U.S. patent application number 11/129612 was filed with the patent office on 2005-12-01 for cell culture environments for the serum-free expansion of mesenchymal stem cells.
This patent application is currently assigned to Becton, Dickinson and Company, Becton, Dickinson and Company. Invention is credited to Chaney, Bryce N., Chen, Chang, Haaland, Perry D., Liebmann-Vinson, Andrea, Mitchell, Matthew, Rowley, Jonathan Allen, Xu, Ruiling.
Application Number | 20050265980 11/129612 |
Document ID | / |
Family ID | 34978975 |
Filed Date | 2005-12-01 |
United States Patent
Application |
20050265980 |
Kind Code |
A1 |
Chen, Chang ; et
al. |
December 1, 2005 |
Cell culture environments for the serum-free expansion of
mesenchymal stem cells
Abstract
Compositions and methods for promoting mesenchymal stem cell
expansion while maintaining a pluripotent phenotype are disclosed.
Serum-free cell culture systems and kits and methods of use for
mesenchymal stem cell expansion are provided. Methods also comprise
the use of the expanded mesenchymal stem cells to treat various
disorders or diseases, particularly those of the cardiovascular
system, bone, or cartilage.
Inventors: |
Chen, Chang; (Raleigh,
NC) ; Liebmann-Vinson, Andrea; (Wake Forest, NC)
; Xu, Ruiling; (Cary, NC) ; Rowley, Jonathan
Allen; (Ann Arbor, MI) ; Haaland, Perry D.;
(Chapel Hill, NC) ; Chaney, Bryce N.; (Durham,
NC) ; Mitchell, Matthew; (Durham, NC) |
Correspondence
Address: |
BECTON, DICKINSON AND COMPANY
ALSTON & BIRD LLP
1 BECTON DRIVE, MC 110
FRANKLIN LAKES
NJ
07417-1880
US
|
Assignee: |
Becton, Dickinson and
Company
Franklin Lakes
NJ
|
Family ID: |
34978975 |
Appl. No.: |
11/129612 |
Filed: |
May 13, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60571212 |
May 14, 2004 |
|
|
|
Current U.S.
Class: |
424/93.7 ;
435/366; 435/404 |
Current CPC
Class: |
C12N 5/0663 20130101;
C12N 2501/105 20130101; C12N 2533/54 20130101; C12N 2501/39
20130101; C12N 2500/25 20130101; C12N 2501/135 20130101; C12N
2501/145 20130101; C12N 2500/42 20130101; C12N 2501/2305 20130101;
C12N 2501/11 20130101; C12N 2501/2311 20130101; C12N 2501/2306
20130101; C12N 2533/70 20130101; C12N 2501/115 20130101; C12N
2501/125 20130101; C12N 2501/15 20130101; C12N 2501/22 20130101;
C12N 2501/26 20130101; C12N 2501/13 20130101; C12N 2501/14
20130101; C12N 2501/21 20130101; C12N 2501/60 20130101; C12N 5/0647
20130101; C12N 2500/36 20130101; C12N 2501/23 20130101; C12N
2501/165 20130101; C12N 2501/155 20130101; C12N 2501/2303 20130101;
A61P 9/00 20180101; C12N 2501/70 20130101; C12N 2533/52 20130101;
A61P 19/08 20180101; C12N 2501/415 20130101; C12N 2501/58 20130101;
C12N 2501/10 20130101; C12N 2501/235 20130101; C12N 2500/90
20130101; C12N 2501/32 20130101; C12N 2501/999 20130101 |
Class at
Publication: |
424/093.7 ;
435/366; 435/404 |
International
Class: |
A61K 045/00; C12N
005/08; C12N 005/00 |
Claims
That which is claimed is:
1. A serum-free cell culture system comprising a serum-free cell
culture medium and a cell culture surface that promotes the
adhesion and expansion of mesenchymal stem cells, wherein at least
one insoluble substrate protein is presented from said cell culture
surface, and wherein said serum-free cell culture medium is
selected from the group consisting of: a) a medium comprising bFGF
in combination with at least one growth factor selected from the
group consisting of a WNT signaling agonist, TGF-.beta., and EGF;
b) a medium comprising a WNT signaling agonist in combination with
at least one growth factor selected from the group consisting of
bFGF, TGF-.beta., and EGF; c) a medium comprising TGF-.beta. in
combination with at least one growth factor selected from the group
consisting of bFGF, a WNT signaling agonist, and EGF; d) a medium
comprising EGF in combination with at least one growth factor
selected from the group consisting of bFGF, a WNT signaling
agonist, and TGF-.beta.; e) a medium comprising fibronectin, SDF-1
.alpha., IL-6, SCF, IL-5, BDNF, PD-ECGF, IL-11, IL-3, EPO,
Flt-3/Flk-2 ligand, BMP-4, thrombospondin, IGF-1, and bFGF; f) a
medium comprising BDNF, bFGF, BIO, BMP-2, BMP-4, DKK-1, EGF, EPO,
fibronectin, thrombospondin, Flt-3/Flk-2 ligand, G-CSF, IGF-1,
IL-11, IL-3, IL-5, IL-6, LIF, PD-ECGF, SCF, SDF-1.alpha., and a WNT
signaling agonist; g) a medium comprising bFGF, BMP-2, EGF, EPO,
fibronectin, thrombospondin, Flt-3/Flk-2 ligand, IGF-1, IL-11,
IL-5, and a WNT signaling agonist; h) a medium comprising bFGF,
BIO, BMP-2, BMP-4, EGF, EPO, Flt-3/Flk-2 ligand, IGF-1, IL-11,
IL-5, and a WNT signaling agonist; i) a medium comprising bFGF,
BMP-4, DKK-1, fibronectin, thrombospondin, Flt-3/Flk-2 ligand,
IL-6, PD-ECGF, SDF-1.alpha., and a WNT signaling agonist; j) a
medium comprising bFGF, BMP-2, fibronectin, thrombospondin,
Flt-3/Flk-2 ligand, IL-11, LIF, and a WNT signaling agonist; k) a
medium comprising bFGF, BMP-2, EGF, IL-11, PD-ECGF, and a WNT
signaling agonist; and l) a medium comprising bFGF, BMP-2, EGF,
fibronectin, thrombospondin, Flt-3/Flk-2 ligand, IL-11, IL-5, LIF,
PD-ECGF, and a WNT signaling agonist.
2. The serum-free cell culture system of claim 1, wherein said WNT
signaling agonist is BIO or WNT-3a.
3. The serum-free cell culture system of claim 1, wherein the
insoluble substrate protein is an extracellular matrix protein.
4. The serum-free cell culture system of claim 3, wherein the
extracellular matrix protein is fibronectin, laminin, HA,
vitronectin, collagen I, collagen II, collagen III, collagen IV,
collagen V, collagen VI, or any combination thereof.
5. The serum-free cell culture system of claim 4, wherein the
extracellular matrix protein is a combination of collagen I and
fibronectin.
6. The serum-free cell culture system of claim 5, wherein the WNT
signaling agonist is BIO or WNT-3a, and wherein the cell culture
surface comprises a cell culture support bound to a cell adhesion
resistant material.
7. A method of expanding a population of mesenchymal stem cells
using the cell culture system of claim 1.
8. The method of claim 7, wherein said WNT signaling agonist is BIO
or WNT-3a, wherein the cell culture surface comprises a cell
culture support bound to a cell adhesion resistant material, and
wherein the insoluble substrate protein is a combination of
collagen I and fibronectin.
9. An expanded population of mesenchymal stem cells that has never
been exposed to serum, wherein said population is produced by the
method of claim 7.
10. A method of transplanting an expanded population of mesenchymal
stem cells, comprising administering the population of claim 9 to a
patient in need thereof, wherein the population is administered in
an amount effective to reconstitute cardiovascular, bone, or
cartilage tissue.
11. A kit comprising the serum-free cell culture system of claim
1.
12. The kit of claim 11, wherein said WNT signaling agonist is BIO
or WNT-3a, wherein the cell culture surface comprises a cell
culture support bound to a cell adhesion resistant material, and
wherein the insoluble substrate protein is a combination of
collagen I and fibronectin.
13. A serum-free cell culture system comprising a serum-free cell
culture medium and a cell culture surface, wherein said cell
culture surface promotes the adhesion and expansion of mesenchymal
stem cells and comprises a cell culture support bound to a cell
adhesion resistant material, and wherein at least one insoluble
substrate protein is presented from the cell culture surface.
14. The serum-free cell culture system of claim 13, wherein the
insoluble substrate protein is an extracellular matrix protein.
15. The serum-free cell culture system of claim 14, wherein the
extracellular matrix protein is fibronectin, laminin, HA,
vitronectin, collagen I, collagen II, collagen III, collagen IV,
collagen V, collagen VI, or any combination thereof.
16. The serum-free cell culture system of claim 15, wherein the
extracellular matrix protein is a combination of collagen I and
fibronectin.
17. The serum-free cell culture system of claim 13, wherein said
serum-free cell culture medium is selected from the group
consisting of: a) a medium comprising bFGF in combination with at
least one growth factor selected from the group consisting of a WNT
signaling agonist, TGF-.beta., and EGF; b) a medium comprising a
WNT signaling agonist in combination with at least one growth
factor selected from the group consisting of bFGF, TGF-.beta., and
EGF; c) a medium comprising TGF-.beta. in combination with at least
one growth factor selected from the group consisting of bFGF, a WNT
signaling agonist, and EGF; d) a medium comprising EGF in
combination with at least one growth factor selected from the group
consisting of bFGF, a WNT signaling agonist, and TGF-.beta.; e) a
medium comprising fibronectin, SDF-1 .alpha., IL-6, SCF, IL-5,
BDNF, PD-ECGF, IL-11, IL-3, EPO, Flt-3/Flk-2 ligand, BMP-4,
thrombospondin, IGF-1, and bFGF; f) a medium comprising BDNF, bFGF,
BIO, BMP-2, BMP-4, DKK-1, EGF, EPO, fibronectin, thrombospondin,
Flt-3/Flk-2 ligand, G-CSF, IGF-1, IL-11, IL-3, IL-5, IL-6, LIF,
PD-ECGF, SCF, SDF-1.alpha., and a WNT signaling agonist; g) a
medium comprising bFGF, BMP-2, EGF, EPO, fibronectin,
thrombospondin, Flt-3/Flk-2 ligand, IGF-1, IL-11, IL-5, and a WNT
signaling agonist; h) a medium comprising bFGF, BIO, BMP-2, BMP-4,
EGF, EPO, Flt-3/Flk-2 ligand, IGF-1, IL-11, IL-5, and a WNT
signaling agonist; i) a medium comprising bFGF, BMP-4, DKK-1,
fibronectin, thrombospondin, Flt-3/Flk-2 ligand, IL-6, PD-ECGF,
SDF-1.alpha., and a WNT signaling agonist; j) a medium comprising
bFGF, BMP-2, fibronectin, thrombospondin, Flt-3/Flk-2 ligand,
IL-11, LIF, and a WNT signaling agonist; k) a medium comprising
bFGF, BMP-2, EGF, IL-11, PD-ECGF, and a WNT signaling agonist; and
l) a medium comprising bFGF, BMP-2, EGF, fibronectin,
thrombospondin, Flt-3/Flk-2 ligand, IL-11, IL-5, LIF, PD-ECGF, and
a WNT signaling agonist.
18. The serum-free cell culture system of claim 17, wherein said
WNT signaling agonist is BIO or WNT-3a.
19. The serum-free cell culture system of claim 17, wherein the
insoluble substrate protein is an extracellular matrix protein and
wherein the extracellular matrix protein is fibronectin, laminin,
HA, vitronectin, collagen I, collagen II, collagen III, collagen
IV, collagen V, collagen VI, or any combination thereof.
20. The serum-free cell culture system of claim 19, wherein the
extracellular matrix protein is a combination of collagen I and
fibronectin.
21. A method of expanding a population of mesenchymal stem cells
using the cell culture system of claim 13.
22. The method of claim 21, wherein the insoluble substrate protein
is a combination of collagen I and fibronectin.
23. An expanded population of mesenchymal stem cells, said
population produced by the method of claim 21, wherein said cells
were unexposed to serum.
24. A method of transplanting an expanded population of mesenchymal
stem cells, comprising administering the population of claim 23 to
a patient in need thereof, wherein the population is administered
in an amount effective to reconstitute cardiovascular, bone, or
cartilage tissue.
25. A kit comprising the serum-free cell culture system of claim
13.
26. The kit of claim 25, wherein the insoluble substrate protein is
a combination of collagen I and fibronectin.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/571,212, filed May 14, 2004, the content of
which is herein incorporated by reference it its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to serum-free cell culture
systems that provide for mesenchymal stem cell expansion while
maintaining a pluripotent phenotype, and methods of use for the
expanded mesenchymal stem cell populations.
BACKGROUND OF THE INVENTION
[0003] Mesenchymal stem cells (MSCs) are present in adult tissues
and constitute a population of cells that can be isolated, expanded
in culture, and characterized in vitro and in vivo (Pittenger and
Martin (2004) Circ. Res. 95:9-20). MSCs are able to differentiate
into multiple cell lineages, including osteoblasts, chondrocytes,
endothelial cells, and neuronal cells, and can express phenotypic
characteristics of endothelial, neural, smooth muscle, skeletal
myoblast, and cardiac myocyte cells (Kassem et al. (2004) Basic
Clin. Pharmacol. Toxicol. 95:209-214; Pittenger and Martin (2004)
Circ. Res. 95:9-20). In recent years, MSCs have generated a high
level of experimental and clinical interest due to their potential
for a range of therapeutic uses including repair of damaged or
diseased tissues (Baksh et al. (2004) J. Cell. Mol. Med. 8:301-316;
Barry and Murphy (2004) Int. J. Biochem. Cell Bio. 36:568-584).
[0004] Typically, MSCs do very poorly in serum-free environments
because they detach and die in culture. These MSCs can be
maintained in an attached state in vitro with minimal serum (e.g.,
<1%), although such an environment provides little stimulation
for MSCs to proliferate and grow. Although serum-free cell culture
environments have been described for MSC expansion (Lennon et al.
(1995) Exp. Cell Res. 219:211-222; U.S. Pat. No. 5,908,782), the
current industry standard still contains a large amount of
serum.
[0005] In addition to the desirability of serum-free environments
for the expansion of MSCs, serum-free media and culture systems
have great utility in the field of cellular therapy. The creation
of highly defined environments for cell expansion is of great
importance for quality purposes, and serum levels are typically
very ill-defined (see, e.g., U.S. Pat. No. 5,908,782). In addition,
there is a risk of Bovine Spongiform Encephalopathy (BSE)
contamination in patients receiving cells cultured in the presence
of serum. Such a risk raises the possibility that the FDA will not
allow therapies involving cells cultured in the presence of animal
sera.
[0006] For the aforementioned reasons, the development of new
serum-free cell culture systems for the expansion of MSCs is
therefore desirable.
SUMMARY OF THE INVENTION
[0007] Compositions and methods for promoting mesenchymal stem cell
(MSC) expansion while maintaining a pluripotent phenotype are
provided. The compositions include serum-free cell culture systems
for MSC expansion that comprise a serum-free cell culture medium
and a two-dimensional or three-dimensional cell culture surface. In
the serum-free cell culture system of the present invention, at
least one insoluble substrate protein is presented from the cell
culture surface. In one embodiment, the cell culture surface
comprises a cell culture support bound to a cell adhesion resistant
(CAR) material, which in turn is bound to at least one insoluble
substrate protein. Insoluble substrate proteins for use in the
present invention include extracellular matrix (ECM) proteins such
as fibronectin, laminin, hyaluronic acid (HA), vitronectin,
collagen proteins such as collagen I, collagen II, collagen III,
collagen IV, collagen V, and collagen VI, or any combination
thereof. In one embodiment, the cell culture surface comprises a
cell culture support bound to a cell adhesion resistant (CAR)
material, which in turn is bound to at least one insoluble
substrate protein, for example, at least one ECM protein. The
serum-free cell culture medium is a solution that comprises a
mixture of soluble MSC growth-promoting factors. Compositions
further include kits comprising serum-free cell culture media and a
two-dimensional or three-dimensional cell culture surface suitable
for MSC expansion.
[0008] Methods of the present invention comprise the use of these
serum-free cell culture systems to promote the expansion of MSCs.
Further methods comprise the use of these serum-free cell culture
systems and expanded MSCs for cell transplantation or to engineer
tissues to treat various disorders or diseases, including those of
the cardiovascular system, muscle, ligament, bone, tendon,
cartilage, nervous system, blood, immune system, liver, or
pancreas. Further methods comprise the use of these serum-free cell
culture systems to promote the expansion of MSCs within primary
aspirates from whole bone marrow such that the MSCs are co-cultured
with non-MSCs present in the primary aspirates.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 shows expansion of total mesenchymal stem cells
(MSCs) over time in the G2 serum-free culture system and collagen
1+fibronectin surface of the present invention (BDT Environment)
versus expansion in Cambrex Biosciences (Baltimore, Md.)
serum-containing medium on tissue culture plastic.
[0010] FIG. 2 shows the adipocyte differentiation capacity of MSCs
expanded in the G2 serum-free medium and collagen 1+fibronectin
surface (BDT Environment) or expanded in Cambrex Biosciences
(Baltimore, Md.) serum-containing medium on tissue culture
plastic.
[0011] FIG. 3 shows the bone differentiation capacity of MSCs
expanded in the G2 serum-free medium collagen 1+fibronectin
surfaces (BDT Environment) or expanded in Cambrex Biosciences
(Baltimore, Md.) serum-containing medium on tissue culture
plastic.
[0012] FIG. 4 shows a comparison of the number of hMSCs after
culture in a serum-free base medium (BDTM) with or without growth
factors on either a tissue culture polystyrene (TCPS) or cell
adhesion resistant (CAR) surface.
[0013] FIG. 5 shows a comparison of various serum-free compositions
and collagen 1+fibronectin surfaces for MSC expansion that are
labeled C6, D3, C2, and G5, compared to G2 medium and a serum
containing medium on tissue culture plastic (10% FBS CM).
[0014] FIG. 6A shows the adipocyte differentiation capacity of MSCs
expanded in D3, C2, and G5 serum-free culture media and collagen
1/fibronectin surfaces or expanded in Cambrex Biosciences
(Baltimore, Md.) serum-containing medium on tissue culture plastic
(10% FBS CM). FIG. 6B shows the bone differentiation capacity of
MSCs expanded in D3, C2, and G5 serum-free culture media and
collagen 1/fibronectin surfaces or expanded in Cambrex Biosciences
(Baltimore, Md.) serum-containing medium on tissue culture plastic
(10% FBS CM).
[0015] FIG. 7 shows a comparison of further refined serum-free
composition for MSC expansion labeled G7, G4, and C8 and cultured
on collagen 1/fibronectin surfaces compared to expansion in Cambrex
Biosciences (Baltimore, Md.) serum-containing medium (10% FBS
CM).
[0016] FIG. 8 shows a comparison of MSC expansion in various media
conditions and collagen 1/fibronectin surfaces, including G4
serum-free medium, G4+TGF.beta., FGF only, EGF only, and TGF.beta.
only, as compared to basal medium and Cambrex Biosciences
(Baltimore, Md.) serum-containing medium on tissue culture plastic
(10% FBS CM).
[0017] FIG. 9 shows a comparison of MSC expansion in various media
conditions where selected growth factor combinations were added to
basal medium, as compared to basal medium alone and Cambrex
Biosciences (Baltimore, Md.) serum-containing medium on tissue
culture plastic (TCPS). Growth factor combinations were
bFGF+TGF-.beta., WNT-3a+bFGF, bFGF+EGF, WNT-3a+TGF-.beta.,
WNT-3a+EGF, EGF+TGF-.beta., WNT-3a alone, bFGF+EGF+TGF-.beta.,
WNT-3a+bFGF+EGF+TGF-.beta., and bFGF+EGF+TGF-.beta.+BIO.
DETAILED DESCRIPTION OF THE INVENTION
[0018] The present invention relates to compositions and methods
for promoting mesenchymal stem cell (MSC) expansion while
maintaining the pluripotent phenotype of these cells. Serum-free
cell culture systems for MSC expansion are provided. These cell
culture systems comprise serum-free cell culture medium and a
two-dimensional or three-dimensional cell culture surface. The
serum-free cell culture medium is a solution that comprises a
mixture of soluble MSC growth-promoting and self-renewal factors.
In the serum-free cell culture system of the present invention, at
least one insoluble substrate protein is presented from the cell
culture surface. In one embodiment, the cell culture surface
comprises a cell culture support bound to a cell adhesion resistant
(CAR) material, which in turn is bound to at least one insoluble
substrate protein. Insoluble substrate proteins may include but are
not limited to extracellular matrix (ECM) proteins such as
fibronectin, laminin, hyaluronic acid (HA), vitronectin, or
collagen proteins such as collagen I, collagen II, collagen III,
collagen IV, collagen V, and collagen VI, or any combination
thereof. Other compositions of the present invention include kits
comprising the serum-free cell culture systems of the invention,
which are suitable for MSC expansion.
[0019] Methods of the present invention are directed to the use of
these serum-free cell culture systems to promote the expansion of
MSCs and to engineer tissues. The expanded MSCs of the present
invention can be used to treat various disorders or diseases,
particularly those of the cardiovascular system, muscle, ligament,
bone, tendon, cartilage, nervous system, blood, immune system,
liver, or pancreas.
[0020] The term "mesenchymal" is intended to mean such cells as
bone marrow, endothelial cells, epithelial cell progenitors,
cardiomyocytes, astrocytes, neurons, chondrocytes, osteoblasts,
pancreatic cells, hepatocytes, and other cells of mesenchymal
origin. As used herein, the term "mesenchymal stem cell" or "MSC"
refers to a cell that gives rise to a cell of mesenchymal
lineage.
[0021] The term "expanded" is intended to mean that the resultant
cell population is derived from ex vivo culture of stem cells in
media compositions comprising mixtures of cytokines, where the
outgoing (cultured) number of cells exceeds the ingoing
(non-cultured) number of cells. The term "expanded" is not to be
construed or limited by any mechanism or theory of cellular origin
and may comprise cells that originate de novo in culture.
[0022] The serum-free cell culture system of the present invention
comprises a serum-free cell culture medium and a cell culture
surface. In one embodiment, at least one insoluble substrate
protein is presented from the cell culture surface, meaning that
the insoluble substrate protein is bound, adsorbed, linked,
attached or in some way associated with the cell culture surface.
Suitable insoluble substrate proteins include the ECM proteins
disclosed herein. In one such embodiment, the insoluble substrate
protein presented from the cell culture surface is a combination of
collagen I and fibronectin. In another embodiment, the cell culture
surface comprises a cell culture support bound to a cell adhesion
resistant material, which in turn is bound to at least one
insoluble substrate protein. The cell culture support may be solid
or porous, polymer, metal, glass, ceramic, or combinations thereof.
Culturing of MSCs in the presence of this cell culture surface and
the serum-free cell culture medium unexpectedly provides for
expansion of these cells while maintaining their pluripotent
phenotype.
[0023] As used herein, the term cell adhesion resistant (CAR)
refers to a material that, when present on a surface, prevents,
inhibits, or reduces the non-specific binding (adhesion) of cells,
proteins, or polypeptides found on cell surfaces. CAR materials are
resistant to adhesion of mammalian cells and also to
microorganisms. CAR materials are sometimes referred to as
non-fouling substrates, inert coatings, low affinity reagents, or
non-adhesive coatings.
[0024] The cell culture support to which is bound the CAR material
may be two- or three-dimensional, solid or porous, and may be
constructed of any of a variety of materials, including natural
polymers, synthetic polymers, hydrogels, metals, ceramics, and
inorganic or organic-inorganic composites. The cell culture support
may be shaped using methods such as, for example, solvent casting,
compression molding, filament drawing, meshing, leaching, weaving,
and coating.
[0025] Examples of suitable polymers and hydrogels include, but are
not limited to, collagen, glycosaminoglycan (GAG)-based materials,
alginate, hyaluronate poly(alpha esters) such as poly(lactate
acid), poly(glycolic acid), polyorthoesters and polyanhydrides and
their copolymers, cellulose ether, cellulose, cellulosic ester,
fluorinated polyethylene, phenolic, poly-4-methylpentene,
polyacrylonitrile, polyamide, polyamideimide, polyacrylate,
polybenzoxazole, polycarbonate, polycyanoarylether,
polyestercarbonate, polyether, polyetheretherketone,
polyetherimide, polyetherketone, polyethersulfone, polyethylene,
polyfluoroolefin, polylmide, polyolefin, polyoxadiazole,
polyphenylene oxide, polyphenylene sulfide, polypropylene,
polystyrene, polysulfide, polysulfone, polytetrafluoroethylene,
polythioether, polytriazole, polyurethane, polyvinylidene fluoride,
regenerated cellulose, urea-formaldehyde, or copolymers or physical
blends of these materials.
[0026] Further examples include synthetic polymers, such as
polylactic acid (PLA), polyglycolic acid (PGA), poly(orthoester),
polysebacic acid polymers (PSA), poly(lactic-co-glycolic) acid
copolymers (PLGA), poly(lactic-co-sebacic) acid copolymers (PLSA),
poly(glycolic-co-sebacic) acid copolymers (PGSA), hydrogels such as
polyhydroxyethylmethacrylate (poly-HEMA) or polyethylene
oxide-polypropylene oxide copolymer, polyethylegylcols (PEG),
polyvinylalchols (PVA), polyvinylpyrrolidone (PVP) and
polyhydroxyalkanoate (PHA). PHAs and their production are described
in, for example, PCT Publication Nos. WO 99/14313, WO 99/32536 and
WO 00/56376. Hybrid materials containing naturally derived and
synthetic polymer materials (e.g., PGA and PLGA) may also be used.
Non-limiting examples of such materials are disclosed in Chen et
al. (2000) Advanced Materials 12:455-457.
[0027] Other polymers useful in the present invention include
polymers or copolymers of caprolactones, carbonates, amides, amino
acids, orthoesters, acetals, cyanoacrylates and degradable
urethanes, as well as copolymers of these with straight chain or
branched, substituted or unsubstituted, alkanyl, haloalkyl,
thioalkyl, aminoalkyl, alkenyl, or aromatic hydroxy- or
di-carboxylic acids. In addition, the biologically important amino
acids with reactive side-chain groups, such as lysine, arginine,
aspartic acid, glutamic acid, serine, threonine, tyrosine and
cysteine, or their enantiomers, may be included in copolymers with
any of the aforementioned materials.
[0028] In one embodiment, polymer surfaces are selected from the
group consisting of polystyrene, polyethylene, polypropylene,
polyethylene terephthalate, polytetrafluoroethylene, polylactide,
and cellulose. Silicone polymers such as polydimethylsiloxane
(PDMS) are also used.
[0029] Inorganic composites include, e.g., calcium phosphate
ceramics, bioglasses and bioactive glass-ceramics, in particular
composites combining calcium hydroxyapatite and silicon stabilized
tricalcium phosphate. Among preferred cell culture supports are
polystyrene (PS), polypropylene, polyethylene, polyethylene
terephthalate, polytri- or tetra-fluoroethylene,
polyhexafluoropropylene, polyvinyl chloride, polyvinylidine
fluoride, polyactide, cellulose, glass, or a ceramic.
[0030] Any suitable CAR material, many of which are known to those
skilled in the art, may be bound to the cell culture support.
Typical CAR materials include hyaluronic acid (HA) or a derivative
thereof, alginic acid (AA) or a derivative thereof, poly-HEMA,
polyethylene glycol (PEG), glyme or a derivative thereof,
polypropylacrylamide, polyisopropylacrylamide, or a combination of
these compounds. In one embodiment, the CAR material is HA.
[0031] In some embodiments, one or more of a proteoglycan, a
biglycan, a glycosaminoglycan, or Matrigel.TM. may be bound to the
CAR material.
[0032] In other embodiments, proteins or other substances may be
bound to the CAR material either covalently or non-covalently, but
are preferably covalently bound. The proteins or other substances
comprise, for example, ECM proteins or polycationic polymers. ECM
proteins for use in the present invention may include fibronectin,
laminin, hyaluronic acid (HA), vitronectin, or collagen proteins
such as collagen I, collagen II, collagen III, collagen IV,
collagen V, and collagen VI, or any combination thereof. Various
types of covalent bonds can form, some of which are discussed in
more detail in co-pending, commonly assigned U.S. Patent
Application Publication Nos. 20050036980, 20040062882, 20030113813,
and 20030113812, all of which are incorporated herein by reference.
These applications also disclose other aspects of making and using
surfaces that include cell culture supports with bound CAR
materials and ECM proteins.
[0033] In one embodiment, one or more ECM proteins as disclosed
herein and/or one or more polycationic polymer are bound to the CAR
material. In one embodiment, a mixture of collagen I and
fibronectin is bound to the CAR material.
[0034] The ECM protein(s) can be in the form of a naturally
occurring polypeptide (protein), a recombinant polypeptide, or a
synthetic or semi-synthetic polypeptide, including any fragment of
the peptide or protein, or combination thereof. The terms
"polypeptide" and "protein" are used interchangeably herein.
[0035] Methods of cloning, expressing, and purifying polypeptides,
such as ECM proteins, are conventional, as are methods of
generating synthetic or semi-synthetic polypeptides. ECM proteins
can also be obtained from commercial sources.
[0036] ECM proteins or polycationic polymers can be bound to the
CAR material either covalently or non-covalently (for example,
passively adsorbed, such as by electrostatic forces, ionic or
hydrogen bonds, hydrophilic or hydrophobic interactions, Van der
Waals forces, etc.). In a preferred embodiment, the binding is
covalent. U.S. Patent Application Publication Nos. 20040062882,
20030113813, and 20030113812 describe such covalent binding of
molecules to CAR surfaces.
[0037] Methods of making surfaces in which a CAR material is bound
to a cell culture support, and in which ECM proteins, polycationic
polymers, or the like, are bound to the CAR material, are described
in detail in U.S. Patent Application Publication Nos. 20040062882,
20030113813, and 20030113812. In brief, one method of attaching a
CAR material to a cell culture support comprises treating the cell
culture support with an oxidizing plasma, and binding the CAR
material to the treated cell culture support. Another method of
attaching a CAR material to a cell culture support comprises
treating the cell culture support with an oxidizing plasma;
exposing the treated cell culture support to a polycationic polymer
with amino groups (such as PEI, PDL, poly-L-lysine (PLL),
poly-L-ornithine (PLO), poly-D-ornithine (PDO), poly(vinylamine)
(PVA) or poly(allylamine) (PAA), in one embodiment, PEI or PDL to
form an intermediate layer, and binding the CAR material to the
intermediate layer. Methods of binding an ECM or a polycationic
polyaminoacid to a CAR material are conventional. These include,
for example, sodium periodate oxidation and reductive amination,
EDC/NHS carbodiimide coupling, or the like.
[0038] This invention also speaks to the use of flexible substrates
in culture. For example, Flexercell culture systems from Flexcell
International Corporation are able to apply tensile, compressive,
or shear stresses to cultured cells. See, for example, U.S. Pat.
Nos. 4,789,601, 4,822,741, 4,839,280, 6,037,141, 6,048,723, and
6,218,178. U.S. Pat. No. 6,057,150 discloses the application of a
biaxial strain to an elastic membrane that may be coated with
extracellular matrix proteins and covered with cultured cells. U.S.
Pat. No. 6,107,081 discloses another system in which a
unidirectional cell stretching device comprising an elastic strip
is coated with an extracellular matrix on which cells are cultured
and stretched. A flexible substrate can be deformed easily and in a
controlled manner, and also supports cell adhesion and growth
comparable to conventional cell culture substrates. Silicones, such
as poly(dimethyl siloxane) (PDMS), are particularly suitable for
this application because they are not only highly flexible but also
provide optical clarity that allows microscopic observation of the
cell cultures.
[0039] Another aspect of the invention is a method of making the
above cell culture surface of the invention, where the method
comprises (a) attaching a CAR material to a cell culture support,
and (b) attaching to the CAR material collagen I and fibronectin,
or biologically active fragment or variant thereof, and,
optionally, one or more other ECM proteins (or a biologically
active fragment or variant of the ECM protein) and/or one or more
polycationic polymers. Any of the ECM proteins or polycationic
polymers disclosed herein, or others, may be used.
[0040] In one embodiment, the CAR material is attached to the cell
culture support by treating the cell culture support with an
oxidizing plasma, and binding the CAR material to the treated cell
culture support. In another embodiment, the CAR material is
attached to the cell culture support by treating the cell culture
support with an oxidizing plasma; exposing the treated cell culture
support to a polycationic polymer with amino groups to form an
intermediate layer; and binding the CAR material to the
intermediate layer. Preferably, the polycationic polymer is
polyethylene imine (PEI) or poly-D-lysine (PDL).
[0041] In a one embodiment, a CAR material, such as HA, is bonded
directly to polymeric nitrogen-containing surfaces. Examples of
such surfaces are ammonia plasma-treated polymers and Primaria
.TM.-treated polystyrene (PS) surfaces, and poly D-lysine coated
surfaces. Polymeric substrates suitable for use in the invention
include polystyrene, polypropylene, polyethylene terephthalate,
polylactide, cellulose, and the like, though polystyrene is
preferred.
[0042] HA is immobilized directly on an intermediate polyamine
layer, e.g., polyethyleneimine, poly-D-Lysine, or poly-L-lysine, or
directly bound to the polymer with the HA bound to that
intermediate layer).
[0043] The use of plasma techniques are familiar to those of skill
in the art (see, for example, Garbassi et al., (1994) Polymer
Surfaces, from Physics to Technology (Wiley, Chichester), and
Inagaki (1996) Plasma Surface Modification and Plasma
Polymerization (Technomic Publishing Company, Lancaster). In the
present invention, the plasma treatment process may be any process
that is capable of causing nitrogen to be incorporated onto the
surface of the polymer article resulting in reactive amine or other
nitrogen-containing groups, including direct as well as remote
plasma treatment methods. Examples of suitable plasma treatments
are ones using reactive gases such as nitrogen, nitrogen oxide,
nitrogen dioxide or ammonia in the gas phase, alone or in mixture
with air, argon, or other inert gases, and may be preceded or
followed by treatments employing argon or other inert gases. The
plasma maybe sustained over the full treatment time or maybe
administered in pulses. Preferably, the plasma gas is ammonia, and
treatment is performed with a power charge of between 1 and 400 W,
preferably between 10 and 150 W, a pressure between 10 mtorr and 10
ton, and a treatment time between 1 second and 1 hour, preferably
between 10 seconds and 30 minutes.
[0044] Plasma-treated polystyrene can be prepared, for example by
pumping the treatment chamber to a 0.3 mTorr base pressure,
establishing a 200 mTorr argon atmosphere, and applying a 60 sec
argon plasma treatment, followed by a 120 sec, 375 mTorr NH.sub.3
plasma treatment at 95 W. Other suitable treatments will be known
to those of skill in the art. Following plasma treatment of the
surface to be coated, the plasma-treated surface may be exposed to
an aqueous solution containing HA or a derivative thereof, or
alginic acid (alginate; AA) in the presence of a condensing agent
such as a carbodiimide, for example, ethyldimethylaminopropyl
carbodiimide (EDC), in aqueous solution, or
dicyclohexylcarbodiimide (DCC), in organic solvents. The term
"expose" or "exposing" as used herein is intended to include any
type of contact made between a liquid and a solid, for example by
pipetting, pouring, spraying, dripping, immersing, pouring,
dipping, injecting, etc., without limitation.
[0045] HA is an anionic polysaccharide composed of repeating units
of beta-1,4-glucuronatebeta-1,3-N-acetylglucosamine. A reactive
--COO.sup.- group is present on every repeat unit of HA that can be
utilized to covalently couple HA to an amine containing surface
using methods described herein. In this manner, a condensing agent
such as EDC activates the --COO.sup.- groups present in HA,
creating a reactive ester intermediate (ester (o-acylisourea)
intermediate). This intermediate is highly unstable and subject to
hydrolysis, leading to the cleaving off of the activated ester
intermediate, forming an isourea, and regenerating the --COO.sup.-
group. To stabilize this reactive ester intermediate, and increase
reaction yield, a molecule able to enhance the reaction promoted by
EDC should also be present. Such stabilizing compounds are
generally selected from the class of N-hydroxysuccinimides and aryl
or heterocyclic derivatives thereof. Preferred
N-hydroxysuccinimides include, but are not limited to,
N-hydroxy-succinimide (NHS), hydroxy-sulfosuccinimide (sulfo-NHS)
or hydroxybenzotriazole hydrate. Although not intended to be bound
to a particular theory, it is believed that attachment of HA to the
amine containing polymer surface occurs through a mechanism wherein
(for example) EDC and NHS combine to create an active ester
polysaccharide with a carboxyl group capable of coupling to an
amine. When coupling occurs, NHS is released. Other compounds known
in the art that are able to react with EDC in this manner and which
serve as reactive intermediate ester stabilizing compounds should
also be effective in the invention. HA covalently bonded to
plasma-treated polystyrene in this way prevents attachment of any
of a number of types of cells, including NIH3T3 and osteoblast
MC3T3 cells as well as MSC's and a variety of primary and stem
cells.
[0046] Suitable derivatives of HA that may be used in the invention
will be known to the skilled artisan, and are described, for
example, in U.S. Pat. No. 4,851,521. These include partial esters
of HA with alcohols of the aliphatic, araliphatic, cycloaliphatic
and heterocyclic series, and salts of such partial esters with
inorganic or organic bases. Similar derivatives of alginic acid
should also be useful. Further, other plasma treatment methods for
producing surfaces with amine and other nitrogen-containing groups
are also suitable, and are known to those of skill in the art.
[0047] The cell culture surfaces for use in the serum-free cell
culture systems include but are not limited to standard tissue
culture vessels and two-dimensional surfaces, including sheets,
slides, culture dishes, culture flasks, bags, culture bottles, or
multiwell dishes. Alternatively, three-dimensional cell culture
surfaces, such as microcarriers or three dimensional (3-D)
scaffolds, including but not limited to foams, hydrogels, or fiber
meshes may be used for generating a three-dimensional cell culture,
tissue, or organ. "Three-dimensional scaffold" refers herein to a
3-D porous template that provides a very high surface area to
volume ratio for cell culture. These scaffolds may be used for
initial cell attachment and expansion, or subsequently for tissue
formation either in vitro or in vivo. A 3-D scaffold according to
this invention comprises base materials (described below), a CAR
layer and bound thereto one or more insoluble substrate proteins,
such as the ECMs disclosed herein, and, optionally, other
substances, which promote or enhance cell attachment, growth,
migration, and/or differentiation. In one embodiment, the scaffold
is seeded with MSCs and contacted with the serum-free cell culture
medium described herein below in order to permit cell growth and
differentiation in a structural environment that more closely
mimics the in vivo setting. Cells derived therefrom can be isolated
from the scaffold or can be implanted with or without the scaffold
into a suitable location in the body of a mammal, preferably a
human patient. However, the use of this technology could easily be
translated to non-human mammals such as cats, dogs, horses, and the
like.
[0048] The shape and dimensions of the 3-D scaffold are determined
based on the organ being replaced or supplemented, and the type of
scaffold material being used to create the construct. For example,
if a polymeric scaffold is used for heart tissue replacement or
supplementation, the dimension of the polymeric scaffold can vary
in terms of width and length of the polymeric scaffold. One of
skill in the art recognizes that the size and dimensions of the
polymeric scaffold will be determined based on the area of the
organ being replaced or supplemented. Furthermore, other suitable
articles may constitute a cell culture support surface of the
present invention, including medical devices, extracorporeal
devices and artificial joints, tubes, sutures, stents, orthopedic
devices, vascular grafts, membranes, films, biosensors, or
microparticles.
[0049] One embodiment includes a method of forming
tissue-engineered constructs using a 3-D scaffold material or other
suitable article that comprises the CAR surface onto which one or
more insoluble substrate proteins, for example, the ECMs disclosed
herein, are attached, either covalently or non-covalently. Such a
scaffold in combination with the serum-free cell culture medium
disclosed herein supports the maturation, development and
differentiation, of additional cultured MSCs in vitro to form
components of adult tissues analogous to their in vivo
counterparts.
[0050] The tissue-engineered constructs, in one embodiment, are
created using scaffold materials disclosed herein as the substrate
onto which cells are deposited and cultured in the presence of the
serum-free cell culture medium described herein below, and on which
cells are grown and adhere. The scaffold allows optimum cell-cell
interactions, thereby allowing a more natural formation of cellular
phenotypes and a tissue microenvironment. The scaffold also allows
MSCs to continue to grow actively, proliferate, and differentiate
to produce a tissue-engineered construct that is also capable of
supporting the growth, proliferation, and differentiation of
additional cultured cell populations, if needed.
[0051] In one embodiment, the scaffold is biocompatible and
conducive to cell attachment and subsequent tissue growth. Other
surface properties can be modified to suit the intended application
without altering other properties of the scaffold such as its
mechanical strength or thermal properties. Useful surface
modifications could include, for example, changes in chemical group
functionality, surface charge, hydrophobicity, hydrophilicity, and
wettability. The CAR surface technology can easily be translated to
the surfaces of 3-D scaffolds. Such surface modifications are well
known in the art. Sterilization is performed prior to seeding the
scaffold with cells. Heat sterilization is often impractical since
the heat treatment could deform the device, especially if the
materials have a melting temperature below that required for the
heat sterilization treatment. For example, cold ethylene oxide gas,
vapor hydrogen peroxide treatments can be used for
sterilization.
[0052] The 3-D scaffolds of the present invention comprise any
suitable base material for construction, including the polymeric
materials disclosed herein above. The polymeric matrix can be
fabricated to have a controlled pore structure that allows
nutrients from the serum-free cell culture medium described herein
below to reach the deposited cell population but prevent cultured
cells from migrating through the pores. In vitro cell attachment
and cell viability can be assessed using scanning electron
microscopy, histology and quantitative assessment with
radioisotopes.
[0053] The polymeric matrix can be shaped into any number of
desirable configurations to satisfy any number of overall system,
geometry, or space restrictions. The polymeric matrix can be shaped
to different sizes to conform to the organs of different sized
patients. Thus, the tissue-engineered construct can be flat,
tubular, or of complex geometry. The shape of the construct will be
decided by its intended use. The construct can be implanted to
repair, supplement, or replace diseased or damaged parts of
organs.
[0054] In one embodiment, the scaffold base material is a hydrogel
composed of crosslinked polymer networks that are typically
insoluble or poorly soluble in water, but can swell to an
equilibrium size in the presence of excess water. For example, the
MSCs can be placed in a hydrogel and the hydrogel injected into
desired locations within the organ. The hydrogel compositions can
include, without limitation, for example, poly(esters),
poly(hydroxy acids), poly(lactones), poly(amides),
poly(ester-amides), poly(amino acids), poly(anhydrides),
poly(ortho-esters), poly(carbonates), poly(phosphazines),
poly(thioesters), polysaccharides and mixtures thereof.
Furthermore, the compositions can also include, for example, a
poly(hydroxy) acid including poly(alpha-hydroxy) acids and
poly(betahydroxy) acids. Such poly(hydroxy) acids include, for
example, polylactic acid, polyglycolic acid, polycaproic acid,
polybutyric acid, polyvaleric acid, and copolymers and mixtures
thereof. Due to the unique properties of hydrogels and their
potential applications in such areas as controlled drug delivery,
various types of hydrogels have been synthesized and characterized.
The matrix materials of the present invention encompass both
conventional foam or sponge materials and the so-called hydrogel
sponges (see, e.g., U.S. Pat. No. 5,451,613).
[0055] Mesenchymal stem cells grown on the scaffold materials in
the presence of the serum-free cell culture medium disclosed herein
in accordance with the present invention may grow in multiple
layers, forming a cellular structure that resembles physiologic
conditions found in vivo. The scaffold can support the
proliferation of different types of cells and the formation of a
number of different tissues. Examples include, but are not limited
to, kidney, heart, skin, liver, pancreas, adrenal and neurological
tissue, as well as tissues of the gastrointestinal and
genitourinary tracts, and the circulatory system.
[0056] The cells grown or expanded on the aforementioned scaffold
can be used, alone or in combination with the scaffold, in a
variety of applications. For example, the scaffold and cells can be
implanted into a subject. Implants can be used to replace or
supplement existing tissue, for example, by treating a subject with
a cardiovascular disorder by replacing or supplementing natural
cardiovascular tissue. The subject can be monitored after
implantation for amelioration of the cardiovascular disorder. A
three-dimensional biocompatible scaffold may be brought into
contact with vasculature-promoting expanded MSCs of the invention
and then brought into contact with a host tissue at a target site
(e.g., within the organ) or where the organ tissue is grown on the
scaffold prior to implantation. The graft is then able to grow and
proliferate within the target site and replace or supplement the
depleted activity of the organ. The construct can be added at a
single location in the host or, alternatively, a plurality of
constructs can be created and added to multiple sites in the
host.
[0057] The term "target site" as used herein refers to a region in
the host or organ that requires replacement or supplementation. The
target site can be a single region in the organ or host, or can be
multiple regions in the organ or host. In some embodiments, the
supplementation or replacement results in the same physiological
response as a normal organ.
[0058] In another embodiment, the scaffold is created using parts
of a natural decellularized organ. Parts of organs can be
decellularized by removing the entire cellular and tissue content
from the organ (see, e.g., U.S. Pat. No. 6,479,064). The term
"decellularized" or "decellularization" as used herein refers to a
biostructure (e.g., an organ, or part of an organ) from which the
cellular and tissue content has been removed leaving behind an
intact acellular infrastructure. The process of decellularization
removes the specialized tissue, leaving behind the complex
three-dimensional network of connective tissue. The connective
tissue infrastructure is primarily composed of collagen. The
decellularized structure provides a matrix material onto which
different cell populations can be infused. Decellularized
biostructures can be rigid, or semi-rigid, having an ability to
alter their shapes. Culture and construction of decellularized
biostructures can be performed, for example, as describe in U.S.
Pat. No. 6,479,064, which is herein incorporated by reference in
its entirety.
[0059] The two-dimensional and three-dimensional cell culture
surfaces serve as a substrate onto which MSCs can be seeded and
cultured in the presence of a serum-free cell culture medium
disclosed herein. In this manner, seeded MSCs can be expanded ex
vivo by contacting them with a serum-free cell culture medium that
comprises a defined cytokine cocktail. In one embodiment, the
defined combinations comprise at least 2, at least 3, at least, 4,
at least 5, at least 6, at least 7, at least 8, at least 9, at
least 10, at least 11, at least 12, at least 13, at least 14, or at
least 15 cytokines from the group comprising brain-derived
neurotrophic factor (BDNF), basic fibroblast growth factor (bFGF),
bone morphogenic protein 2 (BMP-2), bone morphogenic protein 4
(BMP-4), dickkopf 1 (DKK-1), epidermal growth factor (EGF),
erythropoietin (EPO), fibronectin, Flt-3/Flk-2 ligand, granulocyte
colony stimulating factor (G-CSF), insulin-like growth factor
(IGF-1), interleukin-11 (IL-11), interleukin-3 (IL-3),
interleukin-5 (IL-5), interleukin-6 (IL-6), leukemia inhibitory
factor (LIF), platelet-derived endothelial cell growth factor
(PD-ECGF), stem cell factor (SCF), stromal cell derived factor
1-.alpha. (SDF 1-.alpha.), transforming growth factor .beta.
(TGF-.beta.), thrombospondin, and a WNT signaling agonist. WNT
signaling agonists include, but are not limited to
(2'Z,3'E)-6-bromoindirubin-3'-oxime (BIO) and WNT proteins
including WNT-1, WNT-2, WNT-2b, WNT-3, WNT-3a, WNT-4, WNT-5a,
WNT-5b, WNT-6, WNT-7a, WNT-7b, WNT-8a, WNT-8b, WNT-9a, WNT-9b,
WNT-10a, WNT-10b, WNT-11, and WNT-16. In one embodiment, the WNT
protein is WNT-3a.
[0060] In one embodiment, the defined cytokine cocktail is termed
"G2" and comprises a combination of fibronectin, SDF-11, IL-6, SCF,
IL-5, BDNF, PD-ECGF, IL-11, IL-3, EPO, Flt-3/Flk-2 ligand, BMP-4,
thrombospondin, IGF-1, and bFGF.
[0061] In another embodiment, the defined cytokine cocktail is
termed "C6" and comprises a combination of BDNF, bFGF, BIO, BMP-2,
BMP-4, DKK-1, EGF, EPO, fibronectin, thrombospondin, Flt-3/Flk-2
ligand, G-CSF, IGF-1, IL-11, IL-3, IL-5, IL-6, LIF, PD-ECGF, SCF,
SDF-1.alpha., and WNT-3a.
[0062] In another embodiment, the defined cytokine cocktail is
termed "D3" and comprises a combination of bFGF, BMP-2, EGF, EPO,
fibronectin, thrombospondin, Flt-3/Flk-2 ligand, IGF-1, IL-11,
IL-5, and WNT-3a.
[0063] In another embodiment, the defined cytokine cocktail is
termed "C2" and comprises a combination of bFGF, BIO, BMP-2, BMP-4,
EGF, EPO, Flt-3/Flk-2 ligand, IGF-1, IL-11, IL-5, and WNT-3a.
[0064] In another embodiment, the defined cytokine cocktail is
termed "G5" and comprises a combination of bFGF, BMP-4, DKK-1,
fibronectin, thrombospondin, Flt-3/Flk-2 ligand, IL-6, PD-ECGF,
SDF-1.alpha., and WNT-3a.
[0065] In another embodiment, the defined cytokine cocktail is
termed "G7" and comprises a combination of bFGF, BMP-2,
fibronectin, thrombospondin, Flt-3/Flk-2 ligand, IL-11, LIF, and
WNT-3a.
[0066] In another embodiment, the defined cytokine cocktail is
termed "G4" and comprises a combination of bFGF, BMP-2, EGF, IL-11,
PD-ECGF, and WNT-3a.
[0067] In another embodiment, the defined cytokine cocktail is
termed "C8" and comprises a combination of bFGF, BMP-2, EGF,
fibronectin, thrombospondin, Flt-3/Flk-2 ligand, IL-11, IL-5, LIF,
PD-ECGF, and WNT-3a.
[0068] In another embodiment, the cytokine cocktail comprises bFGF
in combination with at least growth factor selected from the group
consisting of a WNT signaling agonist, TGF-.beta., and EGF. In
another embodiment, the cytokine cocktail comprises a WNT signaling
agonist in combination with at least one growth factor selected
from the group consisting of bFGF, TGF-.beta., and EGF. In another
embodiment, the cytokine cocktail comprises TGF-.beta. in
combination with at least one growth factor selected from the group
consisting of bFGF, a WNT signaling agonist, and EGF. In another
embodiment, the cytokine cocktail comprises EGF in combination with
at least one growth factor selected from the group consisting of
bFGF, a WNT signaling agonist, and TGF-.beta.. In another
embodiment, the cytokine cocktail comprises at least two growth
factors selected from the group consisting of bFGF, TGF-.beta.,
EGF, and a WNT signaling agonist. In some of these embodiments, the
WNT signaling agonist is WNT-3a; in other of these embodiments, the
WNT signaling agonist is BIO.
[0069] In one embodiment, adding complexity to a media composition
comprising at least two growth factors selected from the group
consisting of bFGF, TGF-.beta., EGF, and a WNT signaling agonist
with the addition of other factors enhances MSC expansion. For
example, although media containing combinations of bFGF,
TGF-.beta., EGF, and a WNT signaling agonist produce MSC expansion,
the addition of other signaling molecules such as BMPs, soluble
ECMs, and other cytokines and factors can further enhance MSC
expansion.
[0070] The G2 combination of growth factors allows for rapid
expansion of MSCs, at similar or even better than commercially
available serum containing media. In particular, this expansion,
when compared to serum containing media that is greater than 10
days old, will outperform the serum containing media. Furthermore,
the MSCs expanded in G2 media are still pluripotent and have been
demonstrated to differentiate into both bone and fat. The ability
of these MSCs to maintain a pluripotent phenotype is significant as
this is evidence that serum can be removed from the culture during
expansion and the cells have not changed considerably nor have they
been pushed towards specific differentiated phenotypes that would
prevent the use of the expanded cells from being used in different
therapeutic applications.
[0071] The defined cytokine combinations are added to a serum-free
base medium to provide a serum-free culture medium suitable for
expanding MSCs. Any serum-free nutritive medium suitable for the
culture of MSCs may be used. In one embodiment, the base medium is
as described in Lennon et al. (1995) Exp. Cell Res., 219:211-222,
comprising Dulbecco's Modified Eagle's Medium (DMEM), MCDB-201
(Sigma-Aldrich (St. Louis, Mo.)), ascorbic acid 2-phosphate,
dexamethasone, linoleic acid-bovine serum albumin, insulin,
transferrin, and sodium selenite. In a further embodiment, the base
medium comprises a ratio of DMEM to MCDB-201 of 60:40, the
concentration of ascorbic acid 2-phosphate is 1.times.10.sup.4 M,
the concentration of dexamethasone is 1.times.10.sup.-9 M, linoleic
acid-bovine serum albumin is in an amount of about 0.1%, insulin is
in an amount of about 5 .mu.g/mL, transferrin is in an amount of
about 5 .mu.g/mL, and sodium selenite is in an amount of about 5
ng/mL (see Table 2, herein below). Many different base media could
be used, and human or other animal-derived components may be
substituted such as human albumin for bovine albumin, or human
insulin. The ability to substitute non-animal derived components
for animal derived is known in the field, and several of these
components are commercially available, for example from Cambrex
Biosciences (Baltimore, Md.) or Sigma-Aldrich (St. Louis, Mo.).
[0072] The final concentration of cytokines in the serum-free cell
culture medium can range from about 1 femtogram/ml to about 1
picogram/ml to about 1 nanogram/ml to about 1 microgram/ml to about
1 milligram/ml. In some embodiments, the concentration of any one
of the cytokines can be about 1 pg/ml, about 5 pg/ml, about 10
pg/ml, about 15 pg/ml, about 20 pg/ml, about 25 pg/ml, about 30
pg/ml, about 35 pg/ml, about 40 pg/ml, about 45 pg/ml, about 50
pg/ml, about 55 pg/ml, about 60 pg/ml, about 65 pg/ml, about 70
pg/ml, about 75 pg/ml, about 80 pg/ml, about 85 pg/ml, about 90
pg/ml, about 95 pg/ml, about 100 pg/ml, about 110 pg/ml, about 120
pg/ml, about 130 pg/ml, about 140 pg/ml, about 150 pg/ml, about 160
pg/ml, about 170 pg/ml, about 180 pg/ml, about 190 pg/ml, about 200
pg/ml, about 210 pg/ml, about 220 pg/ml, about 230 pg/ml, about 240
pg/ml, about 250 pg/ml, about 260 pg/ml, about 270 pg/ml, about 280
pg/ml, about 290 pg/ml, about 300 pg/ml, about 310 pg/ml, about 320
pg/ml, about 330 pg/ml, about 340 pg/ml, about 350 pg/ml, about 360
pg/ml, about 370 pg/ml, about 380 pg/ml, about 390 pg/ml, about 400
pg/ml, about 410 pg/ml, about 420 pg/ml, about 430 pg/ml, about 440
pg/ml, about 450 pg/ml, about 460 pg/ml, about 470 pg/ml, about 480
pg/ml, about 490 pg/ml, about 500 pg/ml, about 510 pg/ml, about 520
pg/ml, about 530 pg/ml, about 540 pg/ml, about 550 pg/ml, about 560
pg/ml, about 570 pg/ml, about 580 pg/ml, about 590 pg/ml, about 600
pg/ml, about 610 pg/ml, about 620 pg/ml, about 630 pg/ml, about 640
pg/ml, about 650 pg/ml, about 660 pg/ml, about 670 pg/ml, about 680
pg/ml, about 690 pg/ml, about 700 pg/ml, about 710 pg/ml, about 720
pg/ml, about 730 pg/ml, about 740 pg/ml, about 750 pg/ml, about 760
pg/ml, about 770 pg/ml, about 780 pg/ml, about 790 pg/ml, about 800
pg/ml, about 810 pg/ml, about 820 pg/ml, about 830 pg/ml, about 840
pg/ml, about 850 pg/ml, about 860 pg/ml, about 870 pg/ml, about 880
pg/ml, about 890 pg/ml, about 900 pg/ml, about 910 pg/ml, about 920
pg/ml, about 930 pg/ml, about 940 pg/ml, about 950 pg/ml, about 960
pg/ml, about 970 pg/ml, about 980 pg/ml, about 990 pg/ml, about 1
ng/ml, about 1.5 ng/ml, about 2 ng/ml, about 2.5 ng/ml, about 3
ng/ml, about 3.5 ng/ml, about 4 ng/ml, about 4.5 ng/ml, about 5
ng/ml, about 6 ng/ml, about 7 ng/ml, about 8 ng/ml, about 9 ng/ml,
about 10 ng/ml, about 15 ng/ml, about 20 ng/ml, about 25 ng/ml,
about 30 ng/ml, about 35 ng/ml, about 40 ng/ml, about 45 ng/ml,
about 50 ng/ml, about 55 ng/ml, about 60 ng/ml, about 65 ng/ml,
about 70 ng/ml, about 75 ng/ml, about 80 ng/ml, about 85 ng/ml,
about 90 ng/ml, about 95 ng/ml, about 100 ng/ml, about 110 ng/ml,
about 120 ng/ml, about 130 ng/ml, about 140 ng/ml, about 150 ng/ml,
about 160 ng/ml, about 170 ng/ml, about 180 ng/ml, about 190 ng/ml,
about 200 ng/ml, about 210 ng/ml, about 220 ng/ml, about 230 ng/ml,
about 240 ng/ml, about 250 ng/ml, about 260 ng/ml, about 270 ng/ml,
about 280 ng/ml, about 290 ng/ml, about 300 ng/ml, about 310 ng/ml,
about 320 ng/ml, about 330 ng/ml, about 340 ng/ml, about 350 ng/ml,
about 360 ng/ml, about 370 ng/ml, about 380 ng/ml, about 390 ng/ml,
about 400 ng/ml, about 410 ng/ml, about 420 ng/ml, about 430 ng/ml,
about 440 ng/ml, about 450 ng/ml, about 460 ng/ml, about 470 ng/ml,
about 480 ng/ml, about 490 ng/ml, about 500 ng/ml, about 510 ng/ml,
about 520 ng/ml, about 530 ng/ml, about 540 ng/ml, about 550 ng/ml,
about 560 ng/ml, about 570 ng/ml, about 580 ng/ml, about 590 ng/ml,
about 600 ng/ml, about 610 ng/ml, about 620 ng/ml, about 630 ng/ml,
about 640 ng/ml, about 650 ng/ml, about 660 ng/ml, about 670 ng/ml,
about 680 ng/ml, about 690 ng/ml, about 700 ng/ml, about 710 ng/ml,
about 720 ng/ml, about 730 ng/ml, about 740 ng/ml, about 750 ng/ml,
about 760 ng/ml, about 770 ng/ml, about 780 ng/ml, about 790 ng/ml,
about 800 ng/ml, about 810 ng/ml, about 820 ng/ml, about 830 ng/ml,
about 840 ng/ml, about 850 ng/ml, about 860 ng/ml, about 870 ng/ml,
about 880 ng/ml, about 890 ng/ml, about 900 ng/ml, about 910 ng/ml,
about 920 ng/ml, about 930 ng/ml, about 940 ng/ml, about 950 ng/ml,
about 960 ng/ml, about 970 ng/ml, about 980 ng/ml, about 990 ng/ml,
or about 1000 ng/ml.
[0073] In one embodiment, the serum-free cell culture medium
comprises a concentration of BDNF from about 10 pg/ml to about 500
ng/ml. In another embodiment, the serum-free cell culture medium
comprises a concentration of bFGF from about 1 pg/ml to about 500
ng/ml. In another embodiment, the serum-free cell culture medium
comprises a concentration of BIO of about 1 pM to about 1 pM. In
another embodiment, the serum-free cell culture medium comprises a
concentration of BMP-2 from about 10 pg/ml to about 500 ng/ml. In
another embodiment, the serum-free cell culture medium comprises a
concentration of BMP-4 from about 10 pg/ml to about 500 ng/ml. In
another embodiment, the serum-free cell culture medium comprises a
concentration of DKK-1 from about 10 pg/ml to about 10 .mu.g/ml. In
another embodiment, the serum-free cell culture medium comprises a
concentration of EGF from about 10 pg/ml to about 100 ng/ml. In
another embodiment, the serum-free cell culture medium comprises a
concentration of erythropoietin from about 0.0001 units/ml to about
50 units/ml. In another embodiment, the serum-free cell culture
medium comprises a concentration of fibronectin from about 10 pg/ml
to about 100 .mu.g/ml. In another embodiment, the serum-free cell
culture medium comprises a concentration of Flt-3/Flk-2 ligand from
about 1 pg/ml to about 100 ng/ml. In another embodiment, the
serum-free cell culture medium comprises a concentration of G-CSF
from about 1 pg/ml to about 100 ng/ml. In another embodiment, the
serum-free cell culture medium comprises a concentration of IGF-1
from about 10 pg/ml to about 500 ng/ml. In another embodiment, the
serum-free cell culture medium comprises a concentration of IL-11
from about 1 pg/ml to about 100 ng/ml. In another embodiment, the
serum-free cell culture medium comprises a concentration of IL-3
from about 1 pg/ml to about 100 ng/ml. In another embodiment, the
serum-free cell culture medium comprises a concentration of IL-5
from about 1 pg/ml to about 100 ng/ml. In another embodiment, the
serum-free cell culture medium comprises a concentration of IL-6
from about 1 pg/ml to about 100 ng/ml. In another embodiment, the
serum-free cell culture medium comprises a concentration of LIF
from about 10 pg/ml to about 100 ng/ml. In another embodiment, the
serum-free cell culture medium comprises a concentration of PD-ECGF
from about 10 pg/ml to about 500 ng/ml. In another embodiment, the
serum-free cell culture medium comprises a concentration of SCF
from about 10 pg/ml to about 100 ng/ml. In another embodiment, the
serum-free cell culture medium comprises a concentration of
SDF-1.alpha. from about 1 pg/ml to about 100 ng/ml. In another
embodiment, the serum-free cell culture medium comprises a
concentration of TGF-.beta. from about 10 fg/ml to about 100 ng/ml.
In another embodiment, the serum-free cell culture medium comprises
a concentration of thrombospondin from about 10 pg/ml to about 1
.mu.g/ml. In another embodiment, the serum-free cell culture medium
comprises a concentration of WNT-3a from about 10 pg/ml to about
500 ng/ml.
[0074] In one embodiment, the serum-free cell culture medium
comprises BDNF in an amount of about 1 ng/ml; bFGF in an amount of
about 2.5 ng/ml; BMP-4 in an amount of about 0.5 ng/ml;
erythropoietin in an amount of about 0.05 U/ml; fibronectin in an
amount of about 10 ng/ml; Flt-3/Flk-2 ligand in an amount of about
5 ng/ml; IGF-1 in an amount of about 2.5 ng/ml; IL-11 in an amount
of about 0.1 ng/ml; IL-3 in an amount of about 1 ng/ml; IL-5 in an
amount of about 0.1 ng/ml; IL-6 in an amount of about 0.2 ng/ml;
PD-ECGF in an amount of about 2 ng/ml; SCF in an amount of about 2
ng/ml; SDF-1.alpha. in an amount of about 3 ng/ml; and
thrombospondin in an amount of about 10 ng/ml.
[0075] Those skilled in the art will recognize that cytokines for
use in the present invention may be concentrated and, in some
instances, lyophilized before addition to the base medium to obtain
the examples of final concentrations listed above. Those skilled in
the art will also recognize that the mass weight added to culture
will depend on the specific biological activity of the cytokine
preparation. Bioassays to determine the biological potency of
cytokines are well known in the art. Therefore, where the
biological activity is correlated to a mass weight, then biological
"units" as defined by the assay are used.
[0076] Populations of MSCs that have been expanded using the
serum-free cell culture system of the present invention comprise
cells expressing cell surface markers of interest. The term "cell
surface marker" or "marker" is intended to mean a protein that is
expressed on the surface of a cell, which can be detected using
specific antibodies. For example, the expanded MSCs of the present
invention express markers reported for commercial MSCs, including
CD166, CD44, CD105, and CD29. The expanded MSCs of the invention
may also express combinations of these cell markers, and may
express CD73, CD90, CD 106, CD 146, or any combination thereof. In
some instances, the expression of a cell surface marker defines
non-mesenchymal cell populations such as mature blood cells and
hematopoietic stem cells, for example: CD3; CD14; CD19; CD34;
CD42a; CD45; and any combinations of these markers.
[0077] The term "substantially free" is intended to mean that less
than 5%, less than 4%, less than 3%, less than 2%, or less than 1%
of the cells within the population express the marker of interest.
For example, in one embodiment, the expanded cell population is
substantially free of T-cells (expressing the CD3 antigen), B-cells
(expressing the CD 19 antigen), or mature granulocytes, NK
lymphocytes, or macrophages (expressing the CD16 antigen).
[0078] The term "substantial proportion" is intended to mean that
at least 40%, at least 50%, at least 60%, at least 70%, at least
80%, at least 90%, at least 95%, at least 99%, or up to 100% of the
cells express the marker of interest. Conversely, the term
"substantially diminished" is intended to mean that at least less
than 10%, at least less than 5%, or at least less than 1% of cells
express the marker of interest.
[0079] Those skilled in the art recognize that cells with increased
cell surface marker expression can be detected and isolated by any
means including flow cytometric sorting, antibody panning, and the
like. Generally in flow cytometric analysis, one of skill in the
art must first set a detection threshold for fluorescence. In
setting the threshold, a negative control sample population will be
recorded and a gate will be set around the population of interest
according to the desired forward scatter (FSC) and side scatter
(SSC). The detection threshold is then adjusted so that 97% or more
of the cells do not fluoresce. Once the detection threshold is set,
the fluorescence of cell population of interest is recorded. A cell
is considered "positive for expression" when it expresses the
marker of interest, whether a protein or a gene. Any method may be
used to determine expression such as gene expression profiles,
FACS, and the like. The term ".sup.+" indicates that the cell is
positive for expression of the marker of interest. The term
".sup.-" indicates that the cell does not have detectable levels of
expression of the marker of interest.
[0080] The antibodies used to detect various lineages may be
conjugated to different fluorochromes. These include
phycobiliproteins, e.g., phycoerythrin and allophycocyanins;
fluorescein; and Texas red. Dead cells can also be detected using
dyes that selectively accumulate in dead cells (e.g., propidium
iodide and 7-amino actinomycin D).
[0081] These expanded MSCs are characterized by a capacity for
self-renewal. Further, the cells are characterized by an ability to
commit to multilineage development. By "multilineage development"
is intended that the cell is capable of differentiating into cell
types of mesenchymal origin. These cells have a capability of
limited self renewal and are constrained to committed development
to various mesenchymal tissues such as bone, cartilage, fat,
tendon, muscle, and marrow stroma (Pittenger et al. (1999) Science.
284:5411 143).
[0082] Stem cell function can be assayed using both in vitro and in
vivo methods. In vitro testing comprises culturing MSCs in
semi-solid medium similar to hematopoetic stem cells. Along with
colony forming assays, MSCs may be tested for multilineage
potential by culturing these cells in the presence of factors that
send them down specific pathways such as bone, fat, and cartilage.
Media for differentiating expanded MSCs are well known in the art
(Pittenger and Martin (2004) Circ. Res. 95:9-20), and are
commercially available through several sources such as Cambrex
(Baltimore, Md.), Stem Cell Technologies (Vancouver, B. C.,
Canada), and R&D Systems (Minneapolis, Minn.). Expanded cells
are cultured either on surfaces such as culture dishes with these
media for 7-30 days for bone, fat, or muscle phenotypes, or in
pellets in the bottom or tubes or flasks for differentiating down
cartilage phenotypes. The test for function, tissue specific tests
are performed such as alkaline phosphatase enzyme, osteocalcin or
osteopontin for bone, oil O red staining for lipid accumulation in
MSCs that differentiate towards an adipocyte lineage.
[0083] The expanded MSCs of the invention can be analyzed based on
gene expression profiles. In this manner, the multilineage
commitment potential can be determined. As used herein, an
"expression profile" comprises one or more values corresponding to
a measurement of the relative abundance of a gene expression
product. Such values may include measurements of RNA levels or
protein abundance. Thus, the expression profile can comprise values
representing the measurement of the transcriptional state or the
translational state of the gene (see U.S. Pat. Nos. 6,040,138,
5,800,992, 6,020135, 6,344,316, and 6,033,860).
[0084] The transcriptional state of a sample includes the
identities and relative abundance of the RNA species, especially
mRNAs present in the sample. Preferably, a substantial fraction of
all constituent RNA species in the sample are measured, but at
least a sufficient fraction to characterize the transcriptional
state of the sample is measured. The transcriptional state can be
conveniently determined by measuring transcript abundance by any of
several existing gene expression technologies. Translational state
includes the identities and relative abundance of the constituent
protein species in the sample. As is known to those of skill in the
art, the transcriptional state and translational state are
related.
[0085] In one embodiment of the invention, microarrays can be used
to measure the values to be included in the expression profiles.
Microarrays are particularly well suited for this purpose because
of the reproducibility between different experiments. DNA
microarrays provide one method for the simultaneous measurement of
the expression levels of large numbers of genes. Each array
consists of a reproducible pattern of capture probes attached to a
cell culture support. Labeled RNA or DNA is hybridized to
complementary probes on the array and then detected by laser
scanning. Hybridization intensities for each probe on the array are
determined and converted to a quantitative value representing
relative gene expression levels (see U.S. Pat. Nos. 6,040,138,
5,800,992 and 6,020,135, 6,033,860, and 6,344,316). High-density
oligonucleotide arrays are particularly useful for determining the
gene expression profile for a large number of RNAs in a sample.
[0086] "Array" is intended to mean a cell culture support or
substrate with peptide or nucleic acid probes attached to the cell
culture support or substrate. Arrays typically comprise a plurality
of different nucleic acid or peptide capture probes that are
coupled to a surface of a substrate in different, known locations.
These arrays, also described as "microarrays" or colloquially
"chips," have been generally described in the art, for example, in
U.S. Pat. Nos. 5,143,854, 5,445,934, 5,744,305, 5,677,195,
6,040,193, 5,424,186, 6,329,143, and 6,309,831 and Fodor et al.
(1991) Science 251:767-77.
[0087] The methods of the invention comprise culturing or expanding
MSCs from a stem cell source in a serum-free cell culture system
that comprises a serum-free cell culture medium disclosed herein
above, which comprises a cytokine cocktail and base medium. Methods
of cell culture are well known in the art (see, e.g., Waymouth
(1984) "Preparation and Use of Serum-free Culture Media," in Cell
Culture Methods for Molecular and Cell Biology, Vol. 1, Methods for
Preparation of Media, Supplements, and Substrata for Serum-Free
Animal Cell Culture, eds. Barnes et al. (Alan R. Liss), pp. 23-68).
The term "cell-culture" is intended to mean the expansion or
maintenance of cells in an artificial in vitro environment while
maintaining a pluripotent phenotype. It is to be understood that
the term "cell culture" is a generic term and may be used to
encompass the cultivation not only of individual cells but also
tissues, organ systems, or whole organisms.
[0088] In some embodiments, cells derived from a stem cell source
are seeded onto a two-dimensional or three-dimensional cell culture
surface described herein and contacted with the serum-free cell
culture medium of the invention. The medium may initially contain
the cytokine cocktail as well as the base medium, or the cytokine
cocktail can be added later. Cells are then incubated with the
serum-free cell culture medium comprising the cytokine cocktail and
cell culture support at a temperature suitable for cell growth (for
some embodiments about 37.degree. C.), for at least about 24 hours,
at least about 48 hours, at least about 72 hours, at least about 96
hours, at least about 120 hours, at least about 144 hours, at least
about 168 hours, at least about 192 hours, or longer. Cells may be
harvested in any manner known in the art including but not limited
to centrifugation after decanting non-adherent cells, trypsinizing
adherent cells, or scraping cells from the surface of the cell
culture support.
[0089] The MSCs used in culture can include cells derived from any
stem cell source, such as umbilical cord, umbilical cord blood,
placenta, embryonic stem cells, adipose tissue, bone marrow, or
other tissue-specific mesenchyme. These samples may be fresh,
frozen, or refrigerated. Methods of freezing cells are well known
in the art (see, e.g., Doyle et al. (1995) Cell & Tissue
Culture: Laboratory Procedures (John Wiley & Sons,
Chichester)).
[0090] Cryopreservation of stem cells prior to culture or
cryopreservation of expanded cells disclosed herein may be carried
out according to known methods. For example, cells may be suspended
in a "freezing medium" such as, for example, culture medium further
comprising 10% dimethylsulfoxide (DMSO), with or without 5-10%
glycerol, at a density, for example, of about 1-2.times.10.sup.6
cells/ml. The cells may be dispensed into glass or plastic vials,
which are then sealed and transferred to a freezing chamber of a
programmable or passive freezer. The optimal rate of freezing may
be determined empirically. For example, a freezing program that
gives a change in temperature of about -1.degree. C./min through
the heat of fusion may be used. Once vials containing the cells
have reached -80.degree. C., they may be transferred to a liquid
nitrogen storage area. Cryopreserved cells may be stored for a
period of years.
[0091] In some embodiments, freshly isolated cells from any stem
cell source may be cryopreserved to constitute a bank of cells,
portions of which may be withdrawn by thawing and then used to
produce the expanded cells of the invention as needed. Thawing may
be carried out rapidly, for example, by transferring a vial from
liquid nitrogen to a 37.degree. C. water bath. The thawed contents
of the vial may be immediately transferred under sterile conditions
to a culture vessel containing an appropriate medium such as
nutritive medium. Once in culture, the cells may be examined daily,
for example, with an inverted microscope to detect cell
proliferation, and subcultured as soon as they reach an appropriate
density.
[0092] Cells may be withdrawn from a cell bank as needed, and used
for the production of new stem cells or tissue either in vitro, for
example, as a three-dimensional scaffold culture, as described
below, or in vivo, for example, by direct administration of cells
to the site where tissue reconstitution or repair is needed. As
described herein, the expanded MSCs of the invention may be used to
reconstitute or repair tissue in a subject where the cells were
originally isolated from that subject's own bone marrow or other
tissue (i.e., autologous cells). Alternatively, the expanded MSCs
disclosed herein may be used as ubiquitous donor cells to
reconstitute or repair tissue in any subject (i.e., heterologous
cells).
[0093] Methods of MSC isolation from bone marrow are well
established. Prior to culture, a large proportion of
non-mesenchymal linage cells may be removed from a stem cell source
by negative or positive selection. For example, large numbers of
lineage-committed cells can be removed by selective magnetic bead
separations. In some embodiments, at least about 80%, usually at
least about 70% of the differentiated cells will be removed prior
to culture. Mononuclear cells from these tissues may be collected
by density gradient centrifugation and cultured in tissue culture
containers. Furthermore, bone marrow aspirates may be seeded onto
tissue culture plates. Non-adherent cells may be removed after 1 to
3 days. Adherent, spindle shaped fibroblast-like MSCs are kept and
expanded. After several hours to days, non-adherent cells are
washed away and the MSCs remain. It has also been reported that
MSCs may be obtained from cord blood, placenta and adipose
tissues.
[0094] Cultured MSCs can be further isolated using any method known
in the art. Generally, the MSCs are contacted with monoclonal
antibodies directed to cell surface antigens and either positively
or negatively selected. Such techniques for selection are well
known in the art and include sorting by immunomagnetic beads, by
complement mediated lysis, by "panning" with antibody attached to a
solid matrix, agglutination methods, magnetic activated cell
sorting (MACS), or fluorescence activated cell sorting (FACS).
[0095] The expanded MSCs of the invention have broad application in
treating and ameliorating disease and injury. The expanded MSCs of
the invention are useful in many therapeutic applications including
repairing, reconstituting, and regenerating tissue as well as gene
delivery. The MSCs of the invention can comprise both
lineage-committed and uncommitted cells; thus, both cell types can
be used together to accomplish multiple therapeutic goals, even
simultaneously in some embodiments. For example, in some
embodiments, the expanded MSCs of the invention can be used
directly as stem cell transplants or be used in stem cell grafts
either in suspension or on a cell culture support scaffold as noted
herein above.
[0096] The expanded MSCs of the invention can be placed in a
carrier medium before administration. For infusion, expanded MSCs
of the invention can be administered in any physiologically
acceptable medium, intravascularly, including intravenously,
although they may also be introduced into other convenient sites
such as into the bone marrow, where the cells may find an
appropriate site for regeneration and differentiation. Usually, at
least about 1.times.10.sup.5 cells/kg, at least about
5.times.10.sup.5 cells/kg, at least about 1.times.10.sup.6
cells/kg, at least about 2.times.10.sup.6 cells/kg, at least about
3.times.10.sup.6 cells/kg, at least about 4.times.10.sup.6
cells/kg, at least about 5.times.10.sup.6 cells/kg, at least about
6.times.10.sup.6 cells/kg, at least about 7.times.10.sup.6
cells/kg, at least about 8.times.10.sup.6 cells/kg, at least about
9.times.10.sup.6 cells/kg, at least about 10.times.10.sup.6
cells/kg, or more will be administered. See, for example, Ballen et
al. (2001) Transplantation 7:635-645. The MSCs may be introduced by
any method including injection, catheterization, or the like. If
desired, additional drugs or growth factors can be co-administered.
Drugs of interest include 5-fluorouracil and growth factors
including cytokines such as IL-2, IL-3, G-CSF, M-CSF, GM-CSF,
IFN.gamma., and erythropoietin. In addition, the MSCs can be
injected with collagen, Matrigel, alone or with other
hydrogels.
[0097] Administered MSCs may also comprise a mixture of cells
herein described and additional cells of interest. Additional cells
of interest include, without limitation, differentiated liver
cells, differentiated cardiac muscle, differentiated pancreatic
cells, and the like. These combinations are particularly useful
when the expanded MSCs of the invention are seeded on a
three-dimensional scaffold, hydrogel, or without carrier, as
disclosed herein above.
[0098] In one embodiment, the expanded MSC population of the
invention can be used to repair or reconstitute damaged or diseased
mesenchymal tissues, such as the heart, the pancreas, the liver,
fat tissue, bone, cartilage, endothelium, nerves, astrocytes,
dermis, and the like. Once the expanded MSCs migrate to or are
placed at the site of injury, they can differentiate to form new
tissues and supplement organ function. In some embodiments, the
cells are used to promote vascularization and, therefore, improve
oxygenation and waste removal from tissues. In these embodiments,
the expanded MSCs of the invention can be used to increase function
of differentiated tissues and organs such as the ischemic heart as
in cardiac failure or ischemic nerves as in stroke. Therefore, the
expanded MSCs of the invention are useful in any disease where
cellular function or organ function has been decremented.
[0099] "Supplementing a damaged organ" or "supplementing organ
function" is intended to mean increasing, enhancing, or improving
the function of an organ that is operating at less than optimum
capacity. The term is used to refer to a gain in function so that
the organ is operating at a physiologically acceptable capacity for
that subject. For example, the physiological acceptable capacity
for an organ from a child, e.g., a kidney or heart, would be
different from the physiological acceptable capacity of an adult,
or an elderly patient. The entire organ or part of the organ can be
supplemented. Preferably the supplementation results in an organ
with the same physiological response as a non-damaged or
non-diseased organ. In one embodiment, an organ is supplemented in
capacity when it is functioning to at least at about 10% of its
natural capacity.
[0100] The expanded MSCs of the present invention can be used for
implantation by contacting the cells with a tissue-engineered
construct prior to grafting as noted herein above. The construct
containing these cells is then implanted into a host in need of
such a graft. The cells of the invention are particularly useful
for promoting bone and cartilage generation, thereby facilitating
tissue regeneration and repair. These cells may also be used in
applications relating to graft versus host disease. The expanded
MSCs of the invention may also be useful as vasculature-promoting
stem cells. "Vascularization promoting" or "vasculature promoting"
is intended to mean promoting the growth of new vessels
(vasculogenesis) or inducing outgrowth from existing vessels
(angiogenesis), or any combination thereof.
[0101] "Differentiated cells" is intended to mean cells that are
committed to restricted tissue development. In some embodiments,
the expanded cells of the invention may comprise both
lineage-committed and uncommitted MSCs. Thus, in some
tissue-engineered constructs, the MSCs may give rise to both the
differentiated tissue and act as the source for the
vasculature-promoting stem cells. In addition, in some
tissue-engineered constructs, the MSCs may give rise to both the
differentiated tissue and act as the source for the
cardiovascular-promoting stem cells. In addition, in some
tissue-engineered constructs, the MSCs may give rise to both the
differentiated tissue and act as the source for the bone and
cartilage-promoting stem cells. In other embodiments, the source of
differentiated tissues may comprise cells or tissue from the
intended graft recipient or another donor. The cell or tissue
source may be differentiated prior to implantation. For example,
pancreatic beta cells can be differentiated using conditions
described for embryoid body formation as detailed in
Itskovich-Eldor et al. (2000) Mol. Med. 6:88.
[0102] In one specific embodiment, the vasculature-promoting stem
cells may be contacted with angiogenic growth factors such as VEGF
and bFGF. These stem cells may be contacted with angiogenic growth
factors prior to or after seeding onto the scaffold before
engrafting the tissue-engineered construct into a damaged organ. In
some embodiments, cytokine-impregnated polymers can release the
angiogenic growth factor, such as VEGF and bFGF, over time. In
other embodiments, the scaffold may be programmed to drive
expansion and proliferation of seeded cells (see, e.g., U.S. Patent
Application Publication No. 20040063206). In other embodiments,
microspheres or microcarriers may be contacted with the
vasculature-promoting stem cells and placed at a target site.
Microsphere-based scaffolds are well known in the art (see, e.g.,
Mahoney and Saltzman (2001) Nature Biotech. 19:934).
[0103] In one specific embodiment, the cardiovascular- or bone and
cartilage-promoting stem cells may be contacted with growth factors
such as BDNF, bFGF, BMP-2, BMP-4, DKK-1, EGF, EPO, fibronectin,
Flt-3/Flk-2 ligand, G-CSF, IGF-1, IL-11, IL-3, IL-5, IL-6, LIF,
PD-ECGF, SCF, SDF 1-.alpha., TGF-.beta., thrombospondin, and WNT
signaling agonists, including but not limited to BIO and WNT
proteins, WNT-1, WNT-2, WNT-2b, WNT-3, WNT-3a, WNT-4, WNT-5a,
WNT-5b, WNT-6, WNT-7a, WNT-7b, WNT-8a, WNT-8b, WNT-9a, WNT-9b,
WNT-10a, WNT-10b, WNT-11, and WNT-16. In one embodiment, the WNT
protein is WNT-3a.
[0104] The expanded MSCs of the invention can also be used for gene
therapy in patients in need thereof. In some embodiments, more
mature lineage-committed cells will be useful, especially where
transient gene expression is needed or where gene transduction is
facilitated by the maturation and division of the cells. For
example, some retroviral vectors require that the cell be cycling
for the gene to be integrated. Methods for transducing stem and
progenitor cells to deliver new and therapeutic genes are known in
the art.
[0105] Another aspect of the invention is a kit useful for
promoting the attachment, survival, and/or proliferation of MSCs,
comprising a serum-free cell culture system of the invention is
provided. Such a kit comprises a serum-free cell culture medium and
a cell culture surface described herein, and can comprise one or
more other components or reagents suitable for culturing the cell.
In one embodiment, the serum-free cell culture medium comprises any
of the cytokine cocktails described herein, for example, the
cocktail shown in Table 1 below, and a base medium, for example,
the base medium shown in Table 2 below, and a cell culture surface
comprising a cell culture support bound to a CAR, which in turn is
bound to an ECM protein. In some embodiments, the ECM is a mixture
of collagen I and fibronectin. In other embodiments, the kits
comprise these components and also comprise one or more reagents
for measuring cell proliferation in the culture. Such kits have
many uses, which will be evident to one of skill in the art. For
example, they can be used to propagate MSCs to be used in methods
of cell therapy. Such kits could be of commercial use, e.g., in
high-throughput drug studies.
[0106] The following examples are offered by way of illustration
and not by way of limitation.
EXPERIMENTAL
[0107] For these examples, the concentrations of various cytokines
in the culture media are described in Table 1, while the base
medium composition is described in Table 2.
1TABLE 1 Cytokine Cocktail for Serum-Free Cell Culture Medium
Concentration Growth Factor Abbrev. Factor Full Name Vendor (ng/ml)
BDNF Brain-derived Neurotrophic Factor R&D Systems, MN 1 bFGF
Basic Fibroblast Growth Factor BD Biosciences, MA 2.5 BIO
(2'Z,3'E)-6-bromoindirubi- n-3'-oxime Calbiochem 50 nM BMP-2 Bone
Morphogenic Protein 2 R&D Systems, MN 0.5 BMP-4 Bone
Morphogenic Protein 4 R&D Systems, MN 0.5 DKK-1 Dickkopf 1
R&D Systems, MN 10 EGF Epidermal Growth Factor BD Biosciences,
MA 10 Erythropoietin Erythropoietin R&D Systems, MN .05
units/ml Fibronectin Fibronectin BD Biosciences, MA 10 Flt-3/Flk-2
ligand Flt-3/Flk-2 ligand R&D Systems, MN 5 G-CSF Granulocytes
colony stimulating factor R&D Systems, MN 1 IGF-1 Insulin-like
Growth Factor 1 R&D Systems, MN 2.5 IL-11 Interleukin-11
R&D Systems, MN 0.1 IL-3 Interleukin-3 R&D Systems, MN 1
IL-5 Interleukin-5 R&D Systems, MN 0.1 IL-6 Interleukin-6
R&D Systems, MN 0.2 LIF Leukemia Inhibitory Factor Chemicon 10
PD-ECGF Platelet-derived Endothelial Cell Growth Factor R&D
Systems, MN 2 SCF Stem Cell Factor BD Biosciences, MA 2 SDF-1
.alpha. Stromal Cell Derived Factor 1 .alpha. R&D Systems, MN 3
TGF-.beta. Transforming Growth Factor-.beta. BD Biosciences 0.1-1
Thrombospondin Thrombospondin Sigma-Aldrich, MO 10 WNT-3a Wnt
protein 3a R&D Systems, MN 10
[0108]
2TABLE 2 Base Medium for Serum-Free Cell Culture Medium Base Medium
Component Amount DMEM/MCDB-201 Media 60:40 Ascorbic acid
2-phosphate 1 .times. 10.sup.-4 M Dexamethasone 1 .times. 10.sup.-9
M Linoleic Acid-Bovine Serum 0.1% Albumin Insulin 5 .mu.g/mL
Transferrin 5 .mu.g/mL Sodium selenite 5 ng/mL
Example 1
MSC Expansion in Various Serum-Free Media
[0109] MSCs along with complete growth medium were purchased from
Cambrex Biosciences (Baltimore, Md.). Frozen cells were thawed and
cultured following manufacturer's instruction. After reaching
.about.90% confluency, MSCs were washed once with PBS, removed from
the culture surface using Trypsin/EDTA and replated at a density of
1600 cells/well (for 96 well plates) or at a density of 50,000
cells/well (for 6-well plates) which corresponds to the
manufacturers recommended seeding density of 5000
cells/cm.sup.2.
[0110] At day 6 or 7, cells were washed once with PBS, fixed using
4% paraformaldehyde for 15 minutes and then stained using DAPI
according to manufacturer's suggestions (Molecular Probes, Eugene,
Oreg.). For 96-well plate experiments, one image per well was taken
at 4.times. magnification using Molecular Devices' Discovery-1 high
content screening system. Cell nuclei enumeration was determined
using Metamorph Image Analysis Software (Molecular Devices,
Sunnyvale, Calif.). The number of DAPI stained cell nuclei
corresponds directly with the total number of cells per well. For
6-well plates, cell enumeration was determined using Trypan Blue
exclusion and manual counting using a hemocytometer. Multiple
donors (different preps of MSCs) were tested in serum-free
conditions while the cells were also cultured on tissue culture
polystyrene (TCPS) plates using the manufacturer's standard
serum-containing medium as the control.
[0111] Several serum-free expansion conditions were scaled up into
the 6-well plate format (.about.10 cm.sup.2) and cultured for 7
days. Cells were then washed with PBS and then removed from the
6-well plates using Trypsin/EDTA. Cells were then reseeded into
96-well plates at two different densities according to
manufacturer's specifications. MSCs induced to form adipocytes were
seeded at 7000 cells/well and cultured in Stem Cell Technologies
(Vancouver, B. C., Canada) adipogenic medium supplement. MSCs
induced to form osteoblasts were seeded at 1000 cells/well using
Cambrex Biosciences (Baltimore, Md.) osteogenic media supplements.
Two to three weeks after induction, cells were washed once with PBS
and fixed using 4% paraformaldehyde. Fixed cells were then stained
with Oil Red O (Sigma-Aldrich (St. Louis, Mo.)) for adipocytes and
BCIP/NBT (Sigma-Aldrich (St. Louis, Mo.)) for alkaline
phosphatase/osteoblasts. Bright field images at multiple sites per
well were taken of stained cells at 10.times. magnification.
Percentage of differentiation was determined using Metamorph Image
Analysis Software (Molecular Devices, Sunnyvale, Calif.) to
calculate total stained area by threshold image analysis.
[0112] Initial experiments compared serum-free medium expansion of
MSCs in G2 medium on Collagen I (Col 1, Sigma-Aldrich)/Fibronectin
(FN, BD Biosciences) cell adhesion resistant (CAR) surface modified
96-well plates. MSCs were expanded for 7 days in G2 serum-free
medium on the CAR surface and cell nuclei counts were compared to
MSCs expanded in the manufacturer's recommended serum-containing
medium on tissue culture polystyrene (TCPS) 96-well plates. MSCs
expanded in the G2-CAR surface serum free environment were
comparable to the manufacturer's recommended serum-containing
expansion conditions up to 7 days (see FIG. 1).
Example 2
Differentiation Capacity and Surface Marker Characterization of G2
Expanded MSCs
[0113] MSCs expanded in serum-free media of the present invention
on CAR Col 1/FN surface for 7 days remained multipotent. Briefly,
MSCs expanded in culture environments of the present invention were
removed from the serum-free media and then cultured in either
adipogenic (fat) or osteogenic (bone) induction media (per
manufacturer's suggestions). MSCs expanded in the serum-free
environments of the present invention were able to differentiate
towards both adipogenic (see FIG. 2) and osteogenic (see FIG. 3)
lineages. These results demonstrate that the serum-free
environments of the present invention help to maintain stem cell
pluripotency in expanded MSCs at least as well as the industry
standard serum-containing media (Cambrex). This demonstrates that
these cells are not committed towards specific lineages, and that
therefore these MSCs may still be used for a variety or research or
clinical applications.
[0114] MSCs cultured in serum-free environments for 7 days retain
cell surface markers showing equivalency to Cambrex cell
populations expanded in serum-containing media. In addition, MSCs
cultured in serum-free environments lacked CD45 marker expression,
indicating that these cells do not differentiate towards
hematopoietic lineages.
Example 3
Comparison of Media and Cell Culture Surface Conditions
[0115] MSCs cultured in three different media (G2, base medium
without G2 growth factors/cytokines, and in serum-containing
complete medium) all expand better on a Col 1/FN CAR surface as
compared to TCPS surfaces (see FIG. 4). Also, MSCs cultured in G2
serum-free medium expand to equivalent levels as MSCs cultured in
serum-containing medium.
Example 4
Media Optimization
[0116] Media compositions that support the expansion of MSCs were
identified. The G2 serum-free medium served as a control for this
optimization experiment. Screening plates were prepared using
96-well plates containing 30 factors in 60 different compositions.
One thousand six hundred (1600) MSCs were incubated in each well
for 7 days. Best media compositions were selected based on the
average number of live cells obtained from the repeats from each
screen.
[0117] Best well "hits" of the cytokine screen were the C6, D3, C2,
and G5 wells, respectively.
[0118] The C6 well contained cytokines BDNF, bFGF, BIO, BMP-2,
BMP-4, DKK-1, EGF, EPO, fibronectin, thrombospondin, Flt-3/Flk-2
ligand, G-CSF, IGF-1, IL-11, IL-3, IL-5, IL-6, LIF, PD-ECGF, SCF,
SDF-1.alpha., and WNT-3a.
[0119] The D3 well contained cytokines bFGF, BMP-2, EGF, EPO,
fibronectin, thrombospondin, Flt-3/Flk-2 ligand, IGF-1, IL-11,
IL-5, and WNT-3a
[0120] The C2 well contained cytokines bFGF, BIO, BMP-2, BMP-4,
EGF, EPO, Flt-3/Flk-2 ligand, IGF-1, IL-11, IL-5, and WNT-3a.
[0121] The G5 well contained cytokines bFGF, BMP-4, DKK-1,
fibronectin, thrombospondin, Flt-3/Flk-2 ligand, IL-6, PD-ECGF,
SDF-1.alpha., and WNT-3a.
[0122] Cytokine compositions of wells C6, D3, C2, and G5 are
referred to hereinafter as "C6," "D3," "C2," and "G5,"
respectively.
[0123] Media compositions C6, D3, C2, and G5 all outperformed G2
serum free medium controls and 10% serum-containing complete medium
(see FIG. 5). These serum free media compositions contain 22, 11,
11, and 10 growth factor/cytokine additives, respectively. In
addition, serum free media G2 and D3 both performed as well or
better than serum-containing complete medium controls and
significantly outperform a previously published serum free MSC
expansion medium (Lennon et al. (1995) Exp. Cell Res. 219:211-222;
U.S. Pat. No. 5,908,782).
[0124] Three of these serum-free media compositions (D3, C2, and
G5) were used to confirm MSCs retained multipotentiality. MSCs were
cultured in one of the three serum-free media for 7 days. Expanded
MSCs were removed from the serum-free media and then cultured in
either adipogenic (fat) or osteogenic (bone) induction media (per
manufacturer's suggestions). MSCs remained multipotential and
differentiated towards both adipogenic and osteogenic lineages in
all three serum-free media, each comparable to the manufacturer's
recommended serum-containing complete medium controls (see FIGS. 6A
and 6B).
[0125] Further media optimization was performed and the best well
"hits" from this cytokine screen were the G7, G4 and C8 wells,
respectively.
[0126] The G7 well contained cytokines bFGF, BMP-2, fibronectin,
thrombospondin, Flt-3/Flk-2 ligand, IL-11, LIF, and WNT-3a.
[0127] The G4 well contained cytokines bFGF, BMP-2, EGF, IL-11,
PD-ECGF, and WNT-3a.
[0128] The C8 well contained cytokines bFGF, BMP-2, EGF,
fibronectin, thrombospondin, Flt-3/Flk-2 ligand, IL-11, IL-5, LIF,
PD-ECGF, and WNT-3a.
[0129] Cytokine compositions of wells G7, G4, and C8 are referred
to hereinafter as "G7," "G4," and "C8," respectively. Media
compositions G7, G4, and C8 all expanded MSCs as well as or better
than serum-containing controls (see FIG. 7).
[0130] Additional optimization data was collected employing a
variety of media compositions. In one experiment, MSCs were
cultured in: 1) the G4 serum-free medium composition; 2)
G4+TGF-.beta.; 3) bFGF in base medium only; 4) EGF in base medium
only; 5) TGF-.beta. in base medium only; 6) base medium alone; and
7) serum-containing medium. Although MSCs cultured in G4 expanded
as well or better than serum-containing medium, MSCs cultured in
G4+TGF-.beta. far outperformed any other media composition, nearly
triple the serum-containing medium controls (see FIG. 8).
Furthermore, the single factors, while promoting MSC expansion, did
not perform as well as serum in expanding MSCs. However,
synergistic combinations of these factors (e.g., G4) expand MSCs
more than any single factor, and the addition of TGF-.beta.
substantially enhanced expansion. These factor synergies, in
combination with the collagen 1+fibronectin surface, comprise
serum-free environments that substantially outperform anything that
has been established to date. G4+TGF-.beta. serum-free medium was
scaled up and used to expand MSCs in 6-well Col 1/FN CAR plates.
MSCs were seeded on 6-well plates at 50,000 per well (n=3). Cells
in serum containing complete media were seeded onto a 6-well TCPS
plate. At confluency, all cells were removed and cell number was
determined using Trypan Blue exclusion and hemocytometer counting.
Cells were replated at 50,000 per well under the same condition for
a second round of expansion. Cell numbers were again determined as
above. After the first round of expansion (approximately 6 days),
MSCs expanded in serum-free conditions outperformed the
manufacturer's recommended serum-containing medium controls. After
the second round of expansion (approximately 6 days), the MSCs
continued to expand in the serum-free medium at an increased rate
as compared to the serum-containing medium controls. G4+TGFb
provided 5.4 and 6.5 fold expansion in the first and second rounds,
respectively, and regular medium gave 3.5 and 3.1 fold expansion in
the first and second rounds, respectively.
[0131] In another experiment, a serum-free medium comprising base
medium and WNT-3a, bFGF, and TGF-.beta. was compared to media
compositions containing the same components but adding one, two,
three, four, five, six, or seven additional cytokines that include
EGF, fibronectin, thrombospondin, BMP-2, BMP-4, IL-11, and PD-ECGF
in their ability to expand MSCs in culture. Adding one, two, three,
four, five, six, or seven growth factors to the serum free medium
comprising base medium and WNT-3a, bFGF, and TGF-.beta. had a
positive effect on MSC expansion. In particular, the addition of
EGF to the three growth factors already present in the serum-free
medium resulted in approximately a 20% increase in MSC expansion
over compositions that did not contain EGF, and both BMPs on top of
EGF further enhanced expansion. Furthermore, the addition of all
seven cytokines to WNT-3a, bFGF, and TGF-.beta. gave the best
expansion, demonstrating that building complexity into the serum
free environments may further enhance MSC expansion. However, the
synergies between WNT-3a, bFGF, EGF, and TGF-.beta. were found to
have the greatest effects on MSC serum-free expansion on collagen
1+fibronectin surfaces.
[0132] In another experiment, a serum-free medium comprising base
medium was compared to media comprising the base medium plus
various combinations of WNT-3a, BIO, bFGF, EGF, and TGF-.beta. in
their ability to expand MSCs in culture (see FIG. 9). Specifically,
the combinations tested were: base medium alone (no growth
factors), Cambrex Biosciences (Baltimore, Md.) serum-containing
medium on TCPS, base medium+bFGF+TGF-.beta., base
medium+WNT-3a+bFGF, base medium+bFGF+EGF, base
medium+WNT-3a+TGF-.beta., base medium+WNT-3a+EGF, base
medium+EGF+TGF-.beta., base medium+WNT-3a, base
medium+bFGF+EGF+TGF-.beta- ., base
medium+WNT-3a+bFGF+EGF+TGF-.beta., and base
medium+bFGF+EGF+TGF-.beta.+BIO. Although all conditions
outperformed base medium alone, the growth factor combinations that
produced the greatest MSC expansion were base
medium+EGF+TGF-.beta., base medium+bFGF+TGF-.beta., base
medium+bFGF+EGF+TGF-.beta., base medium+WNT-3a+bFGF+EGF+TGF-.beta.,
and base medium+bFGF+EGF+TGF-.beta.+BI- O.
[0133] All publications and patent applications mentioned in the
specification are indicative of the level of those skilled in the
art to which this invention pertains. All publications and patent
applications are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually to be incorporated by reference.
[0134] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
* * * * *