U.S. patent application number 15/553115 was filed with the patent office on 2018-02-08 for cell culture method for mesenchymal stem cells.
This patent application is currently assigned to Mesoblast International Sarl. The applicant listed for this patent is Mesoblast International Sarl. Invention is credited to Paul SIMMONS, Colby SUIRE.
Application Number | 20180037867 15/553115 |
Document ID | / |
Family ID | 55453191 |
Filed Date | 2018-02-08 |
United States Patent
Application |
20180037867 |
Kind Code |
A1 |
SIMMONS; Paul ; et
al. |
February 8, 2018 |
CELL CULTURE METHOD FOR MESENCHYMAL STEM CELLS
Abstract
The present disclosure relates to methods, cell culture medium
and compositions that promote cell proliferation during fetal
bovine serum free cell culture.
Inventors: |
SIMMONS; Paul; (Melbourne,
Victoria, AU) ; SUIRE; Colby; (New York, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Mesoblast International Sarl |
Meyrin |
|
CH |
|
|
Assignee: |
Mesoblast International
Sarl
Meyrin
CH
|
Family ID: |
55453191 |
Appl. No.: |
15/553115 |
Filed: |
March 4, 2016 |
PCT Filed: |
March 4, 2016 |
PCT NO: |
PCT/EP2016/054640 |
371 Date: |
August 23, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2501/11 20130101;
C12N 2501/115 20130101; C12N 2501/135 20130101; C12N 2500/99
20130101; C12N 5/0662 20130101 |
International
Class: |
C12N 5/0775 20060101
C12N005/0775 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 4, 2015 |
AU |
AU 2015900752 |
Mar 5, 2015 |
AU |
AU 2015900777 |
Claims
1. A method of promoting stem cell proliferation in-vitro, the
method comprising: culturing a population of mesenchymal lineage
stem cells in a fetal bovine serum free cell culture medium
comprising platelet derived growth factor (PDGF) and fibroblast
growth factor 2 (FGF2), wherein the level of FGF2 is less than
about 6 ng/ml.
2. A fetal bovine serum free cell culture medium for promoting stem
cell proliferation in-vitro comprising: a basal medium; platelet
derived growth factor (PDGF) fibroblast growth factor 2 (FGF2);
wherein the level of FGF2 is less than about 6 ng/ml.
3. The method of claim 1 or the culture medium of claim 2, wherein
the level of FGF2 is between at least about 2 pg/ml and 40
pg/ml.
4. The method of claim 1 or the culture medium of claim 2, wherein
the level of FGF2 is about 20 pg/ml.
5. The method or culture medium of any one of claims 1 to 4,
wherein the PDGF is PDGF-BB.
6. The method or culture medium of claim 5, wherein the level of
PDGF-BB is between at least about 3.0 ng/ml and about 120
ng/ml.
7. The method or culture medium of claim 5, wherein the level of
PDGF-BB is between about 9.0 ng/ml and about 60 ng/ml.
8. The method or culture medium of claim 5, wherein the level of
PDGF-BB is at least about 30 ng/ml.
9. The method or culture medium of any one of claims 1 to 8,
wherein the cell culture medium further comprises epidermal growth
factor (EGF).
10. The method or culture medium of claim 9, wherein the level of
EGF is between about 0.08 ng/ml and about 7 ng/ml.
11. The method or culture medium of claim 9, wherein the level of
EGF is between about 0.4 ng/ml and about 1.6 ng/ml.
12. The method or culture medium of claim 9, wherein the level of
EGF is at least about 0.8 ng/ml.
13. The method or culture medium of any one of claims 1 to 12,
wherein the cell culture medium comprises alpha-minimal essential
medium or fetal bovine serum free expansion medium.
14. The method or culture medium of any one of claims 1 to 13,
wherein the cell culture medium maintains the stem cells in an
undifferentiated state.
15. The method or culture medium of any one of claims 1 to 14,
wherein the stem cell is a mesenchymal lineage precursor cell.
16. The method of any one of claims 1 to 15, wherein the stem cell
is a mesenchymal stem cell.
Description
TECHNICAL FIELD
[0001] The present disclosure relates to methods, cell culture
media and compositions that promote stem cell proliferation during
fetal bovine serum (FBS) free cell culture.
BACKGROUND
[0002] Multipotent mesenchymal stem cells (MSC) have been proposed
as an attractive candidate for therapeutic applications because of
their high proliferation and differentiation potential as well as
immunoregulatory and other beneficial properties (Caplan A I (2007)
J. Cell Physiol., 213, 341-347; Prockop D J (2007) Clin Pharmacol
Ther., 82, 241-243). Ex vivo propagation of sparse populations of
mesenchymal stem cells (MSC) is often necessary for generating
numbers suitable for therapeutic applications.
[0003] Conventional media used for isolating and expanding MSC
consist of a defined basal medium (e.g. Dulbecco's modified Eagle's
medium (DMEM) or .alpha.-modified minimum essential medium
(.alpha.-MEM)) supplemented with fetal bovine serum because of its
high content of stimulatory growth factors. Although these media
are generally reported to support the proliferation of MSC for
multiple passages, concerns have been raised because of the
potential risks associated with fetal bovine serum (Dimarakis &
Levicar (2006) Stem Cells., 24, 1407-1408; Mannello & Tonti
(2007) Stem Cells., 25, 1603-1609). In particular, fetal bovine
serum may contain harmful contaminants such as prion, viral and
zoonotic agents, and can elicit immune reactions. Moreover, the
poorly defined nature of fetal bovine serum, and its high degree of
batch-to-batch variation, can cause inconsistencies in the
growth-supporting properties of media, and thus make
standardization of a cell production process difficult.
[0004] Human sourced supplements, such as human serum and platelet
lysate, have been investigated as a replacement for fetal bovine
serum. Human serum is not generally considered a suitable
replacement because of its lack of availability and inconsistent
growth-promoting potential. Human platelet-derived supplements such
as platelet lysate (hPL) and platelet-rich plasma have recently
been proposed as a superior alternative (Doucet et al. (2005) J
Cell Physiol., 205, 228-236; Muller et al. (2006), Cytotherapy., 8,
437-444; Capelli et al. (2007) Bone Marrow Transplant., 40, 785-91;
Lange et al. (2007) J Cell Physiol., 213, 18-26; Reinisch et al.
(2007) Regen Med., 2, 371-82). While these studies demonstrated
considerable growth-promoting properties of pooled human platelet
derivatives, their impact on MSC growth is not consistent (Bieback
et al. (2008) Transfus Med Hemother., 35, 286-294). Furthermore,
the high cost of these hPL formulations can be prohibitive for
commercial cell culture.
[0005] Accordingly, there remains an unmet need for cost effective
methods of supporting both the isolation and rapid expansion of MSC
in fetal bovine serum free cell culture.
SUMMARY
[0006] The present inventors have found that platelet derived
growth factor (PDGF) is required for MSC proliferation in fetal
bovine serum free culture conditions. The present inventors have
also found that fibroblast growth factor 2 (FGF2) and PDGF
synergistically promote MSC proliferation in fetal bovine serum
free culture conditions. Surprisingly, this synergistic effect is
maintained when FGF2 is present at very low levels. Accordingly,
the present inventors have found that they are able to increase
in-vitro stem cell proliferation in fetal bovine serum free culture
conditions using a specific combination of growth factors. These
findings suggest that the methods, culture medium and compositions
of the present disclosure may provide a suitable, cost effective
replacement for serum that may also be suitable for increasing the
efficiency of stem cell culture.
[0007] Thus, in one example, the present disclosure relates to a
method of promoting stem cell proliferation in-vitro, the method
comprising culturing a population of mesenchymal lineage stem cells
in a fetal bovine serum free cell culture medium comprising PDGF
and FGF2, wherein the level of FGF2 is less than about 6 ng/ml. In
another example, the present disclosure relates to a method of
promoting mesenchymal lineage precursor cell proliferation
in-vitro. In another example, the present disclosure relates to a
method of promoting mesenchymal stem cell proliferation
in-vitro.
[0008] PDGF, FGF2 and any other culture medium components may be
provided in a stem cell culture medium or in a composition for
addition to a stem cell culture medium for use in the methods of
the present disclosure. Thus, in another example, the present
disclosure provides a fetal bovine serum free cell culture medium,
the cell culture medium comprising:
[0009] a basal medium;
[0010] platelet derived growth factor (PDGF);
[0011] fibroblast growth factor 2 (FGF2);
wherein, the level of FGF2 in the culture medium is less than about
6 ng/ml.
[0012] In another example, the level of FGF2 in the culture medium
is between about 2 pg/ml and 40 pg/ml. In another example, the
level of FGF2 in the culture medium is about 20 pg/ml.
[0013] In another example, the PDGF is PDGF-BB or PDGF-AB. In one
example, the PDGF is PDGF-BB. In another example, the level of
PDGF-BB in the culture medium is between about 3.0 ng/ml and about
120 ng/ml. In another example, level of PDGF-BB in the culture
medium is between about 9 ng/ml and about 60 ng/ml. In another
example, the level of PDGF-BB in the culture medium is at least
about 30 ng/ml.
[0014] The present inventors have also found that fibroblast growth
factor 2 (FGF2) acts synergistically with other growth factors to
promote MSC proliferation. Accordingly, these growth factors can
also be used in the methods, cell culture medium and compositions
of the present disclosure. In an example, the cell culture medium
further comprises epidermal growth factor (EGF). In an example, the
level of EGF in the cell culture medium is between about 0.08 ng/ml
and about 7 ng/ml. For example, the level of EGF in the cell
culture medium is at least about 5 ng/ml. In another example, the
level of EGF in the cell culture medium is between about 0.2 ng/ml
and about 3.2 ng/ml. In another example, the level of EGF in the
cell culture medium is between about 0.4 ng/ml and about 1.6 ng/ml.
In another example, the level of EGF is at least about 0.8
ng/ml.
[0015] In another example, the medium is for the culture of
mesenchymal stem cells. In another example, the medium is for the
culture of mesenchymal lineage precursor cells.
[0016] In an example, the basal medium is alpha-minimal essential
medium or fetal bovine serum free expansion medium such as
StemSpan.TM..
[0017] In another example, the cell culture medium maintains the
stem cells in an undifferentiated state.
[0018] In another example, the present disclosure relates to a
composition for addition to a fetal bovine serum free stem cell
culture medium, the composition comprising:
[0019] platelet derived growth factor (PDGF);
[0020] fibroblast growth factor 2 (FGF2);
wherein the FGF2 is present in the composition in a sufficient
amount to be added to a stem cell culture media at a level less
than about 6 ng/ml. In another example, the FGF2 is present in the
composition in a sufficient amount to be added to a stem cell
culture media at a level between about 5 pg/ml and 40 pg/ml. In
another example, the FGF2 is present in the composition in a
sufficient amount to be added to a stem cell culture media at a
level between about 20 pg/ml.
[0021] In another example, the PDGF in the composition is PDGF-BB
or PDGF-AB. In one example, the PDGF in the composition is PDGF-BB.
In another example, the PDGF-BB is present in the composition in a
sufficient amount to be added to a stem cell culture media at a
level between about 7.5 ng/ml and about 120 ng/ml. In another
example, the PDGF-BB is present in the composition in a sufficient
amount to be added to a stem cell culture media at a level between
about 15 ng/ml and about 60 ng/ml. In another example, the PDGF-BB
is present in the composition in a sufficient amount to be added to
a stem cell culture media at a level about at least 30 ng/ml.
[0022] In another example, the cell composition further comprises
epidermal growth factor (EGF). In one example, the EGF is present
in the composition in a sufficient amount to be added to a stem
cell culture media at a level between about 0.1 ng/ml and about 7
ng/ml. For example, EGF may be present in the composition in a
sufficient amount to be added to a stem cell culture media at a
level of about 5 ng/ml. In one example, the EGF is present in the
composition in a sufficient amount to be added to a stem cell
culture media at a level between about 0.2 ng/ml and about 3.2
ng/ml. In another example, the EGF is present in the composition in
a sufficient amount to be added to a stem cell culture media at a
level between about 0.4 ng/ml and about 1.6 ng/ml. In another
example, the EGF is present in the composition in a sufficient
amount to be added to a stem cell culture media at a level about at
least 0.8 ng/ml.
[0023] In another example, the composition is added to a medium
that is suitable for the culture of mesenchymal lineage precursor
cells. In another example, the composition is added to a medium
that is suitable for the culture of mesenchymal stem cells. In
another example, the composition is added to a medium that is
suitable for the culture of mesenchymal lineage precursor
cells.
[0024] In another example, the composition is added to
alpha-minimal essential medium or fetal bovine serum free expansion
medium such as StemSpan.TM..
BRIEF DESCRIPTION OF DRAWINGS
[0025] FIG. 1: Effect of platelet lysate percentage on MPC
proliferation.
[0026] FIG. 2: Effect of antibody neutralisation of growth factors
on MPC proliferation.
[0027] FIG. 3: Effect of combined antibody neutralisation of growth
factors on MPC proliferation.
[0028] FIG. 4: MPC proliferation following cell culture on tissue
culture plastic using Eagles Alpha MEM supplemented with varying
concentrations of PDGF-BB, EGF and FGF2.
[0029] FIG. 5: MPC proliferation following cell culture on
fibronectin using Eagles Alpha MEM supplemented with varying
concentrations of PDGF-BB, EGF and FGF2.
[0030] FIG. 6: MPC proliferation following cell culture on 3% hPL
using Eagles Alpha MEM supplemented with varying concentrations of
PDGF-BB, EGF and FGF2.
[0031] FIG. 7: MPC proliferation following cell culture on tissue
culture plastic using StemSpan.TM. supplemented with varying
concentrations of PDGF-BB, EGF and FGF2.
[0032] FIG. 8: MPC proliferation following cell culture on
fibronectin using StemSpan.TM. supplemented with varying
concentrations of PDGF-BB, EGF and FGF2.
[0033] FIG. 9: MPC proliferation following cell culture on 3% hPL
using StemSpan.TM. supplemented with varying concentrations of
PDGF-BB, EGF and FGF2.
[0034] FIG. 10: Cell proliferation in media comprising PDGF (P),
FGF (F), EGF (E) or a combination thereof (PEF).
[0035] FIG. 11: Cytokine levels in cell culture medium after cell
propagation.
DETAILED DESCRIPTION
General Techniques and Definitions
[0036] Unless specifically defined otherwise, all technical and
scientific terms used herein shall be taken to have the same
meaning as commonly understood by one of ordinary skill in the art
(e.g., in cell culture, molecular biology, stem cell culture,
protein chemistry, and biochemistry).
[0037] Unless otherwise indicated, cell culture techniques and
assays utilized in the present disclosure are standard procedures,
well known to those skilled in the art. Such techniques are
described and explained throughout the literature in sources such
as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley
and Sons (1984), J. Sambrook et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.
A. Brown (editor), Essential Molecular Biology: A Practical
Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D.
Hames (editors), and F. M. Ausubel et al. (editors), Current
Protocols in Molecular Biology, Greene Pub. Associates and
Wiley-Interscience (1988, including all updates until present), Ed
Harlow and David Lane (editors) Antibodies: A Laboratory Manual,
Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al.
(editors) Current Protocols in Immunology, John Wiley & Sons
(including all updates until present).
[0038] The term "and/or", e.g., "X and/or Y" shall be understood to
mean either "X and Y" or "X or Y" and shall be taken to provide
explicit support for both meanings or for either meaning.
[0039] As used herein, the term "about", unless stated to the
contrary, refers to +/-10%, more preferably +/-5%, of the
designated value.
[0040] The term "level" is used to define the amount of a
particular substance present in the cell culture medium and
compositions of the present disclosure. For example, a particular
concentration, weight, percentage (e.g. v/v %) or ratio can be used
to define the level of a particular substance.
[0041] In the context of the present disclosure, the term
"sufficient" is used to define an amount that provides a specific
concentration when dissolved in a stem cell culture medium. A
"sufficient amount" is dictated by the volume of culture medium
required. For example, if the required concentration of FGF2 in a
stem cell culture medium was about 10 pg/ml and 500 ml of cell
culture media was required, a sufficient amount would be about 5
ng.
[0042] Throughout this specification the word "comprise", or
variations such as "comprises" or "comprising", will be understood
to imply the inclusion of a stated element, integer or step, or
group of elements, integers or steps, but not the exclusion of any
other element, integer or step, or group of elements, integers or
steps.
[0043] Throughout this specification, unless specifically stated
otherwise or the context requires otherwise, reference to a single
step, composition of matter, group of steps or group of
compositions of matter shall be taken to encompass one and a
plurality (i.e. one or more) of those steps, compositions of
matter, groups of steps or group of compositions of matter.
[0044] Those skilled in the art will appreciate that the disclosure
described herein is susceptible to variations and modifications
other than those specifically described. It is to be understood
that the disclosure includes all such variations and modifications.
The disclosure also includes all of the steps, features,
compositions and compounds referred to or indicated in this
specification, individually or collectively, and any and all
combinations or any two or more of said steps or features.
[0045] The present disclosure is not to be limited in scope by the
specific embodiments described herein, which are intended for the
purpose of exemplification only. Functionally-equivalent products,
compositions and methods are clearly within the scope of the
disclosure, as described herein.
[0046] Any example disclosed herein shall be taken to apply mutatis
mutandis to any other example unless specifically stated
otherwise.
Mesenchymal Lineage Precursor Cells
[0047] As used herein, the term "mesenchymal lineage precursor or
stem cells" refers to undifferentiated multipotent cells that have
the capacity to self renew while maintaining multipotentency and
the capacity to differentiate into a number of cell types either of
mesenchymal origin, for example, osteoblasts, chondrocytes,
adipocytes, stromal cells, fibroblasts and tendons, or
non-mesodermal origin, for example, hepatocytes, neural cells and
epithelial cells. For the avoidance of doubt, a "mesenchymal
lineage precursor cell" refers to a cell which can differentiate
into a mesenchymal cell such as bone, cartilage, muscle and fat
cells, and fibrous connective tissue.
[0048] The term "mesenchymal lineage precursor or stem cells"
includes both parent cells and their undifferentiated progeny. The
term also includes mesenchymal precursor cells, multipotent stromal
cells, mesenchymal stem cells (MSCs), perivascular mesenchymal
precursor cells, and their undifferentiated progeny.
[0049] Mesenchymal lineage precursor or stem cells can be
autologous, xenogenic, syngenic or isogenic. Autologous cells are
isolated from the same individual to which they will be
reimplanted. Allogeneic cells are isolated from a donor of the same
species. Xenogenic cells are isolated from a donor of another
species. Syngenic or isogenic cells are isolated from genetically
identical organisms, such as twins, clones, or highly inbred
research animal models.
[0050] Mesenchymal lineage precursor or stem cells reside primarily
in the bone marrow, but have also shown to be present in diverse
host tissues including, for example, cord blood and umbilical cord,
adult peripheral blood, adipose tissue, trabecular bone and dental
pulp.
[0051] In one example the mesenchymal lineage precursor or stem
cells are STRO-1+ mesenchymal precursor cells. As used herein, the
phrase "STRO-1+ multipotential cells" shall be taken to mean
STRO-1+ and/or TNAP+ progenitor cells capable of forming
multipotential cell colonies.
[0052] STRO-1+ multipotential cells are cells found in bone marrow,
blood, dental pulp cells, adipose tissue, skin, spleen, pancreas,
brain, kidney, liver, heart, retina, brain, hair follicles,
intestine, lung, lymph node, thymus, bone, ligament, tendon,
skeletal muscle, dermis, and periosteum; and are capable of
differentiating into germ lines such as mesoderm and/or endoderm
and/or ectoderm. Thus, STRO-1+ multipotential cells are capable of
differentiating into a large number of cell types including, but
not limited to, adipose, osseous, cartilaginous, elastic, muscular,
and fibrous connective tissues. The specific lineage-commitment and
differentiation pathway which these cells enter depends upon
various influences from mechanical influences and/or endogenous
bioactive factors, such as growth factors, cytokines, and/or local
microenvironmental conditions established by host tissues.
[0053] Mesenchymal lineage precursor or stem cells can be isolated
from host tissues and enriched for by selection of STRO-1+ cells.
For example, a bone marrow aspirate from a subject may be further
treated with an antibody to STRO-1 or TNAP to enable selection of
mesenchymal lineage precursor or stem cells. In one example, the
mesenchymal lineage precursor or stem cells can be enriched for by
using the STRO-1 antibody described in (Simmons & Torok-Storb,
1991).
[0054] The terms "enriched", "enrichment" or variations thereof are
used herein to describe a population of cells in which the
proportion of one particular cell type or the proportion of a
number of particular cell types is increased when compared with an
untreated population of the cells (e.g., cells in their native
environment). In one example, a population enriched for STRO-1+
cells comprises at least about 0.1% or 0.5% or 1% or 2% or 5% or
10% or 15% or 20% or 25% or 30% or 50% or 75% STRO-1+ cells. In
this regard, the term "population of cells enriched for STRO-1+
cells" will be taken to provide explicit support for the term
"population of cells comprising X % STRO-1+ cells", wherein X % is
a percentage as recited herein. The STRO-1+ cells can, in some
examples, form clonogenic colonies, e.g. CFU-F (fibroblasts) or a
subset thereof (e.g., 50% or 60% or 70% or 80% or 90% or 95%) can
have this activity.
[0055] In one example, the population of cells is enriched from a
cell preparation comprising STRO-1+ cells in a selectable form. In
this regard, the term "selectable form" will be understood to mean
that the cells express a marker (e.g., a cell surface marker)
permitting selection of the STRO-1+ cells. The marker can be
STRO-1, but need not be. For example, cells (e.g., mesenchymal
precursor cells) expressing STRO-2 and/or STRO-3 (TNAP) and/or
STRO-4 and/or VCAM-1 and/or CD146 and/or 3G5 also express STRO-1
(and can be STRO-1b). Accordingly, an indication that cells are
STRO-1+ does not mean that the cells are selected by STRO-1
expression. In one example, the cells are selected based on at
least STRO-3 expression, e.g., they are STRO-3+(TNAP+).
[0056] Reference to selection of a cell or population thereof does
not necessarily require selection from a specific tissue source. As
described herein STRO-1+ cells can be selected from or isolated
from or enriched from a large variety of sources. That said, in
some examples, these terms provide support for selection from any
tissue comprising STRO-1+ cells (e.g., mesenchymal precursor cells)
or vascularized tissue or tissue comprising pericytes (e.g.,
STRO-1+ pericytes) or any one or more of the tissues recited
herein.
[0057] In one example, the mesenchymal lineage precursor or stem
cells used in the present disclosure express one or more markers
individually or collectively selected from the group consisting of
TNAP+, VCAM-1+, THY-1+, STRO-2+, STRO-4+(HSP-90.beta.), CD45+,
CD146+, 3G5+ or any combination thereof.
[0058] By use of the term "individually" it is meant that the
disclosure encompasses the recited markers or groups of markers
separately, and that, notwithstanding that individual markers or
groups of markers may not be separately listed herein the
accompanying claims may define such marker or groups of markers
separately and divisibly from each other.
[0059] By use of the term "collectively" it is meant that the
disclosure encompasses any number or combination of the recited
markers or groups of markers, and that, notwithstanding that such
numbers or combinations of markers or groups of markers may not be
specifically listed herein the accompanying claims may define such
combinations or sub-combinations separately and divisibly from any
other combination of markers or groups of markers.
[0060] In one example, the STRO-1+ cells are STRO-1.sup.bright
(syn. STRO-1.sup.bri). In another example, the STRO-1.sup.bri cells
are preferentially enriched relative to STRO-1.sup.dim or
STRO-1.sup.intermediate cells. In another example, the
STRO-1.sup.bri cells are additionally one or more of TNAP+,
VCAM-1+, THY-1+, STRO-2+, STRO-4+(HSP-90.beta.) and/or CD146+. For
example, the cells are selected for one or more of the foregoing
markers and/or shown to express one or more of the foregoing
markers. In this regard, a cell shown to express a marker need not
be specifically tested, rather previously enriched or isolated
cells can be tested and subsequently used, isolated or enriched
cells can be reasonably assumed to also express the same
marker.
[0061] In one example, the mesenchymal precursor cells are
perivascular mesenchymal precursor cells as defined in WO
2004/85630, characterized by the presence of the perivascular
marker 3G5.
[0062] A cell that is referred to as being "positive" for a given
marker may express either a low (lo or dim) or a high (bright, bri)
level of that marker depending on the degree to which the marker is
present on the cell surface, where the terms relate to intensity of
fluorescence or other marker used in the sorting process of the
cells. The distinction of lo (or dim or dull) and bri will be
understood in the context of the marker used on a particular cell
population being sorted. A cell that is referred to as being
"negative" for a given marker is not necessarily completely absent
from that cell. This term means that the marker is expressed at a
relatively very low level by that cell, and that it generates a
very low signal when detectably labelled or is undetectable above
background levels, e.g., levels detected using an isotype control
antibody.
[0063] The term "bright" or "bri" as used herein, refers to a
marker on a cell surface that generates a relatively high signal
when detectably labelled. Whilst not wishing to be limited by
theory, it is proposed that "bright" cells express more of the
target marker protein (for example the antigen recognized by
STRO-1) than other cells in the sample. For instance,
STRO-1.sup.bri cells produce a greater fluorescent signal, when
labelled with a FITC-conjugated STRO-1 antibody as determined by
fluorescence activated cell sorting (FACS) analysis, than
non-bright cells (STRO-1.sup.dull/dim). In one example, "bright"
cells constitute at least about 0.1% of the most brightly labelled
bone marrow mononuclear cells contained in the starting sample. In
other examples, "bright" cells constitute at least about 0.5%, at
least about 1%, at least about 1.5%, or at least about 2%, of the
most brightly labelled bone marrow mononuclear cells contained in
the starting sample. In an example, STRO-1.sup.bright cells have 2
log magnitude higher expression of STRO-1 surface expression
relative to "background", namely cells that are STRO-1.sup.-. By
comparison, STRO-1.sup.dim and/or STRO-1.sup.intermediate cells
have less than 2 log magnitude higher expression of STRO-1 surface
expression, typically about 1 log or less than "background".
[0064] As used herein the term "TNAP" is intended to encompass all
isoforms of tissue non-specific alkaline phosphatase. For example,
the term encompasses the liver isoform (LAP), the bone isoform
(BAP) and the kidney isoform (KAP). In one example, the TNAP is
BAP. In one example, TNAP as used herein refers to a molecule which
can bind the STRO-3 antibody produced by the hybridoma cell line
deposited with ATCC on 19 Dec. 2005 under the provisions of the
Budapest Treaty under deposit accession number PTA-7282.
[0065] Furthermore, in one example, the STRO-1+ cells are capable
of giving rise to clonogenic CFU-F.
[0066] In one example, a significant proportion of the STRO-1+
multipotential cells are capable of differentiation into at least
two different germ lines. Non-limiting examples of the lineages to
which the multipotential cells may be committed include bone
precursor cells; hepatocyte progenitors, which are multipotent for
bile duct epithelial cells and hepatocytes; neural restricted
cells, which can generate glial cell precursors that progress to
oligodendrocytes and astrocytes; neuronal precursors that progress
to neurons; precursors for cardiac muscle and cardiomyocytes,
glucose-responsive insulin secreting pancreatic beta cell lines.
Other lineages include, but are not limited to, odontoblasts,
dentin-producing cells and chondrocytes, and precursor cells of the
following: retinal pigment epithelial cells, fibroblasts, skin
cells such as keratinocytes, dendritic cells, hair follicle cells,
renal duct epithelial cells, smooth and skeletal muscle cells,
testicular progenitors, vascular endothelial cells, tendon,
ligament, cartilage, adipocyte, fibroblast, marrow stroma, cardiac
muscle, smooth muscle, skeletal muscle, pericyte, vascular,
epithelial, glial, neuronal, astrocyte and oligodendrocyte
cells.
[0067] In an aspect of the present disclosure, the presently
described mesenchymal lineage precursor or stem cells are MSCs. The
MSCs may be a homogeneous composition or may be a mixed cell
population enriched in MSCs. Homogeneous MSCs cell compositions may
be obtained by culturing adherent marrow or periosteal cells, and
the MSCs may be identified by specific cell surface markers which
are identified with unique monoclonal antibodies. A method for
obtaining a cell population enriched in MSCs is described, for
example, in U.S. Pat. No. 5,486,359. Alternative sources for MSCs
include, but are not limited to, blood, skin, cord blood, muscle,
fat, bone, and perichondrium.
[0068] In another example, the mesenchymal lineage precursor or
stem cells are CD29+, CD54+, CD73+, CD90+, CD102+, CD105+, CD106+,
CD166+, MHC1+ MSCs (e.g. remestemcel-L).
[0069] Isolated or enriched mesenchymal lineage precursor or stem
cells can be expanded in-vitro by culture. Isolated or enriched
mesenchymal lineage precursor or stem cells can be cryopreserved,
thawed and subsequently expanded in-vitro by culture.
[0070] In one example, isolated or enriched mesenchymal lineage
precursor or stem cells are seeded at 50,000 viable cells/cm.sup.2
in culture medium (serum free or serum-supplemented), for example,
cell culture medium according to the present disclosure, and
allowed to adhere to the culture vessel overnight at 37.degree. C.,
20% O.sub.2. The culture medium is subsequently replaced and/or
altered as required and the cells cultured for a further 68 to 72
hours at 37.degree. C., 5% O.sub.2.
[0071] As will be appreciated by those of skill in the art,
cultured mesenchymal lineage precursor or stem cells are
phenotypically different to cells in-vivo. For example, in one
embodiment they express one or more of the following markers, CD44,
NG2, DC146 and CD140b. Cultured mesenchymal lineage precursor or
stem cells are also biologically different to cells in-vivo, having
a higher rate of proliferation compared to the largely non-cycling
(quiescent) cells in-vivo.
[0072] Mesenchymal lineage precursor or stem cells cultured using
the methods of the present disclosure may also be
cryopreserved.
Promoting Cell Proliferation
[0073] An aspect of the present disclosure relates to methods of
promoting stem cell proliferation in in-vitro cell culture. In the
context of the present disclosure, the term "promote" or
"promoting" is used to define an increase or acceleration in cell
proliferation.
[0074] Various methods of identifying increased and/or accelerated
cell proliferation are available to those of skill in the art. For
example, an increase or acceleration in cell proliferation can be
measured based on the number of cells produced over time using, for
example, routinely available cell proliferation assays such as MTT
assay, Bromodeoxyuridine (BrdU) incorporation assay or real time
proliferation assays such as the xCELLigence.TM. system from
Roche.
[0075] Accordingly, one of skill in the art could readily determine
whether cell proliferation is promoted using the methods of the
present disclosure by performing routine proliferation assays known
in the art.
[0076] In one example of identifying whether cell proliferation is
promoted, populations of cells can be cultured in either a cell
culture medium according to the present disclosure or an equivalent
control medium without PDGF and FGF2 (i.e. the medium of the
present disclosure with 0 pg/ml PDGF and FGF2). Cell proliferation
in each of the culture mediums can be assessed daily, over a period
of time in culture (e.g. seven days) or tracked in real-time.
Increased cell numbers or accelerated cell proliferation between
day 0 and day 7 indicates that cell proliferation has been
promoted.
Promoting Cell Derivation
[0077] Stem cells divide asymmetrically to give rise to two
distinct daughter cells: one copy of the original stem cell as well
as a second daughter cell programmed to differentiate into a
non-stem cell fate. For example, pluripotent stem cells can divide
to produce one copy of the original stem cell as well as a
mesenchymal lineage precursor cell programmed to differentiate into
a mesenchymal cell type.
[0078] The present disclosure also encompasses methods of promoting
stem cell derivation in in-vitro cell culture. For example, the
methods of the present disclosure can be used to promote derivation
of mesenchymal lineage precursor cell from pluripotent stem cells.
In the context of promoting cell derivation, the term "promote" or
"promoting" is used to define an increase or acceleration in stem
cell derivation.
[0079] An aspect of the present disclosure relates to a method of
promoting stem cell derivation in-vitro, the method comprising
culturing a population of stem cells in a fetal bovine serum free
cell culture medium comprising platelet derived growth factor
(PDGF) and fibroblast growth factor 2 (FGF2), wherein the level of
FGF2 is less than about 6 ng/ml.
[0080] For example, the methods of the present disclosure relate to
a method of promoting mesenchymal lineage stem cell derivation
in-vitro, the method comprising culturing a population of
pluripotent stem cells in a fetal bovine serum free cell culture
medium comprising platelet derived growth factor (PDGF) and
fibroblast growth factor 2 (FGF2), wherein the level of FGF2 is
less than about 6 ng/ml.
[0081] In another example, the methods of the present disclosure
can be used to promote mesenchymal lineage precursor cell
derivation from pluripotent stem cells and subsequently promote
proliferation of the derived mesenchymal lineage precursor cell
population.
[0082] Various methods of identifying increased and/or accelerated
stem cell derivation are available to those of skill in the art.
For example, increased or accelerated mesenchymal lineage precursor
cell derivation can be measured based on the number of mesenchymal
lineage precursor cells produced from pluripotent stem cells over
time. Mesenchymal lineage precursor cells are characterized by
specific surface markers, the expression of which facilitates their
isolation and purification by immunoselection. Examples of these
markers include STRO-1+, TNAP+, VCAM-1+, THY-1+, STRO-2+,
STRO-4+(HSP-90.beta.), CD146+, 3G5+ or combinations thereof.
Accordingly, one of skill in the art could use immunoselection to
purify and count the mesenchymal lineage precursor cells in a
population. Cell numbers could be used to identify the number of
mesenchymal lineage precursor cells produced over time and
determine whether mesenchymal lineage precursor cell derivation is
increased or accelerated.
[0083] In another example of identifying whether cell derivation is
promoted, populations of pluripotent stem cells can be cultured in
either a cell culture medium according to the present disclosure or
an equivalent control medium without PDGF and FGF2 (i.e. the medium
of the present disclosure with 0 pg/ml PDGF and FGF2). Derivation
of mesenchymal lineage precursor cells in each of the culture
mediums can be assessed daily or over a period of time in culture
(e.g. seven days) by immunoselecting mesenchymal lineage precursor
cells from cell culture and determining the number of cells
produced. Increased mesenchymal lineage precursor cell numbers
between day 0 and day 7 relative to control cell numbers indicates
that cell derivation was promoted.
Cell Culture Medium
[0084] The present disclosure provides fetal bovine serum free stem
cell culture medium comprising growth factors that promote MSC
proliferation. In an embodiment, the present disclosure relates to
a fetal bovine serum free stem cell culture medium, the cell
culture medium comprising:
[0085] a basal medium;
[0086] platelet derived growth factor (PDGF);
[0087] fibroblast growth factor 2 (FGF2).
[0088] The term "medium" or "media" as used in the context of the
present disclosure, includes the components of the environment
surrounding the cells. The media contributes to and/or provides the
conditions suitable to allow cells to grow. Media may be solid,
liquid, gaseous or a mixture of phases and materials. Media can
include liquid growth media as well as liquid media that do not
sustain cell growth. Media also include gelatinous media such as
agar, agarose, gelatin and collagen matrices. Exemplary gaseous
media include the gaseous phase that cells growing on a petri dish
or other solid or semisolid support are exposed to. The term
"medium" also refers to material that is intended for use in a cell
culture, even if it has not yet been contacted with cells.
[0089] The culture media of the present disclosure can be prepared
by using a basal culture medium. In the context of the present
disclosure, "basal culture medium" refers to an unsupplemented
medium which is suitable for exposure to cells, for example MSC.
Basal culture medium includes, for example, Eagles minimal
essential (MEM) culture media, alpha modified MEM culture media,
StemSpan.TM. and mixed culture media thereof, and is not
particularly restricted providing it can be used for culturing of
stem cells.
[0090] Further, the cell culture medium of the present disclosure
can contain any components such as fatty acids or lipids, vitamins,
cytokines, antioxidants, buffering agents, inorganic salts and the
like.
[0091] The cell culture media used in the present disclosure
contains all essential amino acids and may also contain
non-essential amino acids. In general, amino acids are classified
into essential amino acids (Thr, Met, Val, Leu, Ile, Phe, Trp, Lys,
His) and non-essential amino acids (Gly, Ala, Ser, Cys, Gln, Asn,
Asp, Tyr, Arg, Pro).
[0092] Those of skill in the art will appreciate that for optimal
results, the basal medium must be appropriate for the cell line of
interest with key nutrients available at adequate levels to enhance
cell proliferation. For example, it may be necessary to increase
the level of glucose (or other energy source) in the basal medium,
or to add glucose (or other energy source) during the course of
culture, if this energy source is found to be depleted and to thus
limit cell proliferation.
[0093] In an example, the cell culture medium of the present
disclosure contains human derived additives. For example, human
serum and human platelet cell lysate can be added to the cell
culture media used in the methods of the present disclosure.
[0094] In an example, the cell culture medium of the present
disclosure contains only human derived additives. Thus, in an
example, the cell culture media is xeno-free.
Ascorbic Acid
[0095] Ascorbic acid is an essential supplement for the growth and
differentiation of various kinds of cells in culture. It is now
understood that particular ascorbic acid derivatives are "short
acting" because they are not stable in solution, especially under
the normal cell culture conditions of neutral pH and 37.degree. C.
These short acting derivatives rapidly oxidise into oxalic acid or
threonic acid. In culture media (pH 7) at 37.degree. C., oxidation
decreases the level of these short acting ascorbic acid derivatives
by approximately 80-90% in 24 hours. Accordingly, short acting
ascorbic acid derivatives have been replaced with more stable "long
acting" ascorbic acid derivatives in conventional cell culture of
various cell types.
[0096] In the context of the present disclosure the term "short
acting" encompasses ascorbic acid derivatives that are oxidised by
approximately 80-90% following 24 hours of cell culture under
culture conditions of neutral pH and 37.degree. C. In one example,
the short acting L-ascorbic acid derivative is a L-ascorbic acid
salt. For example, in the context of the present disclosure,
L-ascorbic acid sodium salt is a "short acting" ascorbic acid
derivative.
[0097] In contrast, the term "long acting" encompasses ascorbic
acid derivatives that are not oxidised by approximately 80-90%
following 24 hours of cell culture under culture conditions of
neutral pH and 37.degree. C. In one example, in the context of the
present disclosure, L-ascorbic acid-2-phospahte is a "long acting"
ascorbic acid derivative. Other examples of long acting ascorbic
acid derivatives include Tetrahexyldecyl Ascorbate Magnesium
Ascorbyl Phosphate and 2-O-.alpha.-D-Glucopyranosyl-L-ascorbic
acid. The cell culture medium of the present disclosure can contain
short acting ascorbic acid derivatives, long acting ascorbic acid
derivatives or mixtures thereof.
Serum
[0098] Conventionally, stem cells are maintained in cell culture
using media supplemented with at least about 10-15% v/v serum,
generally fetal bovine serum (FBS), also known as fetal calf serum
(FCS). The cell culture medium of the present disclosure is a fetal
bovine serum-free cell culture medium. Thus, in an embodiment, the
cell culture media is supplemented with a non-fetal serum. For
example, the culture media may be supplemented with a neo-natal or
adult serum
[0099] In another embodiment, the cell culture medium is
supplemented with human serum. In an example, the cell culture
media can be supplemented with human non-fetal serum. For example,
the cell culture media can be supplemented with at least about 1%
v/v, at least about 2% v/v, at least about 3% v/v, at least about
4% v/v, at least about 5% v/v, at least about 6% v/v, at least
about 7% v/v, at least about 8% v/v, at least about 9%, at least
about 10%, at least about 11%, at least about 12%, at least about
13%, at least about 14%, at least about 15%, at least about 16%, at
least about 17%, at least about 18%, at least about 19%, at least
about 20%, at least about 21%, at least about 22%, at least about
23%, at least about 24%, at least about 25% v/v human non-fetal
serum.
[0100] In another example, the cell culture medium can be
supplemented with human neo-natal serum. For example, the cell
culture medium can be supplemented with at least about 1% v/v, at
least about 2% v/v, at least about 3% v/v, at least about 4% v/v,
at least about 5% v/v, at least about 6% v/v, at least about 7%
v/v, at least about 8% v/v, at least about 9% v/v human neo-natal
serum. In an example, the human neo-natal serum is obtained from
umbilical cord blood "cord blood".
[0101] In another example, the cell culture medium can be
supplemented with human adult serum. For example, the culture media
can be supplemented with at least about 1% v/v, at least about 2%
v/v, at least about 3% v/v, at least about 4% v/v, at least about
5% v/v, at least about 6% v/v, at least about 7% v/v, at least
about 8% v/v, at least about 9%, at least about 10%, at least about
11%, at least about 12%, at least about 13%, at least about 14%, at
least about 15%, at least about 16%, at least about 17%, at least
about 18%, at least about 19%, at least about 20%, at least about
21%, at least about 22%, at least about 23%, at least about 24%, at
least about 25% v/v human adult serum.
[0102] In an example, the human adult serum is human AB serum. For
example, the cell culture medium can be supplemented with at least
about 1% v/v, at least about 2% v/v, at least about 3% v/v, at
least about 4% v/v, at least about 5% v/v, at least about 6% v/v,
at least about 7% v/v, at least about 8% v/v, at least about 9% v/v
human AB serum. In an example, the cell culture medium is
supplemented with at least about 3% human AB serum.
[0103] The cell culture medium of the present disclosure may also
contain known serum replacements. The serum replacement can be, for
example, albumin (for example, lipid-rich albumin), transferrin,
fatty acid, insulin, collagen precursor, trace element,
2-mercaptoethanol or 3'-thiol glycerol, platelet lysate,
platelet-rich plasma, or those appropriately containing serum
equivalents. Such a serum replacement can be prepared, for example,
by a method described in International Publication WO 93/30679, and
commercially available products can also be used.
Mitogenic Factors
[0104] The present inventors have found that PDGF and FGF2
synergistically promote stem cell proliferation in in-vitro fetal
bovine serum free cell culture.
[0105] PDGF is a regulator of cell growth and division which binds
to platelet derived growth factor receptors (PDGFR). In chemical
terms, PDGF is a dimeric glycoprotein composed of two A (-AA) or
two B (-BB) chains or a combination of the two (-AB). PDGF-AB has
been shown to bind PDGF alpha and beta receptor subunits to form
PDGF alpha beta and alpha alpha receptor dimers. In the context of
the present disclosure PDGF encompasses PDGF-BB and PDGF-AB.
[0106] Basic fibroblast growth factor (FGF2) also known as BFGF,
FGFB, HBGF-2 is a member of the fibroblast growth factor (FGF)
family. FGF2 is also a regulator of cell growth and division. Both
PDGF and FGF2 can be classified as mitogens in that they encourage
a cell to commence cell division.
[0107] In an example, the method of the present disclosure
comprises culturing a population of stem cells in a fetal bovine
serum free cell culture medium comprising platelet derived growth
factor (PDGF) and fibroblast growth factor 2 (FGF2), wherein the
level of FGF2 is less than about 6 ng/ml. For example, the FGF2
level may be less than about 5 ng/ml, less than about 4 ng/ml, less
than about 3 ng/ml, less than about 2 ng/ml, less than about 1
ng/ml. In other examples, the FGF2 level is less than about 0.9
ng/ml, less than about 0.8 ng/ml, less than about 0.7 ng/ml, less
than about 0.6 ng/ml, less than about 0.5 ng/ml, less than about
0.4 ng/ml, less than about 0.3 ng/ml, less than about 0.2
ng/ml.
[0108] In another example, the level of FGF2 is between about 1
pg/ml and 100 pg/ml. In another example, the level of FGF2 is
between about 5 pg/ml and 80 pg/ml. In another example, the level
of FGF2 is between about 10 pg/ml and 40 pg/ml. In another example,
the level of FGF2 is at least about 10 pg/ml. In another example,
the level of FGF2 is at least about 11 pg/ml. In another example,
the level of FGF2 is at least about 12 pg/ml. In another example,
the level of FGF2 is at least about 13 pg/ml. In another example,
the level of FGF2 is at least about 14 pg/ml. In another example,
the level of FGF2 is at least about 15 pg/ml. In another example,
the level of FGF2 is at least about 16 pg/ml. In another example,
the level of FGF2 is at least about 17 pg/ml. In another example,
the level of FGF2 is at least about 18 pg/ml. In another example,
the level of FGF2 is at least about 19 pg/ml. In another example,
the level of FGF2 is at least about 20 pg/ml. In another example,
the level of FGF2 is at least about 21 pg/ml. In another example,
the level of FGF2 is at least about 22 pg/ml. In another example,
the level of FGF2 is at least about 23 pg/ml. In another example,
the level of FGF2 is at least about 24 pg/ml. In another example,
the level of FGF2 is at least about 25 pg/ml. In another example,
the level of FGF2 is at least about 26 pg/ml. In another example,
the level of FGF2 is at least about 27 pg/ml. In another example,
the level of FGF2 is at least about 28 pg/ml. In another example,
the level of FGF2 is at least about 29 pg/ml. In another example,
the level of FGF2 is at least about 30 pg/ml.
[0109] In an example, the PDGF is PDGF-BB. In an example, the level
of PDGF-BB is between about 1 ng/ml and 150 ng/ml. In another
example, the level of PDGF-BB is between about 7.5 ng/ml and 120
ng/ml. In another example, the level of PDGF-BB is between about 15
ng/ml and 60 ng/ml. In another example, the level of PDGF-BB is at
least about 10 ng/ml. In another example, the level of PDGF-BB is
at least about 15 ng/ml. In another example, the level of PDGF-BB
is at least about 20 ng/ml. In another example, the level of
PDGF-BB is at least about 21 ng/ml. In another example, the level
of PDGF-BB is at least about 22 ng/ml. In another example, the
level of PDGF-BB is at least about 23 ng/ml. In another example,
the level of PDGF-BB is at least about 24 ng/ml. In another
example, the level of PDGF-BB is at least about 25 ng/ml. In
another example, the level of PDGF-BB is at least about 26 ng/ml.
In another example, the level of PDGF-BB is at least about 27
ng/ml. In another example, the level of PDGF-BB is at least about
28 ng/ml. In another example, the level of PDGF-BB is at least
about 29 ng/ml. In another example, the level of PDGF-BB is at
least about 30 ng/ml. In another example, the level of PDGF-BB is
at least about 31 ng/ml. In another example, the level of PDGF-BB
is at least about 32 ng/ml. In another example, the level of
PDGF-BB is at least about 33 ng/ml. In another example, the level
of PDGF-BB is at least about 34 ng/ml. In another example, the
level of PDGF-BB is at least about 35 ng/ml. In another example,
the level of PDGF-BB is at least about 36 ng/ml. In another
example, the level of PDGF-BB is at least about 37 ng/ml. In
another example, the level of PDGF-BB is at least about 38 ng/ml.
In another example, the level of PDGF-BB is at least about 39
ng/ml. In another example, the level of PDGF-BB is at least about
40 ng/ml.
[0110] In another example, the PDGF is PDGF-AB. In an example, the
level of PDGF-AB is between about 1 ng/ml and 150 ng/ml. In another
example, the level of PDGF-AB is between about 7.5 ng/ml and 120
ng/ml. In another example, the level of PDGF-AB is between about 15
ng/ml and 60 ng/ml. In another example, the level of PDGF-AB is at
least about 10 ng/ml. In another example, the level of PDGF-AB is
at least about 15 ng/ml. In another example, the level of PDGF-AB
is at least about 20 ng/ml. In another example, the level of
PDGF-AB is at least about 21 ng/ml. In another example, the level
of PDGF-AB is at least about 22 ng/ml. In another example, the
level of PDGF-AB is at least about 23 ng/ml. In another example,
the level of PDGF-AB is at least about 24 ng/ml. In another
example, the level of PDGF-AB is at least about 25 ng/ml. In
another example, the level of PDGF-AB is at least about 26 ng/ml.
In another example, the level of PDGF-AB is at least about 27
ng/ml. In another example, the level of PDGF-AB is at least about
28 ng/ml. In another example, the level of PDGF-AB is at least
about 29 ng/ml. In another example, the level of PDGF-AB is at
least about 30 ng/ml. In another example, the level of PDGF-AB is
at least about 31 ng/ml. In another example, the level of PDGF-AB
is at least about 32 ng/ml. In another example, the level of
PDGF-AB is at least about 33 ng/ml. In another example, the level
of PDGF-AB is at least about 34 ng/ml. In another example, the
level of PDGF-AB is at least about 35 ng/ml. In another example,
the level of PDGF-AB is at least about 36 ng/ml. In another
example, the level of PDGF-AB is at least about 37 ng/ml. In
another example, the level of PDGF-AB is at least about 38 ng/ml.
In another example, the level of PDGF-AB is at least about 39
ng/ml. In another example, the level of PDGF-AB is at least about
40 ng/ml.
[0111] The present inventors have also found that other factors can
be added to the cell culture medium of the present disclosure to
increase cell proliferation. In an example, the method of the
present disclosure comprises culturing a population of stem cells
in a fetal bovine serum free cell culture medium further comprising
EGF. EGF is a growth factor that stimulates cell proliferation by
binding to its receptor EGFR. In an example, the method of the
present disclosure comprises culturing a population of stem cells
in a fetal bovine serum free cell culture medium further comprising
EGF. In an example, the level of EGF is between about 0.1 and 7
ng/ml. For example, the level of EGF can be at least about 5
ng/ml.
[0112] In another example, the level of EGF is between about 0.2
ng/ml and 3.2 ng/ml. In another example, the level of EGF is
between about 0.4 ng/ml and 1.6 ng/ml. In another example, the
level of EGF is between about 0.2 ng/ml. In another example, the
level of EGF is at least about 0.3 ng/ml. In another example, the
level of EGF is at least about 0.4 ng/ml. In another example, the
level of EGF is at least about 0.5 ng/ml. In another example, the
level of EGF is at least about 0.6 ng/ml. In another example, the
level of EGF is at least about 0.7 ng/ml. In another example, the
level of EGF is at least about 0.8 ng/ml. In another example, the
level of EGF is at least about 0.9 ng/ml. In another example, the
level of EGF is at least about 1.0 ng/ml. In another example, the
level of EGF is at least about 1.1 ng/ml. In another example, the
level of EGF is at least about 1.2 ng/ml. In another example, the
level of EGF is at least about 1.3 ng/ml. In another example, the
level of EGF is at least about 1.4 ng/ml.
[0113] In an example, the level of PDGF-BB is at least about 3.2
ng/ml, the level of EGF is at least about 0.8 ng/ml and the level
of FGF2 is at least about 0.002 ng/ml. In another example, the
level of PDGF-BB is at least about 9.6 ng/ml, the level of EGF is
at least about 0.24 ng/ml and the level of FGF2 is at least about
0.006 ng/ml. In another example, the level of PDGF-BB is at least
about 16 ng/ml, the level of EGF is at least about 0.40 ng/ml and
the level of FGF2 is at least about 0.01 ng/ml. In another example,
the level of PDGF-BB is at least about 32 ng/ml, the level of EGF
is at least about 0.80 ng/ml and the level of FGF2 is at least
about 0.01 ng/ml.
[0114] The present disclosure also encompasses cell culture medium
comprising PDGF and FGF2, wherein the level of FGF2 is less than
about 6 ng/ml. For example, the FGF2 level may be less than about 5
ng/ml, less than about 4 ng/ml, less than about 3 ng/ml, less than
about 2 ng/ml, less than about 1 ng/ml. In other examples, the FGF2
level is less than about 0.9 ng/ml, less than about 0.8 ng/ml, less
than about 0.7 ng/ml, less than about 0.6 ng/ml, less than about
0.5 ng/ml, less than about 0.4 ng/ml, less than about 0.3 ng/ml,
less than about 0.2 ng/ml.
[0115] In an example, the cell culture medium contains between
about 1 pg/ml and 100 pg/ml of FGF2. In another example, the cell
culture medium contains between about 5 pg/ml and 80 pg/ml of FGF2.
In another example, the cell culture medium contains between about
10 pg/ml and 40 pg/ml of FGF2. For example, the cell culture medium
may contain at least about 10 pg/ml of FGF2. For example, the cell
culture medium may contain at least about 11 pg/ml of FGF2. For
example, the cell culture medium may contain at least about 12
pg/ml of FGF2. For example, the cell culture medium may contain at
least about 13 pg/ml of FGF2. For example, the cell culture medium
may contain at least about 14 pg/ml of FGF2. For example, the cell
culture medium may contain at least about 15 pg/ml of FGF2. For
example, the cell culture medium may contain at least about 16
pg/ml of FGF2. For example, the cell culture medium may contain at
least about 17 pg/ml of FGF2. For example, the cell culture medium
may contain at least about 18 pg/ml of FGF2. For example, the cell
culture medium may contain at least about 19 pg/ml of FGF2. For
example, the cell culture medium may contain at least about 20
pg/ml of FGF2. For example, the cell culture medium may contain at
least about 21 pg/ml of FGF2. For example, the cell culture medium
may contain at least about 22 pg/ml of FGF2. For example, the cell
culture medium may contain at least about 23 pg/ml of FGF2. For
example, the cell culture medium may contain at least about 24
pg/ml of FGF2. For example, the cell culture medium may contain at
least about 25 pg/ml of FGF2. For example, the cell culture medium
may contain at least about 26 pg/ml of FGF2. For example, the cell
culture medium may contain at least about 27 pg/ml of FGF2. For
example, the cell culture medium may contain at least about 28
pg/ml of FGF2. For example, the cell culture medium may contain at
least about 29 pg/ml of FGF2. For example, the cell culture medium
may contain at least about 30 pg/ml of FGF2.
[0116] In an embodiment the cell culture medium of the present
disclosure is supplemented with PDGF-BB. For example, the cell
culture medium may contain between about 1 ng/ml and 150 ng/ml of
PDGF-BB. In another example, the cell culture medium may contain
between about 7.5 ng/ml and 120 ng/ml of PDGF-BB. In another
example, the cell culture medium may contain between about 15 ng/ml
and 60 ng/ml of PDGF-BB. For example, the cell culture medium may
contain at least about 10 ng/ml of PDGF-BB. For example, the cell
culture medium may contain at least about 15 ng/ml of PDGF-BB. For
example, the cell culture medium may contain at least about 20
ng/ml of PDGF-BB. For example, the cell culture medium may contain
at least about 21 ng/ml of PDGF-BB. For example, the cell culture
medium may contain at least about 22 ng/ml of PDGF-BB. For example,
the cell culture medium may contain at least about 23 ng/ml of
PDGF-BB. For example, the cell culture medium may contain at least
about 24 ng/ml of PDGF-BB. For example, the cell culture medium may
contain at least about 25 ng/ml of PDGF-BB. For example, the cell
culture medium may contain at least about 26 ng/ml of PDGF-BB. For
example, the cell culture medium may contain at least about 27
ng/ml of PDGF-BB. For example, the cell culture medium may contain
at least about 28 ng/ml of PDGF-BB. For example, the cell culture
medium may contain at least about 29 ng/ml of PDGF-BB. For example,
the cell culture medium may contain at least about 30 ng/ml of
PDGF-BB. For example, the cell culture medium may contain at least
about 31 ng/ml of PDGF-BB. For example, the cell culture medium may
contain at least about 32 ng/ml of PDGF-BB. For example, the cell
culture medium may contain at least about 33 ng/ml of PDGF-BB. For
example, the cell culture medium may contain at least at least
about 34 ng/ml of PDGF-BB. For example, the cell culture medium may
contain at least about 35 ng/ml of PDGF-BB. For example, the cell
culture medium may contain at least about 36 ng/ml of PDGF-BB. For
example, the cell culture medium may contain at least about 37
ng/ml of PDGF-BB. For example, the cell culture medium may contain
at least about 38 ng/ml of PDGF-BB. For example, the cell culture
medium may contain at least about 39 ng/ml of PDGF-BB. For example,
the cell culture medium may contain at least about 40 ng/ml of
PDGF-BB.
[0117] In an embodiment the cell culture medium of the present
disclosure is supplemented with PDGF-AB. For example, the cell
culture medium may contain between about 1 ng/ml and 150 ng/ml of
PDGF-AB. In another example, the cell culture medium may contain
between about 7.5 ng/ml and 120 ng/ml of PDGF-AB. In another
example, the cell culture medium may contain between about 15 ng/ml
and 60 ng/ml of PDGF-AB. For example, the cell culture medium may
contain at least about 10 ng/ml of PDGF-AB. For example, the cell
culture medium may contain at least about 15 ng/ml of PDGF-AB. For
example, the cell culture medium may contain at least about 20
ng/ml of PDGF-AB. For example, the cell culture medium may contain
at least about 21 ng/ml of PDGF-AB. For example, the cell culture
medium may contain at least about 22 ng/ml of PDGF-AB. For example,
the cell culture medium may contain at least about 23 ng/ml of
PDGF-AB. For example, the cell culture medium may contain at least
about 24 ng/ml of PDGF-AB. For example, the cell culture medium may
contain at least about 25 ng/ml of PDGF-AB. For example, the cell
culture medium may contain at least about 26 ng/ml of PDGF-AB. For
example, the cell culture medium may contain at least about 27
ng/ml of PDGF-AB. For example, the cell culture medium may contain
at least about 28 ng/ml of PDGF-AB. For example, the cell culture
medium may contain at least about 29 ng/ml of PDGF-AB. For example,
the cell culture medium may contain at least about 30 ng/ml of
PDGF-AB. For example, the cell culture medium may contain at least
about 31 ng/ml of PDGF-AB. For example, the cell culture medium may
contain at least about 32 ng/ml of PDGF-AB. For example, the cell
culture medium may contain at least about 33 ng/ml of PDGF-AB. For
example, the cell culture medium may contain at least about 34
ng/ml of PDGF-AB. For example, the cell culture medium may contain
at least about 35 ng/ml of PDGF-AB. For example, the cell culture
medium may contain at least about 36 ng/ml of PDGF-AB. For example,
the cell culture medium may contain at least about 37 ng/ml of
PDGF-AB. For example, the cell culture medium may contain at least
about 38 ng/ml of PDGF-AB. For example, the cell culture medium may
contain at least about 39 ng/ml of PDGF-AB. For example, the cell
culture medium may contain at least about 40 ng/ml of PDGF-AB.
[0118] In an embodiment, the cell culture medium of the present
disclosure can also be supplemented with EGF. For example, the cell
culture medium may contain between about 0.1 ng/ml and 7 ng/ml of
EGF. For example, the level of EGF can be at least about 5
ng/ml.
[0119] In another example, the cell culture medium may contain
between about 0.2 ng/ml and 3.2 ng/ml of EGF. In another example,
the cell culture medium may contain between about 0.4 ng/ml and 1.6
ng/ml of EGF. For example, the cell culture medium may contain at
least about 0.2 ng/ml of EGF. For example, the cell culture medium
may contain at least about 0.3 ng/ml of EGF. For example, the cell
culture medium may contain at least about 0.4 ng/ml of EGF. For
example, the cell culture medium may contain at least about 0.5
ng/ml of EGF. For example, the cell culture medium may contain at
least about 0.6 ng/ml of EGF. For example, the cell culture medium
may contain at least about 0.7 ng/ml of EGF. For example, the cell
culture medium may contain at least about 0.8 ng/ml of EGF. For
example, the cell culture medium may contain at least about 0.9
ng/ml of EGF. For example, the cell culture medium may contain at
least about 1.0 ng/ml of EGF. For example, the cell culture medium
may contain at least about 1.1 ng/ml of EGF. For example, the cell
culture medium may contain at least about 1.2 ng/ml of EGF. For
example, the cell culture medium may contain at least about 1.3
ng/ml of EGF. For example, the cell culture medium may contain at
least about 1.4 ng/ml of EGF.
[0120] For example, the cell culture medium may contain at least
about 3.2 ng/ml PDGF-BB, at least about 0.08 ng/ml EGF and at least
about 0.002 ng/ml FGF2. In another example, the cell culture medium
may contain at least about 9.6 ng/ml PDGF-BB, at least about 0.24
ng/ml EGF and at least about 0.006 ng/ml FGF2. In another example,
the cell culture medium may contain at least about 16 ng/ml
PDGF-BB, at least about 0.40 ng/ml EGF and at least about 0.01
ng/ml FGF2. In another example, the cell culture medium may contain
at least about 32 ng/ml PDGF-BB, at least about 0.80 ng/ml EGF and
at least about 0.02 ng/ml FGF2.
[0121] Other factors can be added to the cell culture medium of the
present disclosure to increase cell proliferation. For example, the
cell culture media can be supplemented with one or more stimulatory
factors selected from the group consisting of epidermal growth
factor (EGF), 1.alpha.,25-dihydroxyvitamin D3 (1,25D), tumor
necrosis factor .alpha. (TNF-.alpha.), interleukin-1.beta.
(IL-1.beta.) and stromal derived factor 1.alpha. (SDF-1.alpha.). In
another embodiment, cells may also be cultured in the presence of
at least one cytokine in an amount adequate to support growth of
the cells. In another embodiment, cells can be cultured in the
presence of heparin or a derivative thereof. For example, the cell
culture medium may contain about 50 ng/ml of heparin. In other
examples, the cell culture medium contains about 60 ng/ml of
heparin, about 70 ng/ml of heparin, about 80 ng/ml of heparin,
about 90 ng/ml of heparin, about 100 ng/ml of heparin, about 110
ng/ml of heparin, about 110 ng/ml of heparin, about 120 ng/ml of
heparin, about 130 ng/ml of heparin, about 140 ng/ml of heparin,
about 150 ng/ml of heparin or a derivative thereof. In an example,
the heparin derivative is a sulphate). Various forms of heparin
sulphate are known in the art and include heparin sulphate 2 (HS2).
HS2 can be derived from various sources including for example, the
liver of male and/or female mammals. Thus, an exemplary heparin
sulphate includes male liver heparin sulphate (MML HS) and female
liver heparin sulphate (FML HS).
[0122] In another example, the methods and cell culture medium of
the present disclosure promote stem cell proliferation while
maintaining stem cells in an undifferentiated state. Stem cells are
considered to be undifferentiated when they have not committed to a
specific differentiation lineage. As discussed above, stem cells
display morphological characteristics that distinguish them from
differentiated cells. Furthermore, undifferentiated stem cells
express genes that may be used as markers to detect differentiation
status. The polypeptide products may also be used as markers to
detect differentiation status. Accordingly, one of skill in the art
could readily determine whether the methods of the present
disclosure maintain stem cells in an undifferentiated state using
routine morphological, genetic and/or proteomic analysis.
Compositions/Kits
[0123] The cell culture medium of the present disclosure can be
provided as a complete medium wherein the basal medium and the
growth factors have been mixed together prior to cell culture.
Alternatively, the cell culture medium components can be provided
separately and mixed with a suitable basal medium prior to or
during cell culture. Accordingly, in an embodiment, the present
disclosure provides a serum replacement composition for addition to
a stem cell culture medium, the composition comprising:
[0124] platelet derived growth factor (PDGF);
[0125] fibroblast growth factor 2 (FGF2);
wherein, when the composition is added to a stem cell culture
medium and a stem cell population is cultured in the cell culture
medium, the level of FGF2 in the culture medium is less than about
6 ng/ml. In other examples, when the composition is added to a stem
cell culture medium and a stem cell population is cultured in the
cell culture medium the FGF2 level may be less than about 5 ng/ml,
less than about 4 ng/ml, less than about 3 ng/ml, less than about 2
ng/ml, less than about 1 ng/ml. In other examples, the FGF2 level
is less than about 0.9 ng/ml, less than about 0.8 ng/ml, less than
about 0.7 ng/ml, less than about 0.6 ng/ml, less than about 0.5
ng/ml, less than about 0.4 ng/ml, less than about 0.3 ng/ml, less
than about 0.2 ng/ml.
[0126] In an example, FGF2 is present in a sufficient amount to be
added to a stem cell culture media at a level between about 1 pg/ml
and 100 pg/ml. In another example, FGF2 is present in a sufficient
amount to be added to a stem cell culture media at a level between
about 5 pg/ml and 80 pg/ml. In another example, FGF2 is present in
a sufficient amount to be added to a stem cell culture media at a
level between about 10 pg/ml and 40 pg/ml. For example, FGF2 may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 10 pg/ml. For example, FGF2 may be present
in a sufficient amount to be added to a stem cell culture media at
least at about 11 pg/ml. For example, FGF2 may be present in a
sufficient amount to be added to a stem cell culture media at least
at about 12 pg/ml. For example, FGF2 may be present in a sufficient
amount to be added to a stem cell culture media at about least at
13 pg/ml. For example, FGF2 may be present in a sufficient amount
to be added to a stem cell culture media at least at about 14
pg/ml. For example, FGF2 may be present in a sufficient amount to
be added to a stem cell culture media at least at about 15 pg/ml.
For example, FGF2 may be present in a sufficient amount to be added
to a stem cell culture media at least at about 16 pg/ml. For
example, FGF2 may be present in a sufficient amount to be added to
a stem cell culture media at least at about 17 pg/ml. For example,
FGF2 may be present in a sufficient amount to be added to a stem
cell culture media at least at about 18 pg/ml. For example, FGF2
may be present in a sufficient amount to be added to a stem cell
culture media at about 19 pg/ml. For example, FGF2 may be present
in a sufficient amount to be added to a stem cell culture media at
least at about 20 pg/ml. For example, FGF2 may be present in a
sufficient amount to be added to a stem cell culture media at least
at about 21 pg/ml. For example, FGF2 may be present in a sufficient
amount to be added to a stem cell culture media at least at about
22 pg/ml. For example, FGF2 may be present in a sufficient amount
to be added to a stem cell culture media at least at about 23
pg/ml. For example, FGF2 may be present in a sufficient amount to
be added to a stem cell culture media at least at about 24 pg/ml.
For example, FGF2 may be present in a sufficient amount to be added
to a stem cell culture media at least at about 25 pg/ml. For
example, FGF2 may be present in a sufficient amount to be added to
a stem cell culture media at least at about 26 pg/ml. For example,
FGF2 may be present in a sufficient amount to be added to a stem
cell culture media at least at about 27 pg/ml. For example, FGF2
may be present in a sufficient amount to be added to a stem cell
culture media at least at about 28 pg/ml. For example, FGF2 may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 29 pg/ml. For example, FGF2 may be present
in a sufficient amount to be added to a stem cell culture media at
least at about 30 pg/ml.
[0127] In an embodiment PDGF-BB is present in the composition of
the present disclosure. For example, PDGF-BB may be present in a
sufficient amount to be added to a stem cell culture media at about
1 ng/ml and 150 ng/ml. In another example, PDGF-BB may be present
in a sufficient amount to be added to a stem cell culture media at
about 7.5 ng/ml and 120 ng/ml. In another example, PDGF-BB may be
present in a sufficient amount to be added to a stem cell culture
media at about 15 ng/ml and 60 ng/ml. For example, PDGF-BB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 10 ng/ml. For example, PDGF-BB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 15 ng/ml. For example, PDGF-BB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 20 ng/ml. For example, PDGF-BB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 21 ng/ml. For example, PDGF-BB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 22 ng/ml. For example, PDGF-BB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 23 ng/ml. For example, PDGF-BB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 24 ng/ml. For example, PDGF-BB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 25 ng/ml. For example, PDGF-BB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 26 ng/ml. For example, PDGF-BB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 27 ng/ml. For example, PDGF-BB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 28 ng/ml. For example, PDGF-BB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 29 ng/ml. For example, PDGF-BB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 30 ng/ml. For example, PDGF-BB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 31 ng/ml. For example, PDGF-BB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 32 ng/ml. For example, PDGF-BB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 33 ng/ml. For example, PDGF-BB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 34 ng/ml. For example, PDGF-BB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 35 ng/ml. For example, PDGF-BB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 36 ng/ml. For example, PDGF-BB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 37 ng/ml. For example, PDGF-BB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 38 ng/ml. For example, PDGF-BB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 39 ng/ml. For example, PDGF-BB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 40 ng/ml.
[0128] In an embodiment the composition of the present disclosure
comprises PDGF-AB. For example, PDGF-AB may be present in a
sufficient amount to be added to a stem cell culture media at about
1 ng/ml and 150 ng/ml. In another example, PDGF-AB may be present
in a sufficient amount to be added to a stem cell culture media at
about 7.5 ng/ml and 120 ng/ml. In another example, PDGF-AB may be
present in a sufficient amount to be added to a stem cell culture
media at about 15 ng/ml and 60 ng/ml. For example, PDGF-AB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 10 ng/ml. For example, PDGF-AB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 15 ng/ml. For example, PDGF-AB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 20 ng/ml. For example, PDGF-AB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 21 ng/ml. For example, PDGF-AB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 22 ng/ml. For example, PDGF-AB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 23 ng/ml. For example, PDGF-AB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 24 ng/ml. For example, PDGF-AB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 25 ng/ml. For example, PDGF-AB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 26 ng/ml. For example, PDGF-AB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 27 ng/ml. For example, PDGF-AB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 28 ng/ml. For example, PDGF-AB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 29 ng/ml. For example, PDGF-AB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 30 ng/ml. For example, PDGF-AB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 31 ng/ml. For example, PDGF-AB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 32 ng/ml. For example, PDGF-AB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 33 ng/ml. For example, PDGF-AB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 34 ng/ml. For example, PDGF-AB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 35 ng/ml. For example, PDGF-AB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 36 ng/ml. For example, PDGF-AB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 37 ng/ml. For example, PDGF-AB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 38 ng/ml. For example, PDGF-AB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 39 ng/ml. For example, PDGF-AB may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 40 ng/ml.
[0129] In an embodiment, the composition of the present disclosure
further comprises EGF. For example, EGF may be present in a
sufficient amount to be added to a stem cell culture media at about
0.1 ng/ml and 7 ng/ml. For example, EGF may be present in a
sufficient amount to be added to a stem cell culture media at least
at about 5 ng/ml.
[0130] In another example, EGF may be present in a sufficient
amount to be added to a stem cell culture media at about 0.2 ng/ml
and 3.2 ng/ml. In another example, EGF may be present in a
sufficient amount to be added to a stem cell culture media at about
0.4 ng/ml and 1.6 ng/ml. For example, EGF may be present in a
sufficient amount to be added to a stem cell culture media at least
at about 0.2 ng/ml. For example, EGF may be present in a sufficient
amount to be added to a stem cell culture media at least at about
0.3 ng/ml. For example, EGF may be present in a sufficient amount
to be added to a stem cell culture media at least at about 0.4
ng/ml. For example, EGF may be present in a sufficient amount to be
added to a stem cell culture media at least at about 0.5 ng/ml. For
example, EGF may be present in a sufficient amount to be added to a
stem cell culture media at least at about 0.6 ng/ml. For example,
EGF may be present in a sufficient amount to be added to a stem
cell culture media at least at about 0.7 ng/ml. For example, EGF
may be present in a sufficient amount to be added to a stem cell
culture media at least at about 0.8 ng/ml. For example, EGF may be
present in a sufficient amount to be added to a stem cell culture
media at least at about 0.9 ng/ml. For example, EGF may be present
in a sufficient amount to be added to a stem cell culture media at
least at about 1.0 ng/ml. For example, EGF may be present in a
sufficient amount to be added to a stem cell culture media at least
at about 1.1 ng/ml. For example, EGF may be present in a sufficient
amount to be added to a stem cell culture media at least at about
1.2 ng/ml. For example, EGF may be present in a sufficient amount
to be added to a stem cell culture media at least at about 1.3
ng/ml. For example, EGF may be present in a sufficient amount to be
added to a stem cell culture media at least at about 1.4 ng/ml.
[0131] In an example, PDGF-BB, EGF and FGF2 are present in a
sufficient amount to be added to stem cell culture media at least
at about 3.2 ng/ml, 0.08 ng/ml and 0.002 ng/ml respectively. In an
example, PDGF-BB, EGF and FGF2 are present in a sufficient amount
to be added to stem cell culture media at least at about 9.6 ng/ml,
0.24 ng/ml and 0.006 ng/ml respectively. In an example, PDGF-BB,
EGF and FGF2 are present in a sufficient amount to be added to stem
cell culture media at least at about 16 ng/ml, 0.4 ng/ml and 0.02
ng/ml respectively. In an example, PDGF-BB, EGF and FGF2 are
present in a sufficient amount to be added to stem cell culture
media at least at about 32 ng/ml, 0.8 ng/ml and 0.01 ng/ml
respectively.
[0132] In an example, the composition of the present disclosure may
be packaged in or with a suitable solvent or in lyophilised
form.
[0133] The cell culture medium and/or compositions disclosed herein
may optionally be packaged in a suitable container with written
instructions for a desired purpose, such as mixing of the
composition with cell culture media to provide a specific
concentration.
[0134] It will be appreciated by persons skilled in the art that
numerous variations and/or modifications may be made to the
invention as shown in the specific embodiments without departing
from the spirit or scope of the invention as broadly described. The
present embodiments are, therefore, to be considered in all
respects as illustrative and not restrictive.
[0135] All publications discussed and/or referenced herein are
incorporated herein in their entirety.
[0136] Any discussion of documents, acts, materials, devices,
articles or the like which has been included in the present
specification is solely for the purpose of providing a context for
the present invention. It is not to be taken as an admission that
any or all of these matters form part of the prior art base or were
common general knowledge in the field relevant to the present
invention as it existed before the priority date of each claim of
this application.
[0137] The present application claims priority from AU 2015900752
filed 4 Mar. 2015 and AU 2015900777 filed 5 Mar. 2015, the
disclosures of which are incorporated herein by reference.
EXAMPLES
Example 1: Growth Factors in Cell Culture Medium
[0138] To assess serial propagation of mesenchymal lineage
precursor cells (MPC) in human platelet lysate, cells were cultured
in human platelet lysate for seven days before measuring cell
proliferation. Platelet lysate was obtained from two separate
suppliers (supplier A and supplier B). The platelet lysate
percentage ranged from 0 to 10%. MPC proliferation at day seven is
shown in FIG. 1. It was noted that the cell proliferation was
highest when cells were grown in 10% human platelet lysate obtained
from supplier B.
[0139] The concentrations (pg/ml) of growth factors EGF, FGF2,
VEGF, PDGF-AA and PDGF-BB were measured in the human platelet
lysate obtained from supplier A and B. The growth factor
concentrations (pg/ml) are shown in Table 1. It was noted that
supplier B had the highest concentration of EGF (8,036 pg/ml), the
highest concentration of PDGF-BB (46,432 pg/ml) and the lowest
concentration of FGF2 (199 pg/m).
TABLE-US-00001 TABLE 1 EGF FGF-2 VEGF PDGF-AA PDGF-BB Analyte
Sample pg/ml pg/ml pg/ml pg/ml pg/ml Supplier A - lot 1 3,875 266
3,745 31,063 139,300 Supplier A - lot 2 4,197 234 5,252 36,110
199,900 Supplier B - lot 1 8,036 199 3,745 46,432 326,100 Supplier
A - lot 3 5,982 314 2,546 37,852 230,600
Example 2: The Effect of Growth Factors on Cell Proliferation
[0140] To assess the effect of growth factors in hPL on cell
proliferation, cell populations were exposed to antibody
antagonists of growth factors EGF, FGF2, VEGF, PDGF-AA and
PDGF-BB.
[0141] Cell populations were exposed to either a single antibody
antagonist, a combination of all antibody antagonists or an Ig
antibody control. Antibodies were obtained from R&D systems and
were added at saturating concentrations. After the addition of
antibodies, cells were cultured for 5 days in 3% hPL (PLTMax)
before measuring cell proliferation. The level of cell
proliferation in each cell population exposed to the antibody
antagonists was compared to the level of cell proliferation in cell
populations exposed to the Ig control antibody.
[0142] Antibody neutralisation of PDGF-BB was the only condition
that resulted in a significant decline in MPC proliferation of the
single anti-mitogen antibodies tested (FIG. 2).
[0143] The combination of all antibody antagonists resulted in
almost complete suppression of cell proliferation (FIG. 2). Thus,
the stimulation of cell proliferation by hPL is almost completely
encompassed by the five growth factors examined in this initial
experiment.
[0144] These results also demonstrate that one of the other growth
factor(s) was contributing to PDGF-BB driven MPC proliferation.
Example 3: The Effect of Growth Factors on Cell Proliferation
[0145] To assess which other growth factor(s) was contributing to
PDGF-BB driven MPC proliferation, MPC cells were cultured with
various combinations of antibodies as shown in FIG. 3.
[0146] After the addition of antibodies, cells were cultured for 5
days in 3% hPL (PLTMax) before measuring cell proliferation. The
level of cell proliferation in each cell population exposed to the
antibody antagonists was compared to the level of cell
proliferation in cell populations exposed to an Ig control antibody
and cell populations not exposed to an antibody (FIG. 3).
[0147] This study demonstrates that PDGF-BB, EGF and FGF2 stimulate
MPC proliferation by hPL. Furthermore, PDGF-BB and EGF each act
synergistically with FGF2 to promote cell proliferation. It was
noted that the concentration of FGF2 in 3% hPL is approximately 6
pg/ml which is a sub-mitogenic dose.
[0148] As expected, no difference in cell proliferation was
observed between cell populations cultured with the Ig control and
cell populations not exposed to an antibody.
[0149] These data represent the basis for development of a
chemically defined, fully humanised xeno-free cell culture medium
in which MPC proliferation is driven by recombinant mitogens.
Example 4: Development of a Fetal Bovine Serum Free Cell Culture
Medium
[0150] Starting basal cell culture mediums included alpha modified
Eagle's minimum essential media (MEM) or StemSpan.TM..
[0151] The Alpha modification of Eagle's MEM with Eagle's balanced
salts, commonly referred to as Eagle's Alpha MEM, contains
non-essential amino acids, sodium pyruvate, and additional
vitamins. These modifications were first described for use in
growing hybrid mouse and hamster cells (Stanners et al. Nat New
Biol., 230, 52-54, 1971).
[0152] StemSpan.TM. is a fetal bovine serum free, hematopoietic
cell expansion media available commercially from STEMCELL
Technologies. The composition of StemSpan.TM. has not been
disclosed.
[0153] Compositions comprising PDGF-BB, EGF and FGF2 were added to
each basal medium to the following concentrations: [0154] PDGF-BB
(3.2 ng/ml), EGF (0.08 ng/ml) and FGF2 (0.002 ng/ml) [0155] PDGF-BB
(9.6 ng/ml), EGF (0.24 ng/ml) and FGF2 (0.006 ng/ml) [0156] PDGF-BB
(16 ng/ml), EGF (0.4 ng/ml) and FGF2 (0.01 ng/ml) [0157] PDGF-BB
(32 ng/ml), EGF (0.8 ng/ml) and FGF2 (0.02 ng/ml)
[0158] MSC populations were cultured in each of the above mediums
(a-d) on tissue culture plastic (FIG. 4--Alpha MEM; FIG.
7--Stemspan.TM.), fibronectin (FIG. 5--Alpha MEM; FIG.
8--Stemspan.TM.) or 3% hPL (FIG. 6--Alpha MEM; FIG.
9--Stemspan.sup.m).
[0159] Cell proliferation in cell populations cultured in growth
factor supplemented media was compared to cell proliferation in
cell populations cultured in basal media without growth factors.
The greatest increase in cell proliferation was observed in cells
cultured in Alpha MEM or StemSpan.TM. basal media supplemented with
PDGF-BB (32 ng/ml), EGF (0.8 ng/ml) and FGF2 (0.02 ng/ml).
Example 5: Xeno-Free Cell Culture
[0160] Cryopreserved human MPCs were thawed and seeded onto 96 well
plates at 1,000 cells/well in xeno-free media supplemented with
recombinant human growth factors or xeno-free media without growth
factors.
[0161] Growth factors were provided in the following
concentrations: [0162] PDGF-BB (P)-- 10 ng/ml [0163] EGF (E)--5
ng/ml [0164] FGF2 (F)-- 1 ng/ml [0165] PDGF, EGF, FGF2 (PEF)
[0166] Cultures were incubated for about 120 hours inside the
IncuCyte Zoom live imaging microscope (Essen BioScience) fitted
into a humidified NuAire incubator set at 5% CO2, 35-37.degree. C.
Cells were simultaneously imaged every 6 hours to measure the level
of confluence during the culture period. Proliferative kinetics
were calculated as the percent of confluence over-time (FIG.
10).
Example 6: Comparison of Cytokine Levels in Cell Culture Medium
from MPC
[0167] MPC from 3 different donors were serially propagated in
standard medium (alpha-MEM+10% FBS) or a fetal bovine serum free
medium comprising alpha-MEM+3% human AB serum+PDGF-BB, EGF and
FGF2. Cytokine levels were measured in the cell culture medium
after cell propagation (FIG. 11).
[0168] Ang1 levels were elevated in two of three donor cell
populations grown in fetal bovine serum free medium. The Ang1:VEGF
ratio increased in all donor cell populations grown in fetal bovine
serum free medium.
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