PPDCs labeled as cell line A and passage 11
placenta-derived PPDCs labeled as cell line B were sent on dry ice
to CTBR (Senneville, Quebec) to conduct a mixed lymphocyte reaction
using CTBR SOP no. CAC-031. Peripheral blood mononuclear cells
(PBMCs) were collected from multiple male and female volunteer
donors. Stimulator (donor) allogeneic PBMC, autologous PBMC, and
postpartum-derived cell lines were treated with mitomycin C.
Autologous and mitomycin C-treated stimulator cells were added to
responder (recipient) PBMCs and cultured for 4 days. After
incubation, [.sup.3H]thymidine was added to each sample and
cultured for 18 hours. Following harvest of the cells, radiolabeled
DNA was extracted, and [.sup.3H]-thymidine incorporation was
measured using a scintillation counter.
[0475] The stimulation index for the allogeneic donor (SIAD) was
calculated as the mean proliferation of the receiver plus mitomycin
C-treated allogeneic donor divided by the baseline proliferation of
the receiver. The stimulation index of the postpartum-derived cells
was calculated as the mean proliferation of the receiver plus
mitomycin C-treated postpartum-derived cell line divided by the
baseline proliferation of the receiver.
[0476] Results
[0477] Mixed Lymphocyte Reaction-Placenta. Seven human volunteer
blood donors were screened to identify a single allogeneic donor
that would exhibit a robust proliferation response in a mixed
lymphocyte reaction with the other six blood donors. This donor was
selected as the allogeneic positive control donor. The remaining
six blood donors were selected as recipients. The allogeneic
positive control donor and placenta-derived cell lines were treated
with mitomycin C and cultured in a mixed lymphocyte reaction with
the six individual allogeneic receivers. Reactions were performed
in triplicate using two cell culture plates with three receivers
per plate (Table 11-2). The average stimulation index ranged from
1.3 (plate 2) to 3 (plate 1) and the allogeneic donor positive
controls ranged from 46.25 (plate 2) to 279 (plate 1) (Table 11-3).
TABLE-US-00020 TABLE 11-2 Mixed Lymphocyte Reaction Data - Cell
Line B (Placenta) DPM for Proliferation Assay Analytical Culture
Replicates number System 1 2 3 Mean SD CV Plate ID: Plate 1
IM03-7769 Proliferation baseline of receiver 79 119 138 112.0 30.12
26.9 Control of autostimulation (Mitomycin C treated autologous
cells) 241 272 175 229.3 49.54 21.6 MLR allogenic donor IM03-7768
(Mitomycin C treated) 23971 22352 20921 22414.7 1525.97 6.8 MLR
with cell line (Mitomycin C treated cell type B) 664 559 1090 771.0
281.21 36.5 SI (donor) 200 SI (cell line) 7 IM03-7770 Proliferation
baseline of receiver 206 134 262 200.7 64.17 32.0 Control of
autostimulation (Mitomycin C treated autologous cells) 1091 602 524
739.0 307.33 41.6 MLR allogenic donor IM03-7768 (Mitomycin C
treated) 45005 43729 44071 44268.3 660.49 1.5 MLR with cell line
(Mitomycin C treated cell type B) 533 2582 2376 1830.3 1128.24 61.6
SI (donor) 221 SI (cell line) 9 IM03-7771 Proliferation baseline of
receiver 157 87 128 124.0 35.17 28.4 Control of autostimulation
(Mitomycin C treated autologous cells) 293 138 508 313.0 185.81
59.4 MLR allogenic donor IM03-7768 (Mitomycin C treated) 24497
34348 31388 30077.7 5054.53 16.8 MLR with cell line (Mitomycin C
treated cell type B) 601 643 a 622.0 29.70 4.8 SI (donor) 243 SI
(cell line) 5 IM03-7772 Proliferation baseline of receiver 56 98 51
68.3 25.81 37.8 Control of autostimulation (Mitomycin C treated
autologous cells) 133 120 213 155.3 50.36 32.4 MLR allogenic donor
IM03-7768 (Mitomycin C treated) 14222 20076 22168 18822.0 4118.75
21.9 MLR with cell line (Mitomycin C treated cell type B) a a a a a
a SI (donor) 275 SI (cell line) a IM03-7768 Proliferation baseline
of receiver 84 242 208 178.0 83.16 46.7 (allogenic Control of
autostimulation (Mitomycin treated autologous cells) 361 617 304
427.3 166.71 39.0 donor) Cell line Proliferation baseline of
receiver 126 124 143 131.0 10.44 8.0 type B Control of
autostimulation (Mitomycin treated autologous cells) 822 1075 487
794.7 294.95 37.1 Plate ID: Plate 2 IM03-7773 Proliferation
baseline of receiver 908 181 330 473.0 384.02 81.2 Control of
autostimulation (Mitomycin C treated autologous cells) 269 405 572
415.3 151.76 36.5 MLR allogenic donor IM03-7768 (Mitomycin C
treated) 29151 28691 28315 28719.0 418.70 1.5 MLR with cell line
(Mitomycin C treated cell type B) 567 732 905 734.7 169.02 23.0 SI
(donor) 61 SI (cell line) 2 IM03-7774 Proliferation baseline of
receiver 893 1376 185 818.0 599.03 73.2 Control of autostimulation
(Mitomycin C treated autologous cells) 261 381 568 403.3 154.71
38.4 MLR allogenic donor IM03-7768 (Mitomycin C treated) 53101
42839 48283 48074.3 5134.18 10.7 MLR with cell line (Mitomycin C
treated cell type B) 515 789 294 532.7 247.97 46.6 SI (donor) 59 SI
(cell line) 1 IM03-7775 Proliferation baseline of receiver 1272 300
544 705.3 505.69 71.7 Control of autostimulation (Mitomycin C
treated autologous cells) 232 199 484 305.0 155.89 51.1 MLR
allogenic donor IM03-7768 (Mitomycin C treated) 23554 10523 28965
21014.0 9479.74 45.1 MLR with cell line (Mitomycin C treated cell
type B) 768 924 563 751.7 181.05 24.1 SI (donor) 30 SI (cell line)
1 IM03-7776 Proliferation baseline of receiver 1530 137 1046 904.3
707.22 78.2 Control of autostimulation (Mitomycin C treated
autologous cells) 420 218 394 344.0 109.89 31.9 MLR allogenic donor
IM03-7768 (Mitomycin C treated) 28893 32493 34746 32044.0 2952.22
9.2 MLR with cell line (Mitomycin C treated cell type B) a a a a a
a SI (donor) 35 SI (cell line) a
[0478] TABLE-US-00021 TABLE 11-3 Average stimulation index of
placenta cells and an allogeneic donor in a mixed lymphocyte
reaction with six individual allogeneic receivers. Average
Stimulation Index Recipient Placenta Plate 1 (receivers 1-3) 279 3
Plate 2 (receivers 4-6) 46.25 1.3
[0479] Mixed Lymphocyte Reaction--Umbilical cord. Six human
volunteer blood donors were screened to identify a single
allogeneic donor that will exhibit a robust proliferation response
in a mixed lymphocyte reaction with the other five blood donors.
This donor was selected as the allogeneic positive control donor.
The remaining five blood donors were selected as recipients. The
allogeneic positive control donor and umbilical cord-derived cell
lines were mitomycin C-treated and cultured in a mixed lymphocyte
reaction with the five individual allogeneic receivers. Reactions
were performed in triplicate using two cell culture plates with
three receivers per plate (Table 11-4). The average stimulation
index ranged from 6.5 (plate 1) to 9 (plate 2) and the allogeneic
donor positive controls ranged from 42.75 (plate 1) to 70 (plate 2)
(Table 11-5). TABLE-US-00022 TABLE 11-4 Mixed Lymphocyte Reaction
Data- Cell Line A (Umbilical cord) DPM for Proliferation Assay
Analytical Culture Replicates number System 1 2 3 Mean SD CV Plate
ID: Plate1 IM04-2478 Proliferation baseline of receiver 1074 406
391 623.7 390.07 62.5 Control of autostimulation (Mitomycin C
treated autologous cells) 672 510 1402 861.3 475.19 55.2 MLR
allogenic donor IM04-2477 (Mitomycin C treated) 43777 48391 38231
43466.3 5087.12 11.7 MLR with cell line (Mitomycin C treated cell
type A) 2914 5622 6109 4881.7 1721.36 35.3 SI (donor) 70 SI (cell
line) 8 IM04-2479 Proliferation baseline of receiver 530 508 527
521.7 11.93 2.3 Control of autostimulation (Mitomycin C treated
autologous cells) 701 567 1111 793.0 283.43 35.7 MLR allogenic
donor IM04-2477 (Mitomycin C treated) 25593 24732 22707 24344.0
1481.61 6.1 MLR with cell line (Mitomycin C treated cell type A)
5086 3932 1497 3505.0 1832.21 52.3 SI (donor) 47 SI (cell line) 7
IM04-2480 Proliferation baseline of receiver 1192 854 1330 1125.3
244.90 21.8 Control of autostimulation (Mitomycin C treated
autologous cells) 2963 993 2197 2051.0 993.08 48.4 MLR allogenic
donor IM04-2477 (Mitomycin C treated) 25416 29721 23757 26298.0
3078.27 11.7 MLR with cell line (Mitomycin C treated cell type A)
2596 5076 3426 3699.3 1262.39 34.1 SI (donor) 23 SI (cell line) 3
IM04-2481 Proliferation baseline of receiver 695 451 555 567.0
122.44 21.6 Control of autostimulation (Mitomycin C treated
autologous cells) 738 1252 464 818.0 400.04 48.9 MLR allogenic
donor IM04-2477 (Mitomycin C treated) 13177 24885 15444 17835.3
6209.52 34.8 MLR with cell line (Mitomycin C treated cell type A)
4495 3671 4674 4280.0 534.95 12.5 SI (donor) 31 SI (cell line) 8
Plate ID: Plate 2 IM04-2482 Proliferation baseline of receiver 432
533 274 413.0 130.54 31.6 Control of autostimulation (Mitomycin C
treated autologous cells) 1459 633 598 896.7 487.31 54.3 MLR
allogenic donor IM04-2477 (Mitomycin C treated) 24286 30823 31346
28818.3 3933.82 13.7 MLR with cell line (Mitomycin C treated cell
type A) 2762 1502 6723 3662.3 2724.46 74.4 SI (donor) 70 SI (cell
line) 9 IM04-2477 Proliferation baseline of receiver 312 419 349
360.0 54.34 15.1 (allogenic Control of autostimulation (Mitomycin C
treated autologous cells) 567 604 374 515.0 123.50 24.0 donor) Cell
line Proliferation baseline of receiver 5101 3735 2973 3936.3
1078.19 27.4 type A Control of autostimulation (Mitomycin treated
autologous cells) 1924 4570 2153 2882.3 1466.04 50.9
[0480] TABLE-US-00023 TABLE 11-5 Average stimulation index of
umbilical cord-derived cells and an allogeneic donor in a mixed
lymphocyte reaction with five individual allogeneic receivers.
Average Stimulation Index Umbilical Recipient Cord Plate 1
(receivers 1-4) 42.75 6.5 Plate 2 (receiver 5) 70 9
[0481] Antigen Presenting Cell Markers--Placenta. Histograms of
placenta-derived cells analyzed by flow cytometry show negative
expression of HLA-DR, DP, DQ, CD80, CD86, and B7-H2, as noted by
fluorescence value consistent with the IgG control, indicating that
placenta-derived cell lines lack the cell surface molecules
required to directly stimulate allogeneic PBMCs (e.g., CD4.sup.+ T
cells).
[0482] Immuno-modulating Markers--Placenta-derived cells.
Histograms of placenta-derived cells analyzed by flow cytometry
show positive expression of PD-L2, as noted by the increased value
of fluorescence relative to the IgG control, and negative
expression of CD178 and HLA-G, as noted by fluorescence value
consistent with the IgG control (data not shown).
[0483] Antigen Presenting Cell Markers--Umbilical cord-derived
cells. Histograms of umbilical cord-derived cells analyzed by flow
cytometry show negative expression of HLA-DR, DP, DQ, CD80, CD86,
and B7-H2, as noted by fluorescence value consistent with the IgG
control, indicating that umbilical cord-derived cell lines lack the
cell surface molecules required to directly stimulate allogeneic
PBMCs (e.g., CD4.sup.+ T cells).
[0484] Immuno-modulating Markers--Umbilical cord-derived cells.
Histograms of umbilical cord-derived cells analyzed by flow
cytometry show positive expression of PD-L2, as noted by the
increased value of fluorescence relative to the IgG control, and
negative expression of CD178 and HLA-G, as noted by fluorescence
value consistent with the IgG control.
[0485] Summary. In the mixed lymphocyte reactions conducted with
placenta-derived cell lines, the average stimulation index ranged
from 1.3 to 3, and that of the allogeneic positive controls ranged
from 46.25 to 279. In the mixed lymphocyte reactions conducted with
umbilical cord-derived cell lines, the average stimulation index
ranged from 6.5 to 9, and that of the allogeneic positive controls
ranged from 42.75 to 70. Placenta- and umbilical cord-derived cell
lines were negative for the expression of the stimulating proteins
HLA-DR, HLA-DP, HLA-DQ, CD80, CD86, and B7-H2, as measured by flow
cytometry. Placenta- and umbilical cord-derived cell lines were
negative for the expression of immuno-modulating proteins HLA-G and
CD178 and positive for the expression of PD-L2, as measured by flow
cytometry. Allogeneic donor PBMCs contain antigen-presenting cells
expressing HLA-DP, DR, DQ, CD80, CD86, and B7-H2, thereby allowing
for the stimulation of allogeneic PBMCs (e.g., naive CD4.sup.+ T
cells). The absence of antigen-presenting cell surface molecules on
placenta- and umbilical cord-derived cells required for the direct
stimulation of allogeneic PBMCs (e.g., naive CD4.sup.+ T cells) and
the presence of PD-L2, an immuno-modulating protein, may account
for the low stimulation index exhibited by these cells in a MLR as
compared to allogeneic controls.
REFERENCES
[0486] Bruder S P et. al. U.S. Pat. No. 6,355,239 B1 (2002) [0487]
Abbas, A K, Lichtman, A H Cellular and Molecular Immunology 5th Ed.
(2003) Saunders, Philadelphia, p. 171 [0488] Bouteiller P. Le et.
al., (2003) Placenta 24; S10-S15 [0489] Coumans B et. al., (1999)
Journal of Immunological Methods 224, 185-196] [0490] Brown, Julia
et. al. (2003) The Journal of Immunology 170, 1257-1266
Example 12
Secretion of Trophic Factors by Postpartum-Derived Cells
[0491] The secretion of selected trophic factors from placenta- and
umbilical cord-derived PPDCs was measured. Factors were selected
that have angiogenic activity (i.e., hepatocyte growth factor (HGF)
(Rosen et al. (1997) Ciba Found. Symp. 212:215-26), monocyte
chemotactic protein 1 (MCP-1) (Salcedo et al. (2000) Blood 96;
34-40), interleukin-8 (IL-8) (Li et al. (2003) J. Immunol.
170:3369-76), keratinocyte growth factor (KGF), basic fibroblast
growth factor (bFGF), vascular endothelial growth factor (VEGF)
(Hughes et al. (2004) Ann. Thorac. Surg. 77:812-8), tissue
inhibitor of matrix metalloproteinase 1 (TIMP1), angiopoietin 2
(ANG2), platelet derived growth factor (PDGF-bb), thrombopoietin
(TPO), heparin-binding epidermal growth factor (HB-EGF),
stromal-derived factor 1a (SDF-1a)), neurotrophic/neuroprotective
activity (brain-derived neurotrophic factor (BDNF) (Cheng et al.
(2003) Dev. Biol. 258; 319-33), interleukin-6 (IL-6), granulocyte
chemotactic protein-2 (GCP-2), transforming growth factor beta2
(TGFbeta2)), or chemokine activity (macrophage inflammatory protein
1a (MIP1a), macrophage inflammatory protein 1beta (MIP1b), monocyte
chemoattractant-1 (MCP-1), Rantes (regulated on activation, normal
T cell expressed and secreted), I309, thymus and
activation-regulated chemokine (TARC), Eotaxin, macrophage-derived
chemokine (MDC), IL-8).
[0492] Methods & Materials
[0493] Cell culture. PPDCs derived from placenta and umbilical cord
as well as human fibroblasts derived from human neonatal foreskin
were cultured in Growth medium (DMEM-low glucose (Gibco, Carlsbad,
Calif.), 15% (v/v) fetal bovine serum (SH30070.03; Hyclone, Logan,
Utah), 50 Units/milliliter penicillin, 50 micrograms/milliliter
streptomycin (Gibco)) on gelatin-coated T75 flasks. Cells were
cryopreserved at passage 11 and stored in liquid nitrogen. After
thawing of the cells, Growth medium was added to the cells followed
by transfer to a 15 milliliter centrifuge tube and centrifugation
of the cells at 150.times.g for 5 minutes. The supernatant was
discarded. The cell pellet was resuspended in 4 milliliter Growth
medium, and cells were counted. Cells were seeded at 5,000
cells/cm.sup.2 on a T75 flask containing 15 milliliter of Growth
medium and cultured for 24 hours. The medium was changed to a
serum-free medium (DMEM-low glucose (Gibco), 0.1% (w/v) bovine
serum albumin (Sigma), 50 Units/milliliter penicillin, 50
micrograms/milliliter streptomycin (Gibco)) for 8 hours.
Conditioned serum-free media was collected at the end of incubation
by centrifugation at 14,000.times.g for 5 minutes and stored at
-0.degree. C. To estimate the number of cells in each flask, cells
were washed with phosphate-buffered saline (PBS) and detached using
2 milliliter trypsin/EDTA (Gibco). Trypsin activity was inhibited
by addition of 8 milliliter Growth medium. Cells were centrifuged
at 150.times.g for 5 minutes. Supernatant was removed, and cells
were resuspended in 1 milliliter Growth Medium. Cell number was
estimated using a hemocytometer.
[0494] ELISA assay. Cells were grown at 37.degree. C. in 5% carbon
dioxide and atmospheric oxygen. Placenta-derived PPDCs (101503)
also were grown in 5% oxygen or beta-mercaptoethanol (BME). The
amount of MCP-1, IL-6, VEGF, SDF-1a, GCP-2, IL-8, and TGF-beta2
produced by each cell sample was measured by an ELISA assay
(R&D Systems, Minneapolis, Minn.). All assays were performed
according to the manufacturer's instructions. Values presented are
picogram/milliliter/million cells (n=2, sem).
[0495] SearchLight Multiplexed ELISA assay. Chemokines (MIP1a,
MIP1b, MCP-1, Rantes, I309, TARC, Eotaxin, MDC, IL8), BDNF, and
angiogenic factors (HGF, KGF, bFGF, VEGF, TIMP1, ANG2, PDGF-bb,
TPO, HB-EGF.quadrature..quadrature. were measured using SearchLight
Proteome Arrays (Pierce Biotechnology Inc.). The Proteome Arrays
are multiplexed sandwich ELISAs for the quantitative measurement of
two to 16 proteins per well. The arrays are produced by spotting a
2.times.2, 3.times.3, or 4.times.4 pattern of four to 16 different
capture antibodies into each well of a 96-well plate. Following a
sandwich ELISA procedure, the entire plate is imaged to capture
chemiluminescent signal generated at each spot within each well of
the plate. The amount of signal generated in each spot is
proportional to the amount of target protein in the original
standard or sample.
[0496] Results
[0497] ELISA assay. MCP-1 and IL-6 were secreted by placenta- and
umbilical cord-derived PPDCs and dermal fibroblasts (Table 12-1).
Umbilical cord-derived cells secreted at least 10-fold higher
amounts of MCP-1 and IL6 than other cell populations. GCP-2 and
IL-8 were highly expressed by umbilical-derived PPDCs. TGF-beta2
was not detectable. VEGF was detected in fibroblast medium.
[0498] The amount of HGF, FGF, and BDNF secreted from umbilical
cord-derived cells were noticeably higher than fibroblasts and
placenta-derived cells (Tables 12-2 and 12-3). Similarly, TIMP1,
TPO, HBEGF, MCP-1, TARC, and IL-8 were higher in umbilical
cord-derived cells than other cell populations (Table 12-3). No
ANG2 or PDGF-bb were detected. TABLE-US-00024 TABLE 12-1 ELISA
assay results TGF- MCP-1 IL-6 VEGF SDF-1a GCP-2 IL-8 beta2
Fibroblast 17 .+-. 1 61 .+-. 3 29 .+-. 2 19 .+-. 1 21 .+-. 1 ND ND
Placenta 60 .+-. 3 41 .+-. 2 ND ND ND ND ND (042303) Umbilical 1150
.+-. 74 4234 .+-. 289 ND ND 160 .+-. 11 2058 .+-. 145 ND (022803)
Placenta 125 .+-. 16 10 .+-. 1 ND ND ND ND ND (071003) Umbilical
2794 .+-. 84 1356 .+-. 43 ND ND 2184 .+-. 98 2369 .+-. 23 ND
(071003) Placenta 21 .+-. 10 67 .+-. 3 ND ND 44 .+-. 9 17 .+-. 9 ND
(101503) BME Placenta 77 .+-. 16 339 .+-. 21 ND 1149 .+-. 137 54
.+-. 2 265 .+-. 10 ND (101503) 5% O.sub.2, W/O BME Key: ND: Not
Detected.
[0499] TABLE-US-00025 TABLE 12-2 SearchLight Multiplexed ELISA
assay results TIMP1 ANG2 PDGFbb TPO KGF HGF FGF VEGF HBEGF BDNF hFB
19306.3 ND ND 230.5 5.0 ND ND 27.9 1.3 ND P1 24299.5 ND ND 546.6
8.8 16.4 ND ND 3.81.3 ND U1 57718.4 ND ND 1240.0 5.8 559.3 148.7 ND
9.3 165.7 P3 14176.8 ND ND 568.7 5.2 10.2 ND ND 1.9 33.6 U3 21850.0
ND ND 1134.5 9.0 195.6 30.8 ND 5.4 388.6 Key: hFB (human
fibroblasts), P1 (placenta-derived PPDC (042303)), U1 (umbilical
cord-derived PPDC (022803)), P3 (placenta-derived PPDC (071003)),
U3 (umbilical cord-derived PPDC (071003)). ND: Not Detected.
[0500] TABLE-US-00026 TABLE 12-3 SearchLight Multiplexed ELISA
assay results MIP1a MIP1b MCP1 RANTES I309 TARC Eotaxin MDC IL8 hFB
ND ND 39.6 ND ND 0.1 ND ND 204.9 P1 79.5 ND 228.4 4.1 ND 3.8 12.2
ND 413.5 U1 ND 8.0 1694.2 ND 22.4 37.6 ND 18.9 51930.1 P3 ND ND
102.7 ND ND 0.4 ND ND 63.8 U3 ND 5.2 2018.7 41.5 11.6 21.4 ND 4.8
10515.9 Key: hFB (human fibroblasts), P1 (placenta-derived PPDC
(042303)), U1 (umbilical cord-derived PPDC (022803)), P3
(placenta-derived PPDC (071003)), U3 (umbilical cord-derived PPDC
(071003)). ND: Not Detected.
[0501] Summary. Umbilical cord-cells secreted significantly higher
amount of trophic factors than placenta-derived cells and
fibroblasts. Some of these trophic factors, such as HGF, bFGF,
MCP-1 and IL-8, play important roles in angiogenesis. Other trophic
factors, such as BDNF and IL-6, have important roles in neural
regeneration. Under these conditions, the expression of some
factors was confined to umbilical cord-derived cells, such as
MIP1b, Rantes, I309, and FGF.
REFERENCES
[0502] Le Belle J E, Svendsen C N. (2002) Stem cells for
neurodegenerative disorders: where can we go from here? BioDrugs.
16; 389-401 [0503] Rosen E M, Lamszus K, Laterra J, Polverini P J,
Rubin J S, Goldberg I D. (1997) HGF/SF in angiogenesis. Ciba Found
Symp. 212; 215-26. [0504] Salcedo R, Ponce M L, Young H A,
Wasserman K, Ward J M, Kleinman H K, Oppenheim J J, Murphy W J.
(2000) Human endothelial cells express CCR2 and respond to MCP-1:
direct role of MCP-1 in angiogenesis and tumor progression. Blood.
96; 34-40. [0505] Li A, Dubey S, Varney M L, Dave B J, Singh R K
(2003) IL-8 directly enhanced endothelial cell survival,
proliferation, and matrix metalloproteinases production and
regulated angiogenesis. J Immunol. 170; 3369-76 [0506] Hughes G C,
Biswas S S, Yin B, Coleman R E, DeGrado T R, Landolfo C K, Lowe J
E, Annex B H, Landolfo K P. (2004) Therapeutic angiogenesis in
chronically ischemic porcine myocardium: comparative effects of
bFGF and VEGF. Ann Thorac Surg. 77; 812-8. [0507] Cheng A, Wang S,
Cai J, Rao M S, Mattson M P (2003) Nitric oxide acts in a positive
feedback loop with BDNF to regulate neural progenitor cell
proliferation and differentiation in the mammalian brain. Dev Biol.
258; 319-33. [0508] Sebire G, Emilie D, Wallon C, Hery C, Devergne
O, Delfraissy J F, Galanaud P, Tardieu M. (1993) In vitro
production of IL-6, IL-1 beta, and tumor necrosis factor-alpha by
human embryonic microglial and neural cells. J Immunol. 150;
1517-23.
Example 13
Plasma Clotting Assay
[0509] Cell therapy may be injected systemically for certain
applications where cells are able to target the site of action. It
is important that injected cells not cause thrombosis, which may be
fatal. Tissue factor, a membrane-bound procoagulant glycoprotein,
is the initiator of the extrinsic clotting cascade, which is the
predominant coagulation pathway in vivo. Tissue factor also plays
an important role in embryonic vessel formation, for example, in
the formation of the primitive vascular wall (Brodsky et al. (2002)
Exp. Nephrol. 10:299-306). To determine the potential for PPDCs to
initiate clotting, umbilical cord- and placenta-derived PPDCs were
evaluated for tissue factor expression and their ability to
initiate plasma clotting.
[0510] Methods & Materials
[0511] Human Tissue factor. Human tissue factor SIMPLASTIN (Organon
Tekailca Corporation, Durham, N.C.), was reconstituted with 20
milliliter distilled water. The stock solution was serially diluted
(1:2) in eight tubes. Normal human plasma (George King Bio-Medical,
Overland Park, Kans.) was thawed at 37.degree. C. in a water bath
and then stored in ice before use. To each well of a 96-well plate
was added 100 microliter phosphate buffered saline (PBS), 10
microliter diluted Simplastin.RTM. (except a blank well), 30
microliter 0.1 molar calcium chloride, and 100 microliter of normal
human plasma. The plate was immediately placed in a
temperature-controlled microplate reader and absorbance measured at
405 nanometer at 40 second intervals for 30 minutes.
[0512] J-82 and postpartum-derived cells. J-82 cells (ATCC, MD)
were grown in Iscove's modified Dulbecco's medium (IMDM; Gibco,
Carlsbad, Calif.) containing 10% (v/v) fetal bovine serum (FBS;
Hyclone, Logan Utah), 1 millimolar sodium pyruvate (Sigma Chemical,
St. Louis, Mo.), 2 millimolar L-Glutamin (Mediatech Herndon, Va.),
1.times. non-essential amino acids (Mediatech Herndon, Va.). At 70%
confluence, cells were transferred to wells of 96-well plate at
100,000, 50,000, and 25,000 cells/well. Postpartum cells derived
from placenta and umbilical cord were cultured in Growth Medium
(DMEM-low glucose (Gibco), 15% (v/v) FBS, 50 Units/milliliter
penicillin, 50 micrograms/milliliter streptomycin (Gibco), and
0.001% betamercaptoethanol (Sigma)) in gelatin-coated T75 flasks
(Corning, Corning, N.Y.). Placenta-derived cells at passage 5 and
umbilical cord-derived cells at passages 5 and 11 were transferred
to wells at 50,000 cells/well. Culture medium was removed from each
well after centrifugation at 150.times.g for 5 minutes. Cells were
suspended in PBS without calcium and magnesium. Cells incubated
with anti-tissue factor antibody cells were incubated with 20
microgram/milliliter CNTO 859 (Centocor, Malvern, Pa.) for 30
minutes. Calcium chloride (30 microliter) was added to each well.
The plate was immediately placed in a temperature-controlled
microplate reader and absorbance measured at 405 nanometers at 40
second intervals for 30 minutes.
[0513] Antibody Staining. Cells were washed in PBS and detached
from the flask with Trypsin/EDTA (Gibco Carlsbad, Calif.). Cells
were harvested, centrifuged, and re-suspended 3% (v/v) FBS in PBS
at a cell concentration of 1.times.10.sup.7 per milliliter.
Antibody was added to 100 microliter cell suspension as per the
manufacturer's specifications, and the cells were incubated in the
dark for 30 minutes at 4.degree. C. After incubation, cells were
washed with PBS and centrifuged at 150.times.g for 5 minutes to
remove unbound antibody. Cells were re-suspended in 100 microliter
of 3% FBS and secondary antibody added as per the manufacturer's
instructions. Cells were incubated in the dark for 30 minutes at
4.degree. C. After incubation, cells were washed with PBS and
centrifuged to remove unbound secondary antibody. Washed cells were
re-suspended in 500 microliter of PBS and analyzed by flow
cytometry.
[0514] Flow Cytometry Analysis. Flow cytometry analysis was
performed with a FACSCalibur instrument (Becton Dickinson, San
Jose, Calif.).
[0515] Results
[0516] Flow cytometry analysis revealed that both placenta- and
umbilical cord-derived postpartum cells express tissue factor. A
plasma clotting assay demonstrated that tissue factor was active.
Both placenta- and umbilical cord-derived cells increased the
clotting rate as indicated by the time to half maximal absorbance
(T 1/2 to max; Table 13-1). Clotting was observed with both early
(P5) and late (P18) cells. The T 1/2 to max is inversely
proportional to the number of J82 cells. Preincubation of umbilical
cells with CNTO 859, an antibody to tissue factor, inhibited the
clotting reaction, thereby showing that tissue factor was
responsible for the clotting. TABLE-US-00027 TABLE 13-1 The effect
of human tissue factor (SIMPLASTIN), placenta-derived cells (Pla),
and umbilical cord-derived cells (Umb) on plasma clotting was
evaluated. The time to half maximal absorbance (T 1/2 to max) at
the plateau in seconds was used as a measurement unit. T 1/2 to max
(seconds) Simplastin .RTM. Dilution 1:2 61 1:4 107 1:8 147 1:16 174
1:32 266 1:64 317 1:128 378 0 (negative control) 1188 J-82 cells
100,000 122 50,000 172 25,000 275 Pla P5 50,000 757 Umb P5 50,000
833 Umb P18 50,000 443
[0517] Summary. Placenta- and umbilical cord-derived PPDCs express
tissue factor, which can induce clotting. The addition of an
antibody to tissue factor can inhibit tissue factor. Tissue factor
is normally found on cells in a conformation that is inactive but
is activated by mechanical or chemical (e.g., LPS) stress
(Sakariassen et al. (2001) Thromb. Res. 104:149-74; Engstad et al.
(2002) Int. Immunopharmacol. 2:1585-97). Thus, minimization of
stress during the preparation process of PPDCs may prevent
activation of tissue factor. In addition to the thrombogenic
activity, tissue factor has been associated with angiogenic
activity. Thus, tissue factor activity may be beneficial when
umbilical cord- or placenta-derived PPDCs are transplanted in
tissue but should be inhibited when PPDCs are injected
intravenously.
REFERENCES
[0518] Doshi and Marmur, Critical Care Med., 30:S241-S250 (2002)
[0519] Moll and Ortel, Ann. Intern. Med., 127:177-185 (1997)
Example 14
Differentiation of PPDCs to an Osteogenic Phenotype
[0520] Mesenchymal stem cells (MSCs) derived from bone marrow can
differentiate into osteoblast-like cells that mineralize and
express alkaline phosphatase. Additional markers expressed by
osteoblasts, such as osteocalcin and bone sialoprotein, have also
been used to demonstrate differentiation into an osteoblast-like
cell. A determination was made as to whether postpartum-derived
cells can also differentiate into an osteogenic phenotype by
culturing in an osteogenic medium and in the presence of bone
morphogenic proteins (BMP)-2 (Rickard et al., 1994) or -4, and
transforming growth factor beta1.
[0521] Methods & Materials
[0522] Culture of cells. Prior to initiation of osteogenesis,
Mesenchymal Stem Cells (MSC) were grown in Mesenchymal Stem Cell
Growth Medium Bullet kit (MSCGM, Cambrex, Walkerville, Md.). Other
cells were cultured in Growth medium (DMEM-low glucose (Gibco,
Carlsbad, Calif.), 15% (v/v) fetal bovine serum (SH30070.03;
Hyclone, Logan, Utah), 0.001% (v/v) betamercaptoethanol (Sigma, St.
Louis, Mo.), penicillin/streptomycin (Gibco)), in a gelatin-coated
T75 flask were washed with phosphate buffered saline (PBS).
[0523] Osteoblasts (9F1721; Cambrex) were grown in osteoblast
growth medium (Cambrex) and RNA was extracted as described
below.
[0524] Osteogenesis
[0525] Protocol 1. Placenta-derived cells, isolate 1, P3,
placenta-derived cells, isolate 2, P4 (previously karyotyped and
shown to be predominantly neonatal-derived cells), umbilical
cord-derived cells isolate 1, P4, and MSC at P3 were seeded at
5.times.10.sup.3 cells/cm.sup.2 in 24 well plates and 6-well dishes
in Growth medium and incubated overnight. The medium was removed
and replaced with Osteogenic medium (DMEM-low glucose, 10% (v/v)
fetal bovine serum, 10 millimolar betaglycerophosphate (Sigma), 100
nanomolar dexamethasone (Sigma, St. Louis, Mo.), 50 micromolar
ascorbate phosphate salt (Sigma), fungizone (Gibco), penicillin and
streptomycin (Gibco)). Osteogenic medium was supplemented with 20
nanograms/milliliter hTGF-beta1 (Sigma), 40 nanograms/milliliter
hrBMP-2 (Sigma), or 40 nanograms/milliliter hrBMP-4 (Sigma).
Cultures were treated for a total of 14, 21 and 28 days, with media
changes every 3-4 days.
[0526] Protocol 2. Postpartum-derived cells were tested for the
ability to differentiate into an osteogenic phenotype. Umbilical
cord-derived cells (isolate 1, P3 & isolate 2, P4) and
placenta-derived cells (isolate 1; P4 & isolate 2, P4) were
seeded at 30,000 cells/well in 6-well, gelatin-coated plates in
Growth medium. Mesenchymal stem cells (MSC) (isolate 1; P3 &
isolate 2; P4), fibroblasts (1F1853, P11), and ileac crest bone
marrow cells (070203; P3; WO2003025149) were also seeded at 30,000
cells/well in 6-well, gelatin-coated plates (gelatin-coated) in
mesenchymal stem cell growth medium (MSCGM, Cambrex) and Growth
medium, respectively.
[0527] Osteogenic induction was initiated by removing the initial
seeding media (24 h) and replacing it with osteogenic induction
medium (DMEM-low glucose, 10% fetal bovine serum, 10 millimolar
betaglycerophosphate (Sigma), 100 nanomolar dexamethasone (Sigma),
50 micromolar ascorbate phosphate salt (Sigma), penicillin and
streptomycin (Gibco)). In some cases, osteogenic medium was
supplemented with either hrBMP-2 (20 nanograms/milliliter) (Sigma),
hrBMP-4 (Sigma), or with both hrBMP-2 (20 nanograms/milliliter) and
hrBMP-4 (20 nanograms/milliliter) (Sigma). Cultures were treated
for a total of 28 days, with media changes every 3-4 days.
[0528] RNA extraction and Reverse Transcription. Cells were lysed
with 350 microliter buffer RLT containing beta-mercaptoethanol
(Sigma, St. Louis, Mo.) according to the manufacturer's
instructions (RNeasy Mini kit, Qiagen, Valencia, Calif.) and stored
at -80.degree. C. Cell lysates were thawed and RNA extracted
according to the manufacturer's instructions (RNeasy Mini kit,
Qiagen, Valencia, Calif.) with a 2.7 U/sample DNase treatment
(Sigma St. Louis, Mo.). RNA was eluted with 50 microliter
DEPC-treated water and stored at -80.degree. C. RNA was reverse
transcribed using random hexamers with the TaqMan reverse
transcription reagents (Applied Biosystems, Foster City, Calif.) at
25.degree. C. for 10 minutes, 37.degree. C. for 60 minutes and
95.degree. C. for 10 minutes. Samples were stored at -20.degree.
C.
[0529] Polymerase Chain Reaction. PCR was performed on cDNA samples
using Assays-on-Demand.TM. gene expression products bone
sialoprotein (Hs00173720), osteocalcin (Hs00609452) GAPDH (Applied
Biosystems, Foster City, Calif.), and TaqMan Universal PCR master
mix according to the manufacturer's instructions (Applied
Biosystems, Foster City, Calif.) using a 7000 sequence detection
system with ABI Prism 7000 SDS software (Applied Biosystems, Foster
City, Calif.). Thermal cycle conditions were initially 50.degree.
C. for 2 min and 95.degree. C. for 10 min followed by 40 cycles of
95.degree. C. for 15 sec and 60.degree. C. for 1 min.
[0530] von Kossa Staining. Cells were fixed with 10% (v/v) neutral
buffered formalin (Richard-Allan, Kalamazoo, Mich.). After
fixation, the cells were washed in deionized water and incubated in
5% (w/v) silver nitrate (Aldrich Chemical Company, Milwaukee, Wis.)
for one hour in direct sunlight. Cells were then washed in DI water
and incubated in 5% (w/v) sodium thiosulfate (EM Sciences,
Gibbstown, N.J.) for five minutes. Cells were washed in distilled
water and examined by light microscopy.
[0531] Results
[0532] Protocol 1. RNA extracted from osteoblasts was used as a
positive control for the real-time gene expression of osteocalcin
and bone sialoprotein. Osteoblast expression levels relative to
placenta-derived cells grown in growth medium of osteocalcin and
BSP was 2.5- and 8000-fold, respectively. MSCs grown in the
osteogenic medium for 28 days mineralized and were positive for von
Kossa staining. Extensive mineralization was observed in one
placenta isolate that had predominantly neonatal-derived cells.
Also, one placenta isolate show induction of BSP expression levels
in osteogenic media and low levels of osteocalcin induction.
[0533] MSC expression of osteocalcin and BSP was significantly
increased in osteogenic medium at 21 days. The addition of BMP-2
and -4 enhanced BSP expression but had no effect on osteocalcin
expression. TGF-beta1 did not augment the effect of osteogenesis
medium. BMP-4 and TGF-beta1 both increased osteocalcin expression
by a placenta isolate.
[0534] Protocol 2. Osteogenic differentiation, as shown by positive
von Kossa staining for mineralization, was observed with
placenta-derived cells P4 and ICBM (070203), P3 incubated with
osteogenic medium supplemented with BMP2 or 4, and MSCs (092903) P3
incubated with osteogenic medium supplemented with BMP 4 (Table
14-1). None of the other cells differentiated into the osteogenic
phenotype and stained by von Kossa. To ensure that von Kossa
staining was related to the cell and not to the extracellular
matrix, cells were counterstained with nuclear fast red. Large
lipid droplets were observed in some MSCs consistent with an
adipocyte phenotype. This suggests that MSCs do not differentiate
specifically into an osteogenic phenotype in these conditions.
Furthermore, adipogenesis increased when MSCs were incubated in
osteogenic medium supplemented with either BMP2 or BMP4.
TABLE-US-00028 TABLE 14-1 Results of osteogenic differentiation
using von Kossa staining for Protocol 2. Umbilical cord-derived
cells (Umb), placenta-derived cells (Pla), mesenchymal stem cells
(MSC), fibroblasts (Fib), and ileac crest bone marrow cells (ICBM)
cells were cultured in osteogenic medium (OM) alone or supplemented
with BMP2 or BMP2 and BMP4. Number Cell Line Conditions Von Kossa
Comments 1 Umb 071003 Osteogenic Neg O1P3 medium (OM) 2 Umb 071003
OM, BMP2 Neg O1P3 3 Umb 071003 OM, BMP4 Neg O1P3 4 ICBM 070203
Osteogenic Neg Normal O2 O1P3 medium (OM) 5 ICBM 070203 OM, BMP2
Pos Normal O3 O1P3 6 ICBM 070203 OM, BMP4 Pos Normal O4 O1P3 7 MSC
092903 Osteogenic Neg lots of fat medium (OM) 8 MSC 092903 OM, BMP2
Neg lots of fat 9 MSC 092903 OM, BMP4 Pos lots of fat 10 Pla 101603
O1P4 Osteogenic Neg medium (OM) 11 Pla 101603 O1P4 OM, BMP2 Pos 12
Pla 101603 O1P4 OM, BMP4 Pos 13 MSC 012104 Osteogenic Neg Fat O1P4
medium (OM) 14 MSC 012104 OM, BMP2 Neg Fat O1P4 15 MSC 012104 OM,
BMP2, Neg Fat O1P4 BMP4 16 Umb 022803 Osteogenic Neg O1P4 medium
(OM) 17 Umb 022803 OM, BMP2 Neg O1P4 18 Umb 022803 OM, BMP2, Neg
O1P4 BMP4 19 Pla 100703 O1P4 Osteogenic Neg medium (OM) 20 Pla
100703 O1P4 OM, BMP2 Neg 21 Pla 100703 O1P4 OM, BMP2, Neg BMP4 22
Fib 1F1853 Osteogenic Neg O1P11 medium (OM) 23 Fib 1F1853 OM, BMP2
Neg O1P11 24 Fib 1F1853 OM, BMP2, Neg O1P11 BMP4
[0535] Summary. Bone marrow-derived MSCs (Kadiyala et al., 1997) as
well as cells derived from other tissue such adipose (Halvorsen et
al., 2001) have been shown to differentiate into osteoblast-like
cells. MSCs have also been shown to differentiate into adipocytes
or osteoblasts in response to BMPs (Chen et al., 1998) due to
different roles for bone morphogenic protein (BMP) receptor type IB
and IA.
[0536] Neonatal-derived placenta-derived cells and MSCs showed
mineralization as well as induction of osteocalcin and bone
sialoprotein. Under the conditions used, umbilical-derived cells
did not show mineralization or induction of osteoblast genes.
Maternal placenta-derived cells may require addition of BMP-4 or
TGF to the osteogenic medium for mineralization to occur. The
gestational age of the sample may also be a factor in the ability
of cells derived from postpartum tissues to differentiate.
REFERENCES
[0537] Kadiyala S, Young R G, Thiede M A, Bruder S P. (1997)
Culture expanded canine mesenchymal stem cells possess
osteochondrogenic potential in vivo and in vitro. Cell Transplant.
6: 125-34. [0538] Chen D, Ji X, Harris M A, Feng J Q, Karsenty G,
Celeste A J, Rosen V, Mundy G R, Harris S E. (1998) Differential
roles for bone morphogenic protein (BMP) receptor type IB and IA in
differentiation and specification of mesenchymal precursor cells to
osteoblast and adipocyte lineages. J Cell Biol. 142:295-305 [0539]
Halvorsen Y D, Franklin D, Bond A L, Hitt D C, Auchter C, Boskey A
L, Paschalis E P, Wilkison W O, Gimble J M (2001) Extracellular
matrix mineralization and osteoblast gene expression by human
adipose tissue-derived stromal cells. Tissue Eng. 7:729-41. [0540]
Richard D J et al., (1994) Induction of rapid osteoblast
differentiation in rat bone marrow stromal cell cultures by
dexamethasone and BMP-2. Dev Biol 161,:218-228 [0541] WO2003025149
A2 HO, Tony, W.; KOPEN, Gene, C.; RIGHTER, William, F.; RUTKOWSKI,
J., Lynn; HERRING, W., Joseph; RAGAGLIA, Vanessa; WAGNER, Joseph
CELL POPULATIONS WHICH CO-EXPRESS CD49C AND CD90, NEURONYX, INC.
Application No. US0229971 US, Filed 20020920, A2 Published
20030327, A3 Published
Example 15
Chondrogenic Differentiation of Postpartum-Derived Cells
[0542] Cartilage damage and defects lead to approximately 600,000
surgical procedures each year in the United States alone (1). A
number of strategies have been developed to treat these conditions
but these have had limited success. One approach, Cartecel
(Genzyme), uses autologous chondrocytes that are collected from a
patient and expanded in vitro and then implanted into the patient
(1). This approach has the disadvantage of collecting healthy
cartilage and requiring a second procedure to implant the cultured
cells. One novel possibility is a stem cell-based therapy in which
cells are placed at or near the defect site to directly replace the
damaged tissue. Cells may be differentiated into chondrocytes prior
to the application or progenitor cells that can differentiate in
situ may be used. Such transplanted cells would replace the
chondrocytes lost in the defect.
[0543] Candidate cells for this indication should be evaluated for
their ability to differentiate into chondrocytes in vitro. A number
of protocols have been developed for testing the ability of cells
to differentiate and express chondrocyte marker genes.
Postpartum-derived cells were tested for their ability to
differentiate into chondrocytes in vitro in two different assay
systems: the pellet assay culture system and collagen gel cultures.
The pellet culture system has been used successfully with selected
lots of human mesenchymal stem cells (MSC). MSCs grown in this
assay and treated with transforming growth factor-beta3 have been
shown to differentiate into chondrocytes (2). The collagen gel
system has been used to culture chondrocytes in vitro (3).
Chondrocytes grown under these conditions form a cartilage-like
structure.
[0544] Materials and Methods
[0545] Cell Culture
[0546] Postpartum tissue-derived cells. Human umbilical cords and
placenta were received and cells were isolated as described above.
Cells were cultured in Growth medium (Dulbecco's Modified Essential
Media (DMEM) with 15% (v/v) fetal bovine serum (Hyclone, Logan
Utah), penicillin/streptomycin (Invitrogen, Carlsbad, Calif.), and
0.001% (v/v) 2-mercaptoethanol (Sigma, St. Louis, Mo.)) on
gelatin-coated tissue culture plastic flasks. The cultures were
incubated at 37.degree. C. with 5% CO.sub.2. For use in
experiments, cells were between passages 4 and 12.
[0547] Human articular chondrocytes. Human articular chondrocytes
were purchased from Cambrex (Walkersville, Md.) and cultured in the
same media as the postpartum-derived cells. Twenty-four hours
before the experiment, the culture media was changed to a media
containing 1% FBS.
[0548] Human mesenchymal stem cells (hMSC). MSCs were purchased
from Cambrex (Walkersville, Md.) and cultured in MSCGM (Cambrex).
Cells used for experiments were between passages 2 and 4.
[0549] Collagen gel assays. Cultured cells were trypsinized to
remove from culture plate. Cells were washed with centrifugation
twice at 300.times.g for 5 min in DMEM without serum and counted.
Cells were mixed with the following components at the final
concentrations listed. Rat tail collagen (1 milligram/milliliter,
BD DiscoveryLabware, Bedford, Mass.), 0.01 N NaOH and Chondrogenic
medium (DMEM, 100 U/100 microgram Penicillin/Streptomycin, 2
millimolar L-Glutamine, 1 millimolar Sodium Pyruvate, 0.35
millimolar L-Proline, 100 nanomolar dexamethasone, 0.17 millimolar
L-Ascorbic Acid, 1% (v/v) ITS (insulin, transferrin, selenium) (All
components from Sigma Chemical Company)). The cells were gently
mixed with the medium the samples were aliquoted into individual
wells of a 24 well ultra-low cluster plate (Corning, Corning, N.Y.)
at a concentration of either 2.times.10.sup.5 per well or
5.times.10.sup.5 per well. Cultures were placed in an incubator and
left undisturbed for 24-48 hours. Medium was replaced with fresh
chondrogenic medium supplemented with appropriate growth factor
every 24-48 hours. Samples were allowed to culture for up to 28
days at which time they were removed and fixed in 10% (v/v)
formalin (VWR Scientific, West Chester, Pa.) and processed for
histological examination. Samples were stained with Safranin O or
hematoxylin/eosin for evaluation.
[0550] Pellet culture assays. Cultured cells were trypsinized to
remove from culture plate. Cells were washed with centrifugation
twice at 300.times.g for 5 minutes in DMEM without serum and
counted. Cells were resuspended in fresh chondrogenic medium
(described above) at a concentration of 5.times.10.sup.5 cells per
milliliter. Cells were aliquoted into new polypropylene tubes at
2.5.times.10.sup.5 cells per tube. The appropriate samples were
then treated with TGF-beta3 (10 nanograms/milliliter, Sigma) or
GDF-5 (100 nanograms/milliliter; R&D Systems, Minneapolis,
Minn.). Cells were then centrifuged at 150.times.g for 3 minutes.
Tubes were then transferred to the incubator at and left
undisturbed for 24-48 hours at 37.degree. C. and 5% CO.sub.2. Media
was replaced with fresh chondrocyte cell media and growth factor,
where appropriate, every 2-3 days. Samples were allowed to culture
for up to 28 days at which time they were removed and fixed and
stained as described above.
[0551] Results
[0552] Pellets were prepared and cultured and described in Methods.
Pellets were grown in media (Control) or supplemented with
TGF-beta3 (10 nanograms/milliliter) or GDF-5 (100
nanograms/milliliter) that was replaced every 2-3 days. Pellets
collected after 21 days of culture and stained by Safranin O to
test for the presence of glycosoaminoglycans. The pellets treated
with TGFbeta3 and GDF-5 showed some positive Safranin O staining as
compared to control cells. The morphology of the umbilical cord
cells showed some limited chondrocyte-like morphology.
[0553] Safranin O stains of cell pellets from placenta cells showed
similar glycosoaminoglycan expression as compared to the umbilical
cord cells. The morphology of the cells also showed some limited
chondrocyte-like morphology.
[0554] Summary. The results of the present study show that the
postpartum-derived cells partially differentiated into chondrocytes
in vitro in the pellet culture and the collagen gel assay systems.
The postpartum-derived cells showed some indications of
glycosaminoglycan expression by the cells. Morphology showed
limited similarity to cartilage tissue.
REFERENCES
[0555] 1. U.S. Markets for Current and Emerging Orthopedic
Biomaterials Products and Technologies. Medtech Insight L.L.C. 2002
[0556] 2. Johnstone, B, T. M. Hering, A. I. Caplan, V. M. Goldberg
and J. U. Yoo. In Vitro Chondrogenesis of Bone-Marrow-Derived
Mesenchymal Stem Cells. 1998. Exp Cell Res 238:265-272. [0557] 3.
Gosiewska, A., A. Rezania, S. Dhanaraj, M. Vyakarnam, J. Zhou, D.
Burtis, L. Brown, W. Kong, M. Zimmerman and J. Geesin. Development
of a Three-Dimensional Transmigration Assay for Testing
Cell-Polymer Interactions for Tissue Engineering Applications. 2001
Tissue Eng. 7:267-277.
Example 16
Evaluation of Chondrogenic Potential of Cells Derived from
Postpartum Tissue in an In Vitro Pellet Culture Based Assay
[0558] This example describes evaluation of the chondrogenic
potential of cells derived from placental or umbilical tissue using
in vitro pellet culture based assays. Cells from umbilical cord and
placenta at early passage (P3) and late passage (P12) were used.
The chondrogenic potential of the cells was assessed in pellet
culture assays, under chondrogenic induction conditions, in medium
supplemented with transforming growth factor beta-3 (TGFbeta-3),
GDF-5 (recombinant human growth and differentiation factor 5), or a
combination of both.
[0559] Materials & Methods
[0560] Reagents. Dulbecco's Modified Essential Media (DMEM),
Penicillin and Streptomycin, were obtained from Invitrogen,
Carlsbad, Calif. Fetal calf serum (FCS) was obtained from HyClone
(Logan, Utah). Mesenchymal stem cell growth medium (MSCGM) and hMSC
chondrogenic differentiation bullet kit were obtained from
Biowhittaker, Walkersville, Md. TGFbeta-3 was obtained from
Oncogene research products, San Diego, Calif. GDF-5 was obtained
from Biopharm, Heidelberg, Germany (WO9601316 A1, U.S. Pat. No.
5,994,094 A).
[0561] Cells. Human mesenchymal stem cells (Lot# 2F1656) were
obtained from Biowhittaker, Walkersville, Md. and were cultured in
MSCGM according to manufacturer's instructions. This lot has been
tested previously, and was shown to be positive in the
chondrogenesis assays. Human adult and neonatal fibroblasts were
obtained from American Type Culture Collection (ATCC), Manassas,
Va. and cultured in growth medium (Dulbecco's Modified Essential
supplemented with 15% (v/v) fetal bovine serum,
penicillin/streptomycin (100 U/100 milligram, respectively) and
0.001% (v/v) 2-mercaptoethanol (Sigma, St. Louis, Mo.) on
gelatin-coated tissue culture plastic flasks. Postpartum
tissue-derived cells, isolated from human umbilical cords (Lot#
022703Umb) and placenta (Lot# 071003Plac) as described in previous
examples, were utilized. Cells were cultured in Growth medium
similar to fibroblasts. The cell cultures were incubated at
37.degree. C. with 5% CO.sub.2. Cells used for experiments were at
passages 3 and 12.
[0562] Pellet culture assay. For pellet cultures,
0.25.times.10.sup.6 cells were placed in a 15 milliliter conical
tube and centrifuged at 150.times.g for 5 minutes at room
temperature to form a spherical pellet according to protocol for
chondrogenic assay from Biowhittaker. Pellets were cultured in
chondrogenic induction medium containing TGFbeta-3 (10
nanograms/milliliter), GDF-5 (500 nanograms/milliliter), or a
combination of TGFbeta-3 (10 nanograms/milliliter), and GDF-5 (500
nanograms/milliliter) for three weeks. Untreated controls were
cultured in growth medium. During culture, pellets were re-fed with
fresh medium every other day. Treatment groups included the
following:
[0563] Treatment Group
[0564] A. Placenta-derived cells early passage (P EP)+GDF-5
[0565] B. Placenta-derived cells late passage (P LP)+GDF-5
[0566] C. Umbilical cord derived cells early passage (U
EP)+GDF-5
[0567] D. Umbilical cord derived cells late passage (U LP)+GDF-5,
n=2
[0568] E. Human Mesenchymal Stem cells (HMSC)+GDF-5
[0569] F. Human adult fibroblast cells (HAF)+GDF-5
[0570] G. Placenta-derived cells early passage (P EP)+TGFbeta-3
[0571] H. Placenta-derived cells late passage (P LP)+TGFbeta-3
[0572] I. Umbilical cord derived cells early passage (U
EP)+TGFbeta-3
[0573] J. Umbilical cord derived cells late passage (U
LP)+TGFbeta-3, n=2
[0574] K. Human Mesenchymal Stem cells (HMSC)+TGFbeta-3
[0575] L. Human adult fibroblast cells (HAF)+TGFbeta-3
[0576] M. Placenta-derived cells early passage (P
EP)+GDF-5+TGFbeta-3, n=1
[0577] N. Placenta-derived cells late passage (P
LP)+GDF-5+TGFbeta-3
[0578] O. Umbilical cord derived cells early passage (U
EP)+GDF-5+TGFbeta-3
[0579] P. Umbilical cord derived cells late passage (U
LP)+GDF-5+TGFbeta-3, n=2
[0580] Q. Human Mesenchymal Stem cells (HMSC)+GDF-5+TGFbeta-3
[0581] R. Human adult fibroblast cells (HAF)+GDF-5+TGFbeta-3
[0582] S. Human neonatal fibroblast cells (HNF)+GDF-5+TGFbeta-3
[0583] T. Placenta-derived cells early passage (P EP)
[0584] U. Placenta-derived cells late passage (P LP)
[0585] V. Umbilical cord derived cells early passage (U EP)
[0586] W. Umbilical cord derived cells late passage (U LP)
[0587] X. Human Mesenchymal Stem cells (HMSC)
[0588] Y. Human adult fibroblast cells (HAF)
[0589] Histology of in vitro samples. At the end of the culture
period pellets were fixed in 10% buffered formalin and sent to MPI
Research (Mattawan, Mich.) for paraffin embedding, sectioning, and
staining with Hematoxylin/Eosin (H/E) and Safranin O (SO)
staining.
Results
[0590] Placenta- and umbilical cord-derived cells, MSCs. and
fibroblasts formed cell pellets in chondrogenic induction medium
with the different growth factors. The size of the pellets at the
end of culture period varied among the different cell types.
Pellets formed with placenta-derived cells were similar in size, or
slightly larger than, those formed by MSCs and fibroblasts. Pellets
formed with the umbilical cord-derived cells tended to be larger
and looser than the other groups. Pellets formed with all cell
types and cultured in control medium were smaller than pellets
cultured in chondrogenic induction medium.
[0591] Examination of cross sections of pellets stained with
hematoxylin/eosin and Safranin-O showed that umbilical cord-derived
cells at early passage had the potential to undergo chondrogenic
differentiation. Chondrogenesis as assessed by cell condensation,
cell morphology and Safranin O positive staining of matrix was
observed in the umbilical cell pellets cultured in chondrogenic
induction medium supplemented with TGFbeta-3, GDF-5, or both.
Chondrogenesis in pellets was similar for TGFbeta-3, GDF-5, and the
combined treatments. Control pellets cultured in growth medium
showed no evidence of chondrogenesis. Chondrogenic potential of the
umbilical cord derived cells was marginally lower than that
observed with the MSCs obtained from Biowhittaker.
[0592] Umbilical cord derived cells at late passage and
placenta-derived cells did not demonstrate as distinct a
chondrogenic potential as did early passage umbilical cord-derived
cells. However, this may be due to the fact that chondrogenic
induction conditions were optimized for MSCs, not for
postpartum-derived cells. Nonetheless, distinct cell populations
were observed in placenta-derived cells at both passages located
apically or centrally. Some cell condensation was observed with
fibroblast, but it was not associated with Safranin O staining.
Example 17
Endothelial Network Formation Assay
[0593] Angiogenesis, or the formation of new vasculature, is
necessary for the growth of new tissue. Induction of angiogenesis
is an important therapeutic goal in many pathological conditions.
The present study was aimed at identifying potential angiogenic
activity of the postpartum-derived cells in in vitro assays. The
study followed a well-established method of seeding endothelial
cells onto a culture plate coated with MATRIGEL (BD Discovery
Labware, Bedford, Mass.), a basement membrane extract (Nicosia and
Ottinetti (1990) In Vitro Cell Dev. Biol. 26(2):119-28). Treating
endothelial cells on MATRIGEL (BD Discovery Labware, Bedford,
Mass.) with angiogenic factors will stimulate the cells to form a
network that is similar to capillaries. This is a common in vitro
assay for testing stimulators and inhibitors of blood vessel
formation (Ito et al. (1996) Int. J. Cancer 67(1):148-52). The
present studies made use of a co-culture system with the
postpartum-derived cells seeded onto culture well inserts. These
permeable inserts allow for the passive exchange of media
components between the endothelial and the postpartum-derived cell
culture media.
[0594] Material & Methods
[0595] Cell Culture.
[0596] Postpartum tissue-derived cells. Human umbilical cords and
placenta were received and cells were isolated as previously
described (Example 1). Cells were cultured in Growth medium
(Dulbecco's Modified Essential Media (DMEM; Invitrogen, Carlsbad,
Calif.), 15% (v/v) fetal bovine serum (Hyclone, Logan Utah), 100
Units/milliliter penicillin, 100 microgram/milliliter streptomycin
(Invitrogen), 0.001% (v/v) 2-mercaptoethanol (Sigma, St. Louis,
Mo.)) on gelatin-coated tissue culture plastic flasks. The cultures
were incubated at 37.degree. C. with 5% CO.sub.2. Cells used for
experiments were between passages 4 and 12.
[0597] Actively growing postpartum cells were trypsinized, counted,
and seeded onto COSTAR TRANSWELL 6.5 millimeter diameter tissue
culture inserts (Corning, Corning, N.Y.) at 15,000 cells per
insert. Cells were cultured on the inserts for 48-72 hours in
Growth medium at 37.degree. C. under standard growth
conditions.
[0598] Human mesenchymal stem cells (hMSC). hMSCs were purchased
from Cambrex (Walkersville, Md.) and cultured in MSCGM (Cambrex).
The cultures were incubated under standard growth conditions.
[0599] Actively growing MSCs were trypsinized and counted and
seeded onto COSTAR TRANSWELL 6.5 millimeter diameter tissue culture
inserts (Corning, Corning, N.Y.) at 15,000 cells per insert. Cells
were cultured on the inserts for 48-72 hours in Growth medium under
standard growth conditions.
[0600] Human umbilical vein endothelial cells (HUVEC). HUVEC were
obtained from Cambrex (Walkersville, Md.). Cells were grown in
separate cultures in either EBM or EGM endothelial cell media
(Cambrex). Cells were grown on standard tissue cultured plastic
under standard growth conditions. Cells used in the assay were
between passages 4 and 10.
[0601] Human coronary artery endothelial cells (HCAEC). HCAEC were
purchased from Cambrex Incorporated (Walkersville, Md.). These
cells were also maintained in separate cultures in either the EBM
or EGM media formulations. Cells were grown on standard tissue
cultured plastic under standard growth conditions. Cells used for
experiments were between passages 4 and 8.
[0602] Endothelial Network Formation (MATRIGEL) assays. Culture
plates were coated with MATRIGEL (BD Discovery Labware, Bedford,
Mass.) according to manufacturer's specifications. Briefly,
MATRIGEL.TM. (BD Discovery Labware, Bedford, Mass.) was thawed at
4.degree. C. and approximately 250 microliter was aliquoted and
distributed evenly onto each well of a chilled 24-well culture
plate (Corning). The plate was then incubated at 37.degree. C. for
30 minutes to allow the material to solidify. Actively growing
endothelial cell cultures were trypsinized and counted. Cells were
washed twice in Growth medium with 2% FBS by centrifugation,
resuspension, and aspiration of the supernatant. Cells were seeded
onto the coated wells 20,000 cells per well in approximately 0.5
milliliter Growth medium with 2% (v/v) FBS. Cells were then
incubated for approximately 30 minutes to allow cells to
settle.
[0603] Endothelial cell cultures were then treated with either 10
nanomolar human bFGF (Peprotech, Rocky Hill, N.J.) or 10 nanomolar
human VEGF (Peprotech, Rocky Hill, N.J.) to serve as a positive
control for endothelial cell response. Transwell inserts seeded
with postpartum-derived cells were added to appropriate wells with
Growth medium with 2% FBS in the insert chamber. Cultures were
incubated at 37.degree. C. with 5% CO.sub.2 for approximately 24
hours. The well plate was removed from the incubator, and images of
the endothelial cell cultures were collected with an Olympus
inverted microscope (Olympus, Melville, N.Y.).
[0604] Results
[0605] In a co-culture system with placenta-derived cells or with
umbilical cord-derived cells, HUVEC form cell networks (data not
shown). HUVEC cells form limited cell networks in co-culture
experiments with hMSC and with 10 nanomolar bFGF (data not shown).
HUVEC cells without any treatment showed very little or no network
formation (data not shown). These results suggest that the
postpartum-derived cells release angiogenic factors that stimulate
the HUVEC.
[0606] In a co-culture system with placenta-derived cells or with
umbilical cord-derived cells, CAECs form cell networks (data not
shown).
[0607] Table 17-1 shows levels of known angiogenic factors released
by the postpartum-derived cells in Growth medium.
Postpartum-derived cells were seeded onto inserts as described
above. The cells were cultured at 37.degree. C. in atmospheric
oxygen for 48 hours on the inserts and then switched to a 2% FBS
media and returned at 37.degree. C. for 24 hours. Media was
removed, immediately frozen and stored at -80.degree. C., and
analyzed by the SearchLight multiplex ELISA assay (Pierce Chemical
Company, Rockford, Ill.). Results shown are the averages of
duplicate measurements. The results show that the
postpartum-derived cells do not release detectable levels of
platelet-derived growth factor-bb (PDGF-bb) or heparin-binding
epidermal growth factor (HBEGF). The cells do release measurable
quantities of tissue inhibitor of metallinoprotease-1 (TIMP-1),
angiopoietin 2 (ANG2), thrombopoietin (TPO), keratinocyte growth
factor (KGF), hepatocyte growth factor (HGF), fibroblast growth
factor (FGF), and vascular endothelial growth factor (VEGF).
TABLE-US-00029 TABLE 17-1 Potential angiogenic factors released
from postpartum-derived cells. Postpartum-derived cells were
cultured in 24 hours in media with 2% FBS in atmospheric oxygen.
Media was removed and assayed by the SearchLight multiplex ELISA
assay (Pierce). Results are the means of a duplicate analysis.
Values are concentrations in the media reported in picograms per
milliliter of culture media. TIMP1 ANG2 PDGFBB TPO KGF HGF FGF VEGF
HBEGF (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml)
(pg/ml) (pg/ml) Plac 91655.3 175.5 <2.0 275.5 3.0 58.3 7.5 644.6
<1.2 (P4) Plac 1592832.4 28.1 <2.0 1273.1 193.3 5960.3 34.8
12361.1 1.7 (P11) Umb 81831.7 <9.8 <2.0 365.9 14.1 200.2 5.8
<4.0 <1.2 cord (P4) Media <9.8 25.1 <2.0 <6.4
<2.0 <3.2 <5.4 <4.0 <1.2 alone Plac: placenta
derived cells; Umb cord: Umbilical cord derived cells
[0608] Table 17-2 shows levels of known angiogenic factors released
by the postpartum-derived cells. Postpartum-derived cells were
seeded onto inserts as described above. The cells were cultured in
Growth medium at 5% oxygen for 48 hours on the inserts and then
switched to a 2% FBS medium and returned to 5% O.sub.2 incubation
for 24 hours. Media was removed, immediately frozen, and stored at
-80.degree. C., and analyzed by the SearchLight multiplex ELISA
assay (Pierce Chemical Company, Rockford, Ill.). Results shown are
the averages of duplicate measurements. The results show that the
postpartum-derived cells do not release detectable levels of
platelet-derived growth factor-bb (PDGF-BB) or heparin-binding
epidermal growth factor (HBEGF). The cells do release measurable
quantities of tissue inhibitor of metallinoprotease-1 (TIMP-1),
angiopoietin 2 (ANG2), thrombopoietin (TPO), keratinocyte growth
factor (KGF), hepatocyte growth factor (HGF), fibroblast growth
factor (FGF), and vascular endothelial growth factor (VEGF).
TABLE-US-00030 TABLE 17-2 Potential angiogenic factors released
from postpartum-derived cells. Postpartum-derived cells were
cultured in 24 hours in media with 2% FBS in 5% oxygen. Media was
removed and assayed by the SearchLight multiplex ELISA assay
(Pierce). Results are the means of a duplicate analysis. Values are
concentrations in the media reported in picograms per milliter of
culture media. PDGF- TIMP1 ANG2 BB TPO KGF HGF FGF VEGF HBEGF
(pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml)
(pg/ml) Plac 72972.5 253.6 <2.0 743.1 2.5 30.2 15.1 1495.1
<1.2 (P4) Plac 458023.1 55.1 <2.0 2562.2 114.2 2138.0 295.1
7521.3 1.8 (P11) Umb 50244.7 <9.8 <2.0 403.3 10.7 156.8 5.7
<4.0 <1.2 cord (P4) Media <9.8 25.1 <2.0 <6.4
<2.0 <3.2 <5.4 <4.0 <1.2 alone Plac: placenta
derived cells; Umb cord: Umbilical cord derived cells
[0609] Summary. The results of the present study show that
postpartum-derived cells can stimulate both human umbilical vein
and coronary artery endothelial cells to form networks in an in
vitro MATRIGEL.TM. (BD Discovery Labware, Bedford, Mass.) assay.
This effect is similar to that seen with known angiogenic factors
in this assay system. These results suggest that the
postpartum-derived cells are useful for stimulating angiogenesis in
vivo.
Example 18
Transplantion of PPDCs
[0610] Cells derived from the postpartum umbilical cord and
placenta are useful for regenerative therapies. The tissue produced
by postpartum-derived cells transplanted into SCID mice with a
biodegradable material was evaluated. The materials evaluated were
VICRYL non-woven, 35/65 PCL/PGA foam, and RAD 16 self-assembling
peptide hydrogel.
[0611] Methods & Materials
[0612] Cell Culture. Placenta-derived cells and umbilical cord
derived cells were grown in Growth medium (DMEM-low glucose (Gibco,
Carlsbad Calif.), 15% (v/v) fetal bovine serum (Cat. #SH30070.03;
Hyclone, Logan, Utah), 0.001% (v/v) betamercaptoethanol (Sigma, St.
Louis, Mo.), 50 Units/milliliter penicillin, 50
microgram/milliliter streptomycin (Gibco)) in a gelatin-coated
flasks.
[0613] Matrix Preparation. A nonwoven scaffold was prepared using a
traditional needle punching technique as described below. Fibers,
comprised of a synthetic absorbable copolymer of glycolic and
lactic acids (PGA/PLA), sold under the tradename VICRYL were
obtained from Ethicon, Inc. (Somerville, N.J.). The fibers were
filaments of approximately 20 microns in diameter. The fibers were
then cut and crimped into uniform 2-inch lengths to form 2-inch
staple fiber. A dry lay needle-punched nonwoven matrix was then
prepared utilizing the VICRYL staple fibers. The staple fibers were
opened and carded on standard nonwoven machinery. The resulting mat
was in the form of webbed staple fibers. The webbed staple fibers
were needle-punched to form the dry lay needle-punched nonwoven
scaffold. The nonwoven scaffold was rinsed in water followed by
another incubation in ethanol to remove any residual chemicals or
processing aids used during the manufacturing process.
[0614] Foams, composed of 35/65
poly(epsilon-caprolactone)/poly(glycolic acid) (35/65 PCL/PGA)
copolymer, werer formed by the process of lyophilized, as discussed
in U.S. Pat. No. 6,355,699.
[0615] Sample Preparation. One million viable cells were seeded in
15 microliter Growth medium onto 5 millimeter diameter, 2.25
millimeter thick VICRYL non-woven scaffolds (64.33 milligram/cubic
centimeters; Lot#3547-47-1) or 5 millimeter diameter 35/65 PCL/PGA
foam (Lot# 3415-53). Cells were allowed to attach for two hours
before adding more Growth medium to cover the scaffolds. Cells were
grown on scaffolds overnight. Scaffolds without cells were also
incubated in medium.
[0616] RAD16 self-assembling peptides (3D Matrix, Cambridge, Mass.
under a material transfer agreement) was obtained as a sterile 1%
(w/v) solution in water, which was mixed 1:1 with 1.times.10.sup.6
cells in 10% (w/v) sucrose (Sigma, St Louis, Mo.), 10 millimolar
HEPES in Dulbecco's modified medium (DMEM; Gibco) immediately
before use. The final concentration of cells in RAD16 hydrogel was
1.times.10.sup.6 cells/100 microliter.
[0617] Test Material (N=4/Rx)
[0618] 1. VICRYL non-woven+1.times.10.sup.6 umbilical cord-derived
cells
[0619] 2. 35/65 PCL/PGA foam+1.times.10.sup.6 umbilical
cord-derived cells
[0620] 3. RAD 16 self-assembling peptide+1.times.10.sup.6 umbilical
cord-derived cells
[0621] 4. VICRYL non-woven+1.times.10.sup.6 placenta-derived
cells
[0622] 5. 35/65 PCL/PGA foam+1.times.10.sup.6 placenta-derived
cells
[0623] 6. RAD 16 self-assembling peptide+1.times.10.sup.6
placenta-derived cells
[0624] 7. 35/65 PCL/PGA foam
[0625] 8. VICRYL non-woven
[0626] Animal Preparation. The animals utilized in this study were
handled and maintained in accordance with the current requirements
of the Animal Welfare Act. Compliance with the above Public Laws
were accomplished by adhering to the Animal Welfare regulations (9
CFR) and conforming to the current standards promulgated in the
Guide for the Care and Use of Laboratory Animals, 7th edition.
[0627] Mice (Mus Musculus)/Fox Chase SCID/Male (Harlan Sprague
Dawley, Inc., Indianapolis, Ind.), 5 weeks of age. All handling of
the SCID mice took place under a hood. The mice were individually
weighed and anesthetized with an intraperitoneal injection of a
mixture of 60 milligram/kilogram KETASET (ketamine hydrochloride,
Aveco Co., Inc., Fort Dodge, Iowa) and 10 milligram/kilogram ROMPUN
(xylazine, Mobay Corp., Shawnee, Kans.) and saline. After induction
of anesthesia, the entire back of the animal from the dorsal
cervical area to the dorsal lumbosacral area was clipped free of
hair using electric animal clippers. The area was then scrubbed
with chlorhexidine diacetate, rinsed with alcohol, dried, and
painted with an aqueous iodophor solution of 1% available iodine.
Ophthalmic ointment was applied to the eyes to prevent drying of
the tissue during the anesthetic period.
[0628] Subcutaneous Implantation Technique. Four skin incisions,
each approximately 1.0 cm in length, were made on the dorsum of the
mice. Two cranial sites were located transversely over the dorsal
lateral thoracic region, about 5-mm caudal to the palpated inferior
edge of the scapula, with one to the left and one to the right of
the vertebral column. Another two were placed transversely over the
gluteal muscle area at the caudal sacro-lumbar level, about 5-mm
caudal to the palpated iliac crest, with one on either side of the
midline. Implants were randomly placed in these sites. The skin was
separated from the underlying connective tissue to make a small
pocket and the implant placed (or injected for RAD16) about 1-cm
caudal to the incision. The appropriate test material was implanted
into the subcutaneous space. The skin incision was closed with
metal clips.
[0629] Animal Housing. Mice were individually housed in
microisolator cages throughout the course of the study within a
temperature range of 64.degree. F.-79.degree. F. and relative
humidity of 30% to 70%, and maintained on an approximate 12 hour
light/12 hour dark cycle. The temperature and relative humidity
were maintained within the stated ranges to the greatest extent
possible. Diet consisted of Irradiated Pico Mouse Chow 5058 (Purina
Co.) and water fed ad libitum.
[0630] Mice were euthanized at their designated intervals by carbon
dioxide inhalation. The subcutaneous implantation sites with their
overlying skin were excised and frozen for histology.
[0631] Histology. Excised skin with implant was fixed with 10%
neutral buffered formalin (Richard-Allan Kalamazoo, Mich.). Samples
with overlying and adjacent tissue were centrally bisected,
paraffin-processed, and embedded on cut surface using routine
methods. Five-micron tissue sections were obtained by microtome and
stained with hematoxylin and eosin (Poly Scientific Bay Shore,
N.Y.) using routine methods.
[0632] Results
[0633] There was minimal ingrowth of tissue into foams implanted
subcutaneously in SCID mice after 30 days (data not shown). In
contrast there was extensive tissue fill in foams implanted with
umbilical-derived cells or placenta-derived cells (data not
shown).
[0634] There was some tissue in growth in VICRYL non-woven
scaffolds. Non-woven scaffolds seeded with umbilical cord- or
placenta-derived cells showed increased matrix deposition and
mature blood vessels (data not shown).
[0635] Summary. The purpose of this study was to determine the type
of tissue formed by cells derived from human umbilical cord or
placenta in scaffolds in immune deficient mice. Synthetic
absorbable non-woven/foam discs (5.0 millimeter diameter.times.1.0
millimeter thick) or self-assembling peptide hydrogel were seeded
with either cells derived from human umbilical cord or placenta and
implanted subcutaneously bilaterally in the dorsal spine region of
SCID mice. The present study demonstrates that postpartum-derived
cells can dramatically increase good quality tissue formation in
biodegradable scaffolds.
Example 19
Chondrogenic and Osteogenic Potential of Postpartum-Derived Cells
on Implantation in SCID Mice
[0636] The chondrogenic potential of cells derived from umbilical
cord or placenta tissue was evaluated following seeding on
bioresorbable growth factor-loaded scaffolds and implantation into
SCID mice.
Materials & Methods
[0637] Reagents. Dulbecco's Modified Essential Media (DMEM),
Penicillin and Streptomycin, were obtained from Invitrogen,
Carlsbad, Calif. Fetal calf serum (FCS) was obtained from HyClone
(Logan, Utah). Mesenchymal stem cell growth medium (MSCGM) was
obtained from Biowhittaker, Walkersville, Md. TGFbeta-3 was
obtained from Oncogene research products, San Diego, Calif. GDF-5
was obtained from Biopharm, Heidelberg, Germany (International PCT
Publication No. WO96/01316 A1, U.S. Pat. No. 5,994,094A).
Chondrocyte growth medium comprised DMEM-High glucose supplemented
with 10% fetal calf serum (FCS), 10 milliMolar HEPES, 0.1
milliMolar nonessential amino acids, 20 microgram/milliliter
L-proline, 50 microgram/milliliter ascorbic acid, 100
Unit/milliliter penicillin, 100 microgram/milliliter streptomycin,
and 0.25 microgram/milliliter amphotericin B. Bovine fibrinogen was
obtained from Calbiochem.
[0638] Cells. Human mesenchymal stem cells (hMSC, Lot# 2F1656) were
obtained from Biowhittaker, Walkersville, Md. and were cultured in
MSCGM according to the manufacturer's instructions. This lot was
tested in the laboratory previously in in vitro experiments and was
shown to be positive in the chondrogenesis assays. Human adult
fibroblasts were obtained from American Type Culture Collection
(ATCC), Manassas, Va. and cultured in Growth Medium on
gelatin-coated tissue culture plastic flasks. Postpartum-derived
cells isolated from human umbilical cords (Lot# 022703Umb) and
placenta (Lot# 071003Plac) were prepared as previously described
(Example 1). Cells were cultured in Growth medium on gelatin-coated
tissue culture plastic flasks. The cell cultures were incubated in
standard growth conditions. Cells used for experiments were at
passages 5 and 14.
[0639] Scaffold. 65/35 Polyglycolic acid (PGA)/Polycaprolactone
(PCL) foam scaffolds [4.times.5 centimeters, 1 millimeter thick,
Ethylene Oxide (ETO) sterilized] reinforced with Polydioxanone
(PDS) mesh (PGA/PCL foam-PDS mesh) were obtained from Center for
Biomaterials and Advanced Technologies (CBAT, Somerville, N.J.).
Punches (3.5 millimeters) made from scaffolds were loaded with
either GDF-5 (3.4 micrograms/scaffold), TGFbeta-3 (10
nanograms/scaffold), a combination of GDF-5 and TGFbeta-3, or
control medium, and lyophilized.
[0640] Cell seeding on scaffolds. Placenta- and umbilical
cord-derived cells were treated with trypsin, and cell number and
viability was determined. 0.75.times.10.sup.6 cells were
resuspended in 15 microliter of Growth Medium and seeded onto 3.5
millimeter scaffold punches in a cell culture dish. The cell-seeded
scaffold was incubated in a cell culture incubator in standard air
with 5% CO.sub.2 at 37.degree. C. for 2 hours after which they were
placed within cartilage explant rings.
[0641] Bovine Cartilage Explants. Cartilage explants 5 millimeter
in diameter were made from cartilage obtained from young bovine
shoulder. Punches (3 millimeter) were excised from the center of
the explant and replaced with cells seeded 3.5 millimeter
resorbable scaffold. Scaffolds with cells were retained within the
explants using fibrin glue (60 microliter of bovine fibrinogen, 3
milligram/milliliter). Samples were maintained in chondrocyte
growth medium overnight, rinsed in Phosphate Buffered Saline the
following day, and implanted into SCID mice.
[0642] Animals. SCID mice ((Mus musculus)/Fox Chase SCID/Male), 5
weeks of age, were obtained from Harlan Sprague Dawley, Inc.
(Indianapolis, Ind.) and Charles River Laboratories (Portage,
Mich.). Animals used in the study were selected without any
apparent systematic bias. A tag was placed on each individual
animal cage listing the accession number, implantation technique,
animal number, species/strain, surgery date, in vivo period, and
date of euthanasia. The animals were identified by sequential
numbers marked on the ear with an indelible ink marker.
[0643] Experimental Design. A total of 42 mice were tested. Two
scaffolds were implanted subcutaneously in each mouse as described
below; 42 mice for subcutaneous implantation; 28 treatments with
n-value of 3 per treatment. The study corresponds to IACUC Approval
Number: Skillman IACUC 01-037. The study lasted six weeks.
[0644] SCID Implantation.
[0645] A. Body Weights
[0646] Each animal was weighed prior to being anesthetized and at
necropsy.
[0647] B. Anesthesia and Surgical Preparation:
[0648] All handling of the SCID mice occurred under a hood. The
mice were individually weighed and anesthetized with an
intraperitoneal injection of a mixture of KETASET (ketamine
hydrochloride [60 milligram/kilogram]), ROMPUN (xylazine [10
milligram/kilogram]), and saline.
[0649] After induction of anesthesia, the entire back of the animal
from the dorsal cervical area to the dorsal lumbosacral area was
clipped free of hair using electric animal clippers. The area was
scrubbed with chlorhexidine diacetate, rinsed with alcohol, dried,
and painted with an aqueous iodophor solution of 1% available
iodine. Ophthalmic ointment was applied to the eyes to prevent
drying of the tissue during the anesthetic period. The anesthetized
and surgically prepared animal was placed in the desired recumbent
position.
[0650] C. Subcutaneous Implantation Technique:
[0651] An approximate 2-cm skin incision was made just lateral to
the thoracic spine parallel to the vertebral column. The skin was
separated from the underlying connective tissue via blunt
dissection. Each SCID mouse received 2 treatments that were placed
in subcutaneous pockets created by blunt dissection in each
hemithorax through one skin incision. Tacking sutures of 5-0
ETHIBOND EXCEL (polyester) were used to tack the skin to
musculature around each scaffold to prevent subcutaneous migration.
Scaffolds were implanted for 6 weeks and then harvested. The
experimental design is outlined in Table 19-1. TABLE-US-00031 TABLE
19-1 Experimental Design: Treatment (N = 3 per treatment) A. 65/35
PGA/PCL Foam + PDS mesh cultured with Placenta-derived cells, EP,
TGFbeta3 B. 65/35 PGA/PCL Foam + PDS mesh cultured with
Placenta-derived cells, EP, rhGDF-5 C. 65/35 PGA/PCL Foam + PDS
mesh cultured with Placenta-derived cells, EP, rhGDF- 5 + TGFbeta3
D. 65/35 PGA/PCL Foam + PDS mesh cultured with Placenta-derived
cells, EP, control E. 65/35 PGA/PCL Foam + PDS mesh cultured with
Placenta-derived cells, LP, TGFbeta3 F. 65/35 PGA/PCL Foam + PDS
mesh cultured with Placenta-derived cells, LP, rhGDF-5 G. 65/35
PGA/PCL Foam + PDS mesh cultured with Placenta-derived cells, LP,
rhGDF- 5 + TGFbeta3 H. 65/35 PGA/PCL Foam + PDS mesh cultured with
Placenta-derived cells, LP, control I. 65/35 PGA/PCL Foam + PDS
mesh cultured with Umbilical cord-derived cells, EP, TGFbeta3 J.
65/35 PGA/PCL Foam + PDS mesh cultured with Umbilical cord-derived
cells, EP, rhGDF-5 K. 65/35 PGA/PCL Foam + PDS mesh cultured with
Umbilical cord-derived cells, EP, rhGDF- 5 + TGFbeta3 L. 65/35
PGA/PCL Foam + PDS mesh cultured with Umbilical cord-derived cells,
EP, control M. 65/35 PGA/PCL Foam + PDS mesh cultured with
Umbilical cord-derived cells, LP, TGFbeta3 N. 65/35 PGA/PCL Foam +
PDS mesh cultured with Umbilical cord-derived cells, LP, rhGDF-5 O.
65/35 PGA/PCL Foam + PDS mesh cultured with Umbilical cord-derived
cells, LP, rhGDF- 5 + TGFbeta3 P. 65/35 PGA/PCL Foam + PDS mesh
cultured with Umbilical cord-derived cells, LP, control Q. 65/35
PGA/PCL Foam + PDS mesh cultured with hMSC, TGFbeta3 R. 65/35
PGA/PCL Foam + PDS mesh cultured with hMSC, rhGDF-5 S. 65/35
PGA/PCL Foam + PDS mesh cultured with hMSC, rhGDF-5 + TGFbeta3 T.
65/35 PGA/PCL Foam + PDS mesh cultured with hMSC, control U. 65/35
PGA/PCL Foam + PDS mesh cultured with fibroblasts, Adult TGFbeta3
V. 65/35 PGA/PCL Foam + PDS mesh cultured with fibroblasts, Adult
rhGDF-5 W. 65/35 PGA/PCL Foam + PDS mesh cultured with fibroblasts,
Adult rhGDF-5 + TGFbeta3 X. 65/35 PGA/PCL Foam + PDS mesh cultured
with fibroblasts, Adult control Y. 65/35 PGA/PCL Foam + PDS mesh,
TGFbeta3 Z. 65/35 PGA/PCL Foam + PDS mesh, rhGDF-5 AA. 65/35
PGA/PCL Foam + PDS mesh, rhGDF-5 + TGFbeta3 BB. 65/35 PGA/PCL Foam
+ PDS mesh, control
[0652] D. Necropsy and Histologic Preparation
[0653] Gross examination was performed on any animals that died
during the course of the study or were euthanized in moribund
condition. Selected tissues were saved at the discretion of the
study director and/or pathologist.
[0654] Mice were euthanized by CO.sub.2 inhalation at their
designated intervals. Gross observations of the implanted sites
were recorded. Samples of the subcutaneous implantation sites with
their overlying skin were excised and fixed in 10% buffered
formalin. Each implant was bisected into halves, and one half was
sent to MPI Research (Mattawan, Mich.) for paraffin embedding,
sectioning, and staining with Hematoxylin & Eosin (H&E) and
Safranin O (SO).
[0655] The data obtained from this study were not statistically
analyzed.
[0656] Results
[0657] New cartilage and bone formation was observed in the
majority of the samples including growth factor-loaded, cell-seeded
scaffolds, cell-seeded control scaffolds, and scaffolds loaded with
growth factor alone. The extent of new cartilage and bone formation
varied within the treatment and control groups.
[0658] Early and Late passage placenta-derived cell seeded
scaffolds showed new cartilage and bone formation within the
scaffolds. No obvious differences in new cartilage and bone
formation was observed between the different growth factor-loaded,
cell-seeded scaffolds and scaffolds seeded with cells alone.
Compared to control scaffolds (without growth factors and without
cells), it appeared that there was greater extent of new cartilage
formation in cell-seeded scaffolds both with and without growth
factors and in growth factor-loaded scaffolds alone. New cartilage
formation with placenta-derived cell-seeded scaffolds was similar
to MSC- and fibroblast-seeded scaffolds.
[0659] In growth factor-treated and control scaffolds seeded with
umbilical cord-derived cells at early and late passage, new
cartilage and bone formation were observed. The extent of cartilage
formation appeared to be less than that seen with placenta-derived
cells. No one sample showed extensive cartilage formation as seen
with the placenta-derived cells. Bone formation appeared to be
higher in scaffolds seeded with umbilical cord-derived cells on
scaffolds containing both TGFbeta-3 and rhGDF-5.
[0660] hMSC-loaded scaffolds also showed new cartilage and bone
formation. The extent of new cartilage and bone formation was
similar for all the hMSC treatment groups. Human adult fibroblast
seeded scaffolds also demonstrated new cartilage and bone
formation. Results were similar to those obtained with
placenta-derived cells and hMSCs
[0661] In the control group, in which growth factor-loaded
scaffolds or scaffold alone were placed in cartilage rings and
implanted, new cartilage and bone formation were also observed. Not
surprisingly, the extent of new cartilage formation was greater in
scaffolds with growth factor than in scaffolds without growth
factor. Increased bone formation was present in the control with
the combination of the two tested growth factors.
[0662] New cartilage formation was observed adjacent to the
cartilage explant rings as well as within the scaffolds. New
cartilage formation within the scaffolds adjacent to the cartilage
rings could be a result of chondrocyte migration. Cartilage
formation seen as islands within the scaffolds may be a result of
either migration of chondrocytes within the scaffolds,
differentiation of seeded cells or differentiation of endogenous
mouse progenitor cells. This observation stems from the fact that
in control growth factor-loaded scaffolds with no seeded cells,
islands of chondrogenic differentiation were observed. New bone
formation was observed within the scaffolds independently and also
associated with chondrocytes. Bone formation may have arisen from
osteoblast differentiation as well as endochondral
ossification.
[0663] It is difficult to separate new cartilage and bone formation
associated with chondrocytes that migrated versus that from any
chondrogenic and osteogenic differentiation of seeded cells that
may have occurred. Staining of sections with specific human
antibodies may distinguish the contribution of the seeded cells to
the observed chondrogenesis and osteogenesis. It is also possible
that placenta-derived cells and umbilical cord-derived cells
stimulated chondrocyte migration.
[0664] Abundant new blood vessels were observed with the scaffolds
loaded with placenta-derived cells and umbilical cord-derived
cells. Blood vessels were abundant in areas of bone formation. New
blood vessels were also observed within the hMSC- and
fibroblast-seeded scaffolds associated with new bone formation.
[0665] Systemic effects of the adjacent scaffold (with growth
factor (GF)) on the control scaffolds (no GF, no cells) on
promoting new cartilage and bone formation cannot be ruled out.
Analysis of new cartilage and bone formation in scaffolds, taking
into consideration the scaffolds implanted adjacent to it in SCID
mice, showed no clear pattern of systemic effect of growth factor
from the adjacent scaffold.
[0666] Summary. Results showed that new cartilage and bone
formation were observed in growth factor and control scaffolds
seeded with placenta- and umbilical cord-derived cells. Results
with placenta-derived cells were similar to that seen with human
mesenchymal stem cells, while the extent of new cartilage like
tissue formation was slightly less pronounced in umbilical
cord-derived cells. Growth factor-loaded scaffolds implanted
without cells also demonstrated new cartilage and bone formation.
These data indicate that new cartilage formation within the
scaffolds may arise from chondrocytes that migrated from the bovine
explants, from chondrogenic differentiation of endogenous
progenitor cells, and from chondrogenic differentiation of seeded
cells.
[0667] These results suggest that placenta- and umbilical
cord-derived cells undergo chondrogenic and osteogenic
differentiation. These results also suggest that placenta- and
umbilical cord-derived cells may promote migration of chondrocytes
from the cartilage explant into the scaffolds. Abundant new blood
vessels were also observed in the scaffolds especially associated
with new bone formation.
[0668] While the present invention has been particularly shown and
described with reference to the presently preferred embodiments, it
is understood that the invention is not limited to the embodiments
specifically disclosed and exemplified herein. Numerous changes and
modifications may be made to the preferred embodiment of the
invention, and such changes and modifications may be made without
departing from the scope and spirit of the invention as set forth
in the appended claims.
Sequence CWU 1
1
10 1 22 DNA Artificial Synthetic Construct 1 gagaaatcca aagagcaaat
gg 22 2 21 DNA Artificial Synthetic Construct 2 agaatggaaa
actggaatag g 21 3 20 DNA Artificial Synthetic Construct 3
tcttcgatgc ttcggattcc 20 4 21 DNA Artificial Synthetic Construct 4
gaattctcgg aatctctgtt g 21 5 21 DNA Artificial Synthetic Construct
5 ttacaagcag tgcagaaaac c 21 6 22 DNA Artificial Synthetic
Construct 6 agtaaacatt gaaaccacag cc 22 7 20 DNA Artificial
Synthetic Construct 7 tctgcagctc tgtgtgaagg 20 8 22 DNA Artificial
Synthetic Construct 8 cttcaaaaac ttctccacaa cc 22 9 17 DNA
Artificial Synthetic Construct 9 cccacgccac gctctcc 17 10 19 DNA
Artificial Synthetic Construct 10 tcctgtcagt tggtgctcc 19
* * * * *