U.S. patent application number 12/581981 was filed with the patent office on 2010-02-18 for culture medium containing kinase inhibitors, and uses thereof.
This patent application is currently assigned to The University Court of the University of Edingburgh. Invention is credited to Austin Gerard Smith, Qi-Long Ying.
Application Number | 20100041137 12/581981 |
Document ID | / |
Family ID | 38050545 |
Filed Date | 2010-02-18 |
United States Patent
Application |
20100041137 |
Kind Code |
A1 |
Smith; Austin Gerard ; et
al. |
February 18, 2010 |
CULTURE MEDIUM CONTAINING KINASE INHIBITORS, AND USES THEREOF
Abstract
Pluripotent cells are maintained in a self-renewing state in
serum-free culture medium comprising a MEK inhibitor, a GSK3
inhibitor and, optionally, an antagonist of an FGF receptor.
Pluripotent cells are also maintained in a self-renewing state in
serum-free culture medium comprising a MEK inhibitor and an
antagonist of an FGF receptor.
Inventors: |
Smith; Austin Gerard;
(Cambridge, GB) ; Ying; Qi-Long; (Los Angeles,
CA) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER;LLP
901 NEW YORK AVENUE, NW
WASHINGTON
DC
20001-4413
US
|
Assignee: |
The University Court of the
University of Edingburgh
Edingburgh
GB
|
Family ID: |
38050545 |
Appl. No.: |
12/581981 |
Filed: |
October 20, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11694351 |
Mar 30, 2007 |
|
|
|
12581981 |
|
|
|
|
Current U.S.
Class: |
435/354 ;
435/366 |
Current CPC
Class: |
C12N 2501/115 20130101;
C12N 2500/90 20130101; C12N 5/0606 20130101; C12N 2501/727
20130101 |
Class at
Publication: |
435/354 ;
435/366 |
International
Class: |
C12N 5/071 20100101
C12N005/071 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 30, 2006 |
GB |
0606392.9 |
Aug 1, 2006 |
GB |
0615327.4 |
Claims
1. A method of culturing mouse or human pluripotent cells,
comprising culturing the mouse or human pluripotent cells in the
presence of (i) a MEK1 inhibitor, and (ii) CHIR99021, wherein the
mouse or human pluripotent cells undergo self-renewal.
2. The method of claim 1, wherein the MEK1 inhibitor is selected
from PD184352 and PD98059.
3. A method of obtaining pluripotent cells, comprising culturing
cells in a culture medium comprising a MEK inhibitor and a GSK3
inhibitor, thereby obtaining pluripotent cells.
4. The method of claim 3, wherein the culture medium further
comprises an antagonist of an FGF receptor.
5. The method of claim 3, wherein the cells comprise mouse
cells.
6. The method of claim 3, wherein the cells comprise human
cells.
7. The method of claim 3, wherein the cells comprise rat cells.
8. The method of claim 3, comprising culturing the cells in the
absence of serum.
9. The method of claim 3, wherein the MEK inhibitor is a MEK1
inhibitor.
10. The method of claim 9, wherein the MEK inhibitor is selected
from PD184352 and PD98059.
11. The method of claim 3, wherein the GSK3 inhibitor is an
inhibitor of GSK-3.beta..
12. The method of claim 11, wherein the GSK3 inhibitor is
CHIR99021.
13. The method of claim 4, wherein the antagonist of an FGF
receptor is SU5402 or PD173074.
14. The method of claim 3, wherein the cells are provided as a
blastocyst.
15. A method of culturing pluripotent cells, comprising culturing
the pluripotent cells in a culture medium comprising a MEK
inhibitor and a GSK3 inhibitor, wherein the pluripotent cells
undergo self-renewal.
16. The method of claim 15, wherein the culture medium further
comprises an antagonist of an FGF receptor.
17. The method of claim 15, wherein the cells comprise mouse
cells.
18. The method of claim 15, wherein the cells comprise human
cells.
19. The method of claim 15, wherein the cells comprise rat
cells.
20. The method of claim 15, comprising culturing the cells in the
absence of serum.
21. The method of claim 15, wherein the MEK inhibitor is a MEK1
inhibitor.
22. The method of claim 15, wherein the MEK inhibitor is selected
from PD184352 and PD98059.
23. The method of claim 15, wherein the GSK3 inhibitor is an
inhibitor of GSK-3.beta..
24. The method of claim 23, wherein the GSK3 inhibitor is
CHIR99021.
25. The method of claim 16, wherein the antagonist of an FGF
receptor is SU5402 or PD173074.
Description
[0001] This application claims the benefit under 35 U.S.C.
.sctn.120 and is a division of U.S. application Ser. No.
11/694,351, filed Mar. 30, 2007, which claims foreign priority
under 35 U.S.C. .sctn.119 of Patent Application No. GB 0606392.9,
filed Mar. 30, 2006, in Great Britain and Patent Application No. GB
0615327.4, filed Aug. 1, 2006, in Great Britain, all of which are
incorporated by reference herein.
[0002] The present invention relates to maintenance of a self
renewing phenotype in pluripotent stem cells. The methods and
compositions provided are suitable for culturing and isolating
pluripotent stem cells such as embryonic stem (ES) cells,
especially mammalian, including rat, mouse, bovine, ovine, porcine,
and human, stem cells. In particular this invention relates to
self-renewing cultures of rat, mouse and human pluripotent cells
and to methods and compositions therefor.
[0003] The establishment and maintenance of in vitro pluripotent
stem cell cultures in the presence of medium containing serum and
Leukaemia Inhibitory Factor (LIF) is well known (Smith et al.
(1988) Nature 336: 688-90). Such methods have been used to maintain
pluripotent embryonic stem (ES) cells from "permissive" strains of
mice over many passages. Maintenance and self renewal of
pluripotent stem cell cultures is further supported where the stem
cells are cultured in the presence of feeder cells or extracts
thereof, usually mouse fibroblast cells. Under such conditions it
is possible to maintain human ES cells in a pluripotent state over
many passages in culture.
[0004] In many cases ES cells can only be maintained, or are best
maintained, using medium that contains serum or serum extract, and
hence is undefined, or using cell culture conditions that require
the presence of other cells, such as the fibroblast feeder cells
used to maintain human ES cells. But any undefined component,
whether in the medium or produced by e.g. the feeder cells,
potentially interferes with or hinders research into ES cell
propagation and differentiation. This prevents development of good
manufacturing practices for therapeutic and other applications of
ES cells and their progeny. Some defined ES cell media are known
but alternative and/or improved defined media are needed.
[0005] In prior applications by the applicants, WO-A-03/095628 and
a later as yet unpublished application, culturing pluripotent stem
cells, such as ES cells, in serum-free media comprising (1)
agonists of gp130 (e.g. LIF) and (2) agonists of the TGF-.beta.
superfamily (e.g. BMP4) or Id signalling pathways is used to
promote self renewal of the stem cells for multiple passages. In
the presence of gp130 signalling, an agonist of the TGF-.beta.
superfamily or the Id signalling pathway surprisingly provided a
self renewal stimulus rather than a pro-differentiation signal.
Nevertheless, ever improved efficiencies in maintaining pluripotent
cells in a self renewing state and media for transferring
pluripotent cells away from feeder cells or away from
feeder-conditioned medium is desired.
[0006] Sato N, et al, Nat. Med. 2004, January 10(1) pp 55-63
describe the effects of a Glycogen Synthase Kinase 3 (GSK3)
inhibitor, 6-bromoindirubin-3'-oxime, on mouse and human ES cells
in serum containing medium. These effects, however, were observed
only over a very short time frame, too short for firm conclusions
to be drawn, and the influence of unknown factors in the undefined
media used in that study may be significant. The inventors of the
present invention have tried but failed to repeat the results, and
have in fact found effects opposite to those described.
[0007] For preparation of ES cell culture media it is desired to
provide individual media components in as pure a form as possible.
However, most media components are cytokines the purity of which is
compromised by the need to manufacture them in cellular systems and
then remove potential contaminants from the production broth.
Another problem with some cytokines is that they have a narrow
range of concentration over which they are effective and non-toxic.
Media components which have a broader range and/or are less toxic
at higher concentrations would be highly useful. Cytokines can also
have limited stability in storage, and more stable media components
are sought.
[0008] An object of the invention is to overcome or at least
ameliorate problems in the art, e.g. to provide alternative or
improved, methods of culturing and culture media suitable for
pluripotent stem cells, which are capable of supporting
self-renewal of said stem cells for many passages. A further object
of the invention is to provide an alternative culturing system that
permits maintenance of a pluripotent stem cell culture in vitro
until differentiation of the cells can be induced in a controlled
manner. A still further object of the invention is to provide
methods and compositions that enhance the derivation and isolation
of pluripotent stem cells and facilitate their derivation and
isolation from organisms refractory to ES cell isolation or from
which pluripotent stem cells have not yet been isolated.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0009] In accordance with the present invention, pluripotent stem
cells, such as ES cells, are cultured in medium comprising a MEK
inhibitor and a GSK3 inhibitor, or a MEK inhibitor and an
antagonist of an FGF receptor. In some embodiments, the medium is
serum-free. In some embodiments, the medium comprises a MEK
inhibitor, a GSK3 inhibitor and an antagonist of a FGF receptor
(e.g. a small molecule GSK3 inhibitor and a small molecule MEK
inhibitor and a small molecule FGFR antagonist). Self renewal of
the stem cells for multiple passages is thereby promoted. Hence,
inhibition of GSK3 and MEK, inhibition of MEK and FGF receptor
signaling, or inhibition of GSK3, MEK and FGF receptor signalling
in the pluripotent cells provides a self renewal stimulus.
[0010] The invention has a number of applications. A combination of
GSK3 and MEK, MEK and FGFR or GSK3, MEK and FGFR inhibition can be
used to grow pluripotent cells, especially ES cells, and, where
they have been derived or grown on feeder cells, to adapt
pluripotent cells, especially ES cells, to grow without feeder
cells or a layer of feeder cells, often referred to as feeders or
feeder cells. A method of expanding stem cells in culture comprises
culturing the cells in the presence of a GSK3 inhibitor and a MEK
inhibitor, in the presence of a MEK inhibitor and an antagonist of
an FGF receptor. In some embodiments, the method comprises
culturing the cells in the presence of a GSK3 inhibitor, a MEK
inhibitor and an antagonist of a FGF receptor. Culture medium can
be prepared containing one or more GSK3 inhibitors and MEK
inhibitors, one or more MEK inhibitors and FGFR antagonists and,
optionally, one or more MEK inhibitors, GSK3 inhibitors and FGFR
antagonists. ES cells can be derived using GSK3 inhibitors and MEK
inhibitors, using MEK inhibitors and FGFR antagonists, or using
GSK3 inhibitors, MEK inhibitors and FGFR antagonists.
[0011] According to a first aspect of the present invention,
inhibition of GSK3 and MEK, or inhibition of all of GSK3 and MEK
and a FGF receptor, in a pluripotent cell is used to promote
self-renewal of the cell.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIGS. 1A-1E show analysis of the effect of PD184352 in the
formation of pluripotent ES-NS hybrid colonies. (FIGS. 1A-1C)--FACS
analysis for red and green fluorescence of RHxNS TGFP fusions.
(FIG. 1A) Fusion mixture 24 hours after PEG treatment; (FIG. 1B)
Purity check of FACS sorted hybrids gated in A. (FIG. 1C) Hybrids
sorted in A were plated and the formed colonies were scored as
percentage of colonies per plated hybrid. These scores take into
account the purity of the FACS sorted cells. (FIG. 1D) Summary of
data. (FIG. 1E) Examples of hybrid colony morphology.
[0013] FIG. 2 shows mouse ES cells derived and maintained according
to the invention and shows high efficiency of chimera contribution
by these ES cells. FIG. 2A shows ES cells derived and maintained in
N2B27 medium with the three inhibitors and no serum, no feeder
cells, and no LIF present from the very beginning of the culture.
FIG. 2B shows high efficiency of chimera contribution.
[0014] FIG. 3 shows passage 4 mouse ES cells grown in accordance
with the invention. Mouse ES cells were grown from single sorted
LIF receptor knock-out ES cell in N2B27 medium plus 3 .mu.M
CHIR99021, 1 .mu.M PD184352, and 2.5 .mu.M SU5402.
[0015] FIG. 4 shows mouse ES cells, grown in accordance with the
invention, are Oct4 positive. From sorted single Oct4GIP ES cells,
passage 5 total 29 days in culture. The culture medium is N2B27
plus 3 .mu.M CHIR99021, 1 .mu.M PD184352 and 2.5 .mu.M SU5402. The
GFP is driven by Oct4 promoter, so the GFP positive cells are Oct4
positive ES cells. Bar=100 .mu.M
[0016] Reference to pluripotent cells includes but is not limited
to reference to embryonic stem (ES) cells. Characteristic
properties of pluripotent cells, including ES cells, include the
expression of multiple genes associated with the pluripotent stage
of development, the ability to differentiate into cells
representative of any and all tissue types present in the source
animal, the ability to contribute to chimeras and, particularly,
the ability to contribute to the germ line of chimeras. For example
true pluripotent cells, such as ES cells, would be expected to
express many, if not all, of the pluripotency-associated genes
Nanog, Oct4, FGF4, Sox-2 and alkaline phosphatase. In particular,
expression of Nanog, Oct4 and Sox-2 is widely regarded as providing
a definitive initial indication that a cell is an ES cell. Germ
line transmission in chimeras and the ability to generate teratomas
or teratocarcinomas comprising differentiated cells from all three
primary germ layers (i.e. endoderm, mesoderm and ectoderm) are also
widely regarded as definitive indications of a cell being an ES
cell.
[0017] Reference to GSK3 inhibition refers to inhibition of one or
more GSK3 enzymes. Thus a GSK3 inhibitor can inhibit one member,
several members or all members of the family of GSK3 enzymes. The
family of GSK3 enzymes is well-known and includes but is not
limited to GSK3-.alpha. and GSK3-.beta.. A number of variants have
been described (see e.g. Schaffer et al.; Gene 2003; 302(1-2):
73-81). In specific embodiments GSK3-.beta. is inhibited.
GSK3-.alpha. inhibitors are also suitable, and in general
inhibitors for use in the invention inhibit both. A wide range of
GSK3 inhibitors are known including but not limited to the
inhibitors CHIR 98014, CHIR 99021, AR-AO144-18, TDZD-8, SB216763
and SB415286. Other inhibitors are known and useful in the
invention. In addition, the structure of the active site of
GSK3-.beta. has been characterised and key residues that interact
with specific and non-specific inhibitors have been identified
(Bertrand et al.; J Mol Biol. 2003; 333(2): 393-407). This
structural characterisation allows additional GSK inhibitors to be
readily identified.
[0018] The inhibitors of certain embodiments are specific for
GSK3-.beta. and GSK3-.alpha., substantially do not inhibit erk2 and
substantially do not inhibit cdc2. In some embodiments the
inhibitors have at least 100 fold, at least 200 fold, or at least
400 fold selectivity for human GSK3 over mouse erk2 and/or human
cdc2, measured as ratio of IC.sub.50 values; here, reference to
GSK3IC.sub.50 values refers to the mean values for human
GSK3-.beta. and GSK3-.alpha.. Good results have been obtained with
CHIR 99021 and CHIR 98014, which are both specific for GSK3.
Examples of GSK3 inhibitors are described in Bennett C, et al, J.
Biol. Chem., vol. 277, no. 34, Aug. 23, 2002, pp 30998-31004 and in
Ring D B, et al Diabetes, vol. 52, March 2003, pp 588-595. Suitable
concentrations for use of CHIR 99021 are in the range 0.01 to 100,
for example 0.1 to 20, or 0.3 to 10 micromolar.
[0019] GSK3 inhibition can also be conveniently achieved using RNA
mediated interference (RNAi). Typically, a double-stranded RNA
molecule complementary to all or part of a GSK3 gene is introduced
into pluripotent cells, thus promoting specific degradation of
GSK3-encoding mRNA molecules. This post-transcriptional mechanism
results in reduced or abolished expression of the targeted GSK3
gene. Suitable techniques and protocols for achieving GSK3
inhibition using RNAi are known.
[0020] Reference to a MEK inhibitor herein refers to MEK inhibitors
in general. Thus, reference to a MEK inhibitor refers to any
inhibitor a member of the MEK family of protein kinases, including
MEK1, MEK2 and MEK3. Reference is also made to MEK1, MEK2 and MEK3
inhibitors. A MEK inhibitor can inhibit one member, several members
or all members of the family of MEK kinases. Examples of suitable
MEK inhibitors, already known in the art, include but are not
limited to the MEK1 inhibitors PD184352 and PD98059, inhibitors of
MEK1 and MEK2 U0126 and SL327, and those discussed in Davies et al
(2000) (Davies S P, Reddy H, Caivano M, Cohen P. Specificity and
mechanism of action of some commonly used protein kinase
inhibitors. Biochem J. 351, 95-105). In particular, PD184352 has
been found to have a high degree of specificity and potency when
compared to other known MEK inhibitors. Other MEK inhibitors and
classes of MEK inhibitors are described in Zhang et al. (2000)
Bioorganic & Medicinal Chemistry Letters; 10:2825-2828.
[0021] Inhibition of MEK kinases can also be conveniently achieved
using RNA-mediated interference (RNAi). Typically, a
double-stranded RNA molecule complementary to all or part of a MEK
gene is introduced into pluripotent cells, thus promoting specific
degradation of MEK-encoding mRNA molecules. This
post-transcriptional mechanism results in reduced or abolished
expression of the targeted MEK gene. Suitable techniques and
protocols for achieving MEK inhibition using RNAi are known.
[0022] A number of assays for identifying kinase inhibitors,
including GSK3 inhibitors and MEK inhibitors, are known. For
example, Davies et al (2000) describe kinase assays in which a
kinase is incubated in the presence of a peptide substrate and
radiolabelled ATP. Phosphorylation of the substrate by the kinase
results in incorporation of the label into the substrate. Aliquots
of each reaction are immobilised on phosphocellulose paper and
washed in phosphoric acid to remove free ATP. The activity of the
substrate following incubation is then measured and provides an
indication of kinase activity. The relative kinase activity in the
presence and absence of candidate kinase inhibitors can be readily
determined using such an assay. Downey et al. (1996) J Biol Chem.;
271(35): 21005-21011 also describes assays for kinase activity
which can be used to identify kinase inhibitors.
[0023] Reference to an antagonist of fibroblast growth factor (FGF)
receptor (FGFR) refers to a polypeptide or small molecule or other
antagonist of a FGF receptor, typically inhibiting FGFR1 and/or
FGFR2. Thus, a FGF receptor antagonist can be an antagonist of one,
several or all members of the FGF receptor family, including but
not limited to FGFR1, FGFR2, FGFR3 and FGFR4. Members of the FGF
receptor family typically comprise three immunoglobulin-like
domains and present a region of acidic amino acids (the acidic box)
which can participate in the binding of a member of the FGF family
to a FGF receptor. In some cases, molecules comprising only two
immunoglobulin-like domains can also function as FGF receptors. A
number of FGFR antagonists are known, including but not limited to
SU5402 and PD173074. Suitable concentrations of SU5402 are in the
micromolar range, such as from 0.1-20 .mu.M. Some embodiments use
concentrations in the range 0.5-10 .mu.M, especially in the range
1-5 .mu.M. We have found that PD173074 can substitute for SU5402
and is fully effective at about 100-fold lower concentrations,
consistent with its higher affinity for the FGF receptor. Thus,
suitable concentrations for PD173074 are in the range 1-200 nM. In
some embodiments the concentration is in the range 5-100 nM,
especially in the range 10-50 nM. It is also known to inhibit FGR
receptor signalling by transgene expression of a dominant negative
mutant FGF receptor. In embodiments of the invention a small
molecule antagonist and not a transgenic based antagonism is
used.
[0024] Suitable assays for identifying antagonists of FGF receptors
are known. For example, a cell line in which signalling via a FGF
receptor activates expression of a reporter gene can be used to
assess the activity of a potential antagonist.
[0025] It has advantageously been found that the use of a MEK
inhibitor in combination with a GSK3 inhibitor and optionally also
an antagonist of the FGF receptor improves the propagation of ES
cells.
[0026] In some embodiments between around 0.1 .mu.M and around 25
.mu.M MEK inhibitor are used. In other embodiments, between around
0.1 .mu.M and around 5 .mu.M MEK inhibitor are used, for example
from 0.2 .mu.M to 2 .mu.M.
[0027] In some embodiments media according to the invention
comprise 0.8 .mu.M PD184352, 3 .mu.M CHIR99021 and/or 3 .mu.M
SU5402. In other embodiments the medium comprises 0.8 .mu.M
PD184352, 3 .mu.M CHIR99021 and 3 .mu.M SU5402, for example in
N2B27 medium. The concentration of SU5402 can be optimized to suit
different pluripotent cell lines, typically in the range 1-5 .mu.M
(e.g. 2 .mu.M).
[0028] In examples below, we have cultured mouse ES cells in the
presence of a GSK3 inhibitor together with a MEK inhibitor and, in
a specific example, an antagonist of the FGF receptor to promote
self renewal. In other specific examples, a method of promoting
self-renewal of mouse pluripotent cells in culture comprises
inhibiting GSK3 and MEK or inhibiting GSK3, MEK and an FGF
receptor.
[0029] Optionally, activating gp130 downstream signalling can also
be employed to further enhance the promotion of self renewal by
inhibiting GSK3 and MEK. Molecules that activate gp130 downstream
signalling are sometimes referred to as gp130 activators or gp130
agonists. Activation of one or more gp130 downstream signalling
pathways can be achieved by use of a cytokine acting through gp130,
for example a cytokine or other agonist of the LIF receptor.
Cytokines capable of acting through gp130, and thus of activating
gp130 signal transduction, include but are not limited to LIF,
ciliary neurotrophic factor (CNTF), cardiotrophin, oncostatin M,
IL-6 plus sIL-6 receptor, hyper IL-6 and IL-11. Suitable cytokines
include mimetics, fusion proteins or chimaeras that can bind to
and/or activate signalling though gp130. The role of cytokines
acting through gp130 in the presence of serum is well established,
but the capacity of those cytokines to sustain undifferentiated
cells in the absence of serum is limited.
[0030] An advantage of the invention is that in the presence of a
GSK3 inhibitor, a MEK inhibitor and, optionally, an antagonist of
the FGF receptor, pluripotent cells can be grown in defined medium.
A particular advantage associated with using the combination of a
GSK3 inhibitor, a MEK inhibitor and an antagonist of the FGF
receptor is that it is not necessary for the medium to contain
other growth factors, such as insulin, N2B27, or a gp130 agonist
(e.g. LIF). The present invention therefore enables alternative
and/or improved culture of ES cells in medium that is free of
serum, serum extract, feeder cells and feeder cell extract.
[0031] Purported embryonic stem cells have been reported from a
number of mammalian sources including mouse (Bradley et al (1984)
Nature 309: 255-56), American mink (Mol Reprod Dev (1992) December;
33(4):418-31), pig and sheep (J Reprod Fertil Suppl (1991);
43:255-60), hamster (Dev Biol (1988) May; 127(1):224-7) and cow
(Roux Arch Dev Biol (1992); 201: 134-141). Specific examples herein
use mouse and human ES cells and also rat cell from primary
outgrowths. It will be appreciated that the methods and
compositions of the present invention are suitable for adaptation
to culturing of other mammalian pluripotent cell cultures, thus
including primate, especially human, rodent, especially mouse and
rat, and avian pluripotent stem cells, especially ES cells.
[0032] A second aspect of the invention provides a method of
culture of pluripotent cells, especially ES cells, so as to promote
self renewal, comprising maintaining the cells in medium
containing:
[0033] a) an inhibitor of GSK3; and
[0034] b) an inhibitor of MEK.
[0035] In some embodiments, the method comprises maintaining the
cells in medium containing:
[0036] a) an inhibitor of GSK3;
[0037] b) an inhibitor of MEK; and
[0038] c) an antagonist of an FGF receptor.
[0039] Methods of the invention can be used generally for growing
pluripotent cells, including growing ES cells in medium which is
free of serum and free of serum extract, which cells have
previously been passaged in the presence of serum or serum extract.
Such methods can also be carried out in the absence of feeder cells
and/or feeder cell extracts. For example, culture of ES cells can
be carried out comprising the steps of:
[0040] a) maintaining the ES cells in a pluripotent state in
culture, optionally on a layer of feeder cells;
[0041] b) passaging the ES cells at least once;
[0042] c) withdrawing the serum or the serum extract from the
medium and withdrawing the feeder cells (if present), so that the
medium is free of feeder cells, serum and serum extract; and
[0043] d) subsequently maintaining ES cells in a pluripotent state
in the presence of an inhibitor of GSK3, a MEK inhibitor and,
optionally, an FGFR antagonist.
[0044] Further optionally, the cells can be maintained in a
pluripotent state in the presence of a MEK inhibitor, a GSK3
inhibitor and an activator of a gp130 downstream signalling
pathway.
[0045] The present invention also provides a method of obtaining a
transfected population of ES cells, comprising:
[0046] a) transfecting ES cells with a construct encoding a
selectable marker;
[0047] b) plating the ES cells;
[0048] c) culturing the ES cells in the presence of a MEK
inhibitor, a GSK3 inhibitor and, optionally, an FGFR antagonist;
and
[0049] d) selecting for cells that express the selectable
marker.
[0050] Further optionally, the cells are cultured in the presence
of a MEK inhibitor, a GSK3 inhibitor and an activator of a gp130
downstream signalling pathway.
[0051] The selectable marker may encode antibiotic resistance, a
cell surface marker or another selectable marker as described e.g.
in EP-A-0695351, and, in some embodiments, comprises a nucleotide
sequence encoding the selectable marker operatively linked to a
promoter which preferentially expresses the selectable marker in
desired cells.
[0052] In a further embodiment, the present invention provides a
method of culture of pluripotent, especially ES, cells, comprising
the steps of transferring an individual cell to a culture vessel,
such as an individual well on a plate, and culturing the cell in
the presence of a GSK3 inhibitor, a MEK inhibitor and, optionally,
an FGFR antagonist, so as to obtain a clonal population of
pluripotent, especially ES, cells, all of which are progeny of a
single cell. Optionally, the cells may also be cultured in the
presence of an activator of gp130 downstream signalling
pathways.
[0053] Once a stable, homogenous culture of ES cells is obtained,
the culture conditions can be altered to direct differentiation of
the cells into one or more cell types selected from ectodermal,
mesodermal or endodermal cell fates. Addition of, or withdrawal of
cytokines and signalling factors, can enable the derivation of
specific differentiated cell populations at high efficiency.
Differentiation of an ES cell towards a non-neuroectodermal fate
may be achieved by maintaining the ES cell in the presence of a
cytokine acting through gp130, a MEK inhibitor and a GSK3 inhibitor
and then withdrawing the cytokine whilst maintaining the GSK3
inhibitor and MEK inhibitor and/or adding a further signalling
molecule capable of directing differentiation. Alternatively, the
cells may be maintained in the presence of a MEK inhibitor and a
GSK3 inhibitor and then differentiation directed by withdrawing one
or both of the inhibitors and/or adding a signalling molecule
capable of directing differentiation. The methods described above
all optionally include the step of obtaining and/or isolating a
differentiated cell which is the product of the process.
[0054] Further aspects of the invention provide for cell culture
media. One medium is for self-renewal of pluripotent, especially
ES, cells, the medium comprising an inhibitor of GSK3, an inhibitor
of MEK and, optionally, an FGFR antagonist. The medium may also
optionally comprise an activator of a gp130 downstream signalling
pathway. Another medium of the invention is a stem cell culture
medium, comprising an inhibitor of GSK3, a MEK inhibitor and,
optionally, an FGFR antagonist. All media can further comprise
basal medium. In some embodiments, all media are free of an agonist
of gp130, and are for example free of LIF.
[0055] The invention provides medium that is free of serum and
serum extract. One such medium comprises:
[0056] a) basal medium;
[0057] b) a MEK inhibitor;
[0058] c) a GSK3 inhibitor; and
[0059] d) an iron-transporter; wherein the medium is optionally
free of serum and serum extract.
[0060] In some embodiments the medium also comprises an FGFR
antagonist. The medium may also optionally comprise an activator of
a gp130 downstream signalling pathway.
[0061] Medium for pluripotent stem cells, especially rat or mouse
cells, may be free of serum and of gp130 agonists and comprises a
MEK inhibitor, a GSK3 inhibitor, an antagonist of an FGF receptor.
Substitutions of media components can be made as described
herein.
[0062] Basal medium is medium that supplies essential sources of
carbon and/or vitamins and/or minerals for the cells. The basal
medium is generally free of protein and incapable on its own of
supporting self-renewal of cells. The iron transporter provides a
source of iron or provides the ability to take up iron from the
culture medium. Suitable iron transporters include transferrin and
apotransferrin. In some embodiments the medium further comprises
one or more of insulin or insulin-like growth factor and albumin
(which can be recombinant) or albumin substitute, and is free of
feeder cells and feeder cell extract. The medium may also comprise
an inhibitor of apoptosis or any other component that promotes the
maintenance of pluripotent cells in culture.
[0063] A particular medium of the invention comprises MEK
inhibitor, GSK3 inhibitor, insulin, albumin and transferrin, with
or without additional basal medium. In this medium, LIF can be
optionally included and can be substituted by other activators of
gp130 signalling. Thus, in some embodiments the medium comprises
the gp130 receptor binding cytokine, LIF, suitable concentrations
of which are generally between 10 U/ml and 1000 U/ml, for example
between 50 U/ml and 500 U/ml, e.g. in the region of 100 U/ml. The
GSK3 and MEK inhibitors are generally as described herein in more
detail.
[0064] The invention further provides a method of deriving a
pluripotent cell from a blastocyst, comprising:
[0065] a) obtaining a blastocyst;
[0066] b) culturing the blastocyst in the presence of a MEK
inhibitor, a GSK3 inhibitor and, optionally, an antagonist of an
FGF receptor, to obtain an inner cell mass;
[0067] c) dissociating the inner cell mass;
[0068] d) isolating a cell or cells from the dissociated inner cell
mass; and
[0069] e) culturing the isolated cell or cells in the presence of a
MEK inhibitor, a GSK3 inhibitor and, optionally, an antagonist of
an FGF receptor.
[0070] Optionally, the isolated cell or cells are cultured in the
presence of a MEK inhibitor, a GSK3 inhibitor and an activator of
gp130 downstream signalling. An antagonist of an FGF receptor may
also be present.
[0071] In some embodiments, the method comprises culturing the
blastocyst in LIF, for example for a period of from 2 to 4 days. In
some embodiments the isolated cell or cells are cultured in serum
free medium. Typically, the cells are replated as clumps. In some
embodiments the blastocyst is also cultured in serum free medium,
optionally in the absence of an agonist of the BMP receptor.
[0072] In some embodiments of the invention, the culture of cells
is carried out in an adherent culture, which may be promoted by the
inclusion of a cell adhesion protein on culture substrate. In some
embodiments the culture of pluripotent cells according to the
invention is in monolayer culture, though it is optional for cells
to be grown in suspension culture or as pre-cell aggregates; cells
can also be grown on beads or on other suitable scaffolds such as
membranes or other 3-dimensional structures.
[0073] Culture medium used in some of the examples of the invention
also comprises serum albumin. This can be used in purified or
preferably recombinant form, and if in a recombinant form this has
the advantage of absence of potential contaminating factors,
cytokines etc. The culture medium does not need to contain serum
albumin and this component can be omitted or replaced by another
bulk protein or by a synthetic polymer (polyvinyl alcohol) as
described by Wiles et al.
[0074] In some embodiments the medium is one that is fully defined.
This medium does not contain any components which are undefined,
that is to say components whose content is unknown or which may
contain undefined or varying factors that are unspecified. An
advantage of using a fully defined medium is that efficient and
consistent protocols for culture and subsequent manipulation of
pluripotent cells can be derived. Further, it is found that
maintenance of cells in a pluripotent state is achievable with
higher efficiency and greater predictability and that when
differentiation is induced in cells cultured using a defined medium
the response to the differentiation signal is more homogenous than
when undefined medium is used.
[0075] The invention also provides concentrates which can be used
as additives for culture medium, and kits of components, for
preparation of culture medium, the resultant medium being in
accordance with the invention. One kit of the invention comprises
first and second containers, the first containing a MEK inhibitor
and the second containing a GSK3 inhibitor. In some embodiments,
the kit comprises a third container containing an antagonist of a
FGF receptor. The kit may also, optionally, comprise a further
container containing an activator of gp130 downstream signalling.
In some embodiments the kits are formulated so that the contents of
each container can be added to culture medium so as to obtain a
culture medium of the invention. Thus, in some embodiments the kits
contain concentrated stock solutions of their respective
components.
[0076] Methods of the invention also include a method of obtaining
a differentiated cell comprising culturing a pluripotent cell as
described and allowing or causing the cell to differentiate,
wherein the cell contains a selectable marker which is capable of
differential expression in the desired differentiated cell compared
with other cell-types, including pluripotent stem cells, whereby
differential expression of the selectable marker results in
preferential isolation and/or survival and/or division of the
desired differentiated cells. The selectable marker may be
expressed in the desired differentiated cells but not expressed in
other cell types, or the level of expression may differ between
desired differentiated cells and other cell types, thereby allowing
selection for expression of the selectable marker. The
differentiated cell can be a tissue stem or progenitor cell, and
may be a terminally differentiated cell.
[0077] Generally also, the invention extends to a cell obtained by
following any of the methods of the invention described herein.
Cells of the invention can be used in assays for drug discovery.
Cells of the invention may also be used for cell therapy, and thus
a method of the invention comprises using a combination of
inhibition of MEK and inhibition of GSK3 and, optionally,
antagonism of FGF signalling to derive and/or maintain pluripotent
cells, deriving cells for cell therapy therefrom and using those
cells in cell therapy. Optionally, the combination is used in the
absence of an activator of gp130 downstream signalling.
[0078] Further aspects of the invention relate to the use of
inhibition of MEK and an FGF receptor, optionally in combination
with inhibition of GSK3 for promoting self-renewal of pluripotent
cells. We have found that the combination of a MEK inhibitor and an
antagonist of an FGF receptor is effective in supporting the growth
of pluripotent cells in serum-free medium in the absence of added
cytokines or growth factors.
[0079] Accordingly, a further aspect of the invention provides a
culture medium, comprising a MEK inhibitor and an antagonist of an
FGF receptor. The MEK inhibitor and the antagonist of an FGF
receptor are as described in relation to other aspects of the
invention. Similarly, the culture medium may further comprise
additional components or factors as described herein in relation to
other aspects of the invention.
[0080] Yet another aspect of the invention provides use of a MEK
inhibitor and an antagonist of an FGF receptor in manufacture of a
culture medium for pluripotent cells.
[0081] The invention also provides methods for culturing
pluripotent cells and obtaining transfected populations of
pluripotent cells, which may be conveniently carried out as
described for other aspects of the invention. Accordingly, a
further aspect of the invention provides a method of culture of
pluripotent cells so as to promote self renewal, comprising
maintaining the cells in medium comprising a MEK inhibitor and an
antagonist of an FGF receptor.
[0082] A related aspect of the invention provides a method of
culture of pluripotent cells, comprising the steps of:
[0083] a) maintaining the ES cells in a pluripotent state in
culture, optionally on feeder cells,
[0084] b) passaging the ES cells at least once;
[0085] c) withdrawing serum or serum extract (if present) from the
medium and withdrawing the feeder cells (if present), so that the
medium is free of feeder cells, serum and serum extract; and
[0086] d) subsequently maintaining ES cells in a pluripotent state
in the presence of a MEK inhibitor and an inhibitor of an FGF
receptor.
[0087] A further aspect of the invention provides a method of
obtaining a transfected population of ES cells, comprising:
[0088] a) transfecting ES cells with a construct encoding a
selectable marker;
[0089] b) plating the ES cells;
[0090] c) culturing the ES cells in the presence of a MEK inhibitor
and an FGF receptor antagonist and
[0091] d) selecting for cells that express the selectable
marker.
[0092] Also provided is a cell culture medium that is free of serum
and serum extract and comprises:
[0093] a) basal medium;
[0094] b) a MEK inhibitor;
[0095] c) an antagonist of an FGF receptor; and
[0096] d) an iron-transporter.
[0097] The combination of a MEK inhibitor and an antagonist of an
FGF receptor is also useful for deriving new pluripotent cell
lines. Accordingly, a further aspect of the invention provides a
method of deriving a pluripotent cell from a blastocyst,
comprising:
[0098] a) obtaining a blastocyst;
[0099] b) culturing the blastocyst in the presence of a MEK
inhibitor and an antagonist of an FGF receptor, to obtain an inner
cell mass;
[0100] c) dissociating the inner cell mass;
[0101] d) isolating a cell or cells from the dissociated inner cell
mass; and
[0102] e) culturing the isolated cell or cells in the presence of a
MEK inhibitor and an antagonist of an FGF receptor.
[0103] The invention also includes kits comprising first and second
containers, the first containing a MEK inhibitor and the second
containing an antagonist of an FGF receptor. The kit may also
comprise other containers and/or components as described
herein.
[0104] Further aspects of the invention provide use of a MEK
inhibitor and an antagonist of an FGF receptor in promoting
self-renewal of pluripotent stem cells, especially pluripotent stem
cells expressing Nanog. A related aspect provides a method of
expanding a stem cell population, comprising culturing the stem
cells in the presence of a MEK inhibitor and an antagonist of an
FGF receptor.
[0105] A number of advantages of the invention are described above
or apparent. Cell culture components may be identified which are
relatively non-toxic and cell permeable. The MEK inhibitors, GSK3
inhibitors and FGFR antagonists used in specific embodiments of the
invention can be purified easily, especially compared to, say,
purification of protein cytokines. Recombinant proteins can be
expensive to make and the small molecule medium components may be
more cheaply produced and more stable in storage, with a wider
effective concentration range.
[0106] Specific embodiments set out below used a combination of
CHIR 99021, PD184352 and, optionally, SU5402 in a serum-free, fully
defined medium and gave improved self renewal of mouse ES cells
with very little differentiation. It is occasionally reported when
culturing ES cells in the presence of BMP that there is some
neurogenesis. This was not seen in the examples of the
invention.
[0107] The invention is now further described in specific examples,
illustrated by drawings. In the examples the term 2i medium or 2i
is used to indicate medium comprising a MEK inhibitor and an
antagonist of an FGF receptor. The term 3i medium or 3i is used to
indicate medium comprising a MEK inhibitor, a GSK3 inhibitor and an
antagonist of an FGF receptor.
EXAMPLES
GSK-3.beta. Inhibitors, MEK Inhibitors, Culture Medium and ES Cell
Self-Renewal
[0108] Mouse and human ES cells were grown under various
conditions, using N2B27 medium unless otherwise stated and in the
presence or absence of the GSK-3.beta. inhibitors CHIR99021,
AR-AO144-18, SB216763 and SB415286 and the MEK inhibitor
PD184352.
[0109] Preparation of N2B27 Medium:
[0110] N2 100.times. stock solution. For 10 ml: mix 1 ml insulin
(final concentration 2.5 mg/ml) with 1 ml apo-transferrin (final
concentration 10 mg/ml), 0.67 ml BSA (final concentration 5 mg/ml),
33 .mu.l progesterone (final concentration 2 .mu.g/ml), 100 .mu.l
putrescine (final concentration 1.6 mg/ml), 10 .mu.l sodium
selenite (final concentration 3 .mu.M) and 7.187 ml DMEM/F12. Store
at 4.degree. C. and use within 1 month.
[0111] DMEM/F12-N2 medium: to 100 ml of DMEM/F12, add 1 ml of N2
100.times. stock solution. The final concentration of each
component of N2 in the DMEM/F12 medium is: insulin, 25 .mu.g/ml;
apo-transferrin, 100 .mu.g/ml; progesterone, 6 ng/ml; putrescine,
16 .mu.g/ml; sodium selenite, 30 nM; BSA 50 .mu.g/ml. Store at
4.degree. C. and use within 1 month.
[0112] Neurolbasal/B27 medium: to 100 ml of Neurolbasal.TM. Medium,
add 2 ml of B27 and 0.5-1 ml of 200 mM L-glutamine. Store at
4.degree. C. and use within 1 month.
[0113] N2B27 medium: mix DMEM/F12-N2 medium with Neurolbasal/B27
medium in the ratio of 1:1. Add .beta.-mercaptoethanol to a final
concentration of 0.1 mM from the 0.1M stock. Store at 4.degree. C.
and use within 1 month.
Example 1
[0114] In serum-free medium a MEK inhibitor plus a GSK-3.beta.
inhibitor was sufficient to sustain mouse ES cell self-renewal in
both (1) N2B27 medium, and (2) fully defined medium
(DMEM/F12-N2)--data not shown. Self renewal of ES cells was
improved further in medium containing a MEK inhibitor, a
GSK-3.beta. inhibitor and LIF (data not shown).
Example 2
[0115] It was shown that PD184352, an inhibitor of MEK, increases
the levels of Nanog in ES cells (data not shown). In addition, it
was shown that Nanog -/- ES cells treated with PD184352 failed to
show enhancing of ES cell self-renewal (data not shown). In fact,
these cells differentiated. This demonstrated that the enhancing of
ES self-renewal phenotype by PD184352 is mediated by Nanog.
[0116] The effect of PD184352 in reprogramming was also
investigated by determining the conversion of NS cells to
pluripotency in the context of cell fusion.
[0117] RH ES cells, which express constitutively the dsRed
fluorescent protein and hygromycin resistance, were fused to foetal
derived Neural Stem cells (NS TGFP) that express the fusion protein
TauGFP linked via an IRES to puromycin resistance. In one of the
fusions RH cells were treated for 3 days prior and after fusion
with 3 .mu.M PD184352. In the control no PD184352 was added.
Treated and untreated primary hybrids were sorted 24 hours after
fusion and then plated (FIG. 1A-C). Hygromycin and puromycin
selection were added to the ES medium 3 days later. Colonies
expressing dsRed2 and GFP fluorescence and exhibiting ES cell
morphology were scored (FIGS. 1D and E). Results showed that
PD184352 enhanced ES-NS hybrid colony formation by 45-fold.
Interestingly, the percentage of hybrid colonies formed per plated
hybrid in PD184352 treated RH cells was just 2-fold lower compared
to Nanog overexpressing ES cells (2.25% vs 4%). This result shows
that PD184352 not only enhances ES cell self-renewal but also
enhances reprogramming in the cell fusion context. This effect is
likely to be mediated by the increased levels of Nanog in treated
RH cells. Accordingly, if Nanog is endogenously expressed then the
MEK inhibitor can be used to upregulate Nanog and achieve
associated effects, such as increased reprogramming.
Example 3
[0118] Human ES cells were cultured in media supplemented with the
GSK-3 inhibitor CHIR99021 and the MEK inhibitor PD184352.
[0119] The addition of LIF to the culture media further improved
propagation of the cells (data not shown).
Example 4
[0120] Mouse ES cells were cultured in media supplemented with the
GSK-3 inhibitor CHIR99021 and the MEK inhibitor PD184352.
[0121] The addition of LIF to the culture media further improved
propagation of the cells (data not shown).
Example 5
[0122] Mouse and human ES cells were grown in medium containing
CHIR99021, PD184352 and SU5402, prepared as follows: --
[0123] Concentrations of the Three Inhibitors/Antagonist:
TABLE-US-00001 Final Concentration Initial when added to Compound
concentration Dilutions media CHIR99021 10 mM Aliquot stock in 20ul
aliquots. 3 .mu.M Store at -20 Initial 1:10 dilution with N2B27
This >1 yr media = 1 mM. concentration Store at 4.degree. C. Add
diluted stock to was used for all media at 1:333 to make 3 .mu.M
final. cell lines PD184352 10 mM Aliquot stock in 10ul aliquots.
0.8 .mu.M Store at -20 Initial 1:100 dilution in N2B27 = Some cell
lines >1 yr 1 ml of 100uM, store at 4.degree. C. Add to were
grown in media at 1:125 for 0.8 .mu.M final. concentrations varying
in the range = 0.5-1 .mu.M SU5402 5 mM Initial 1:10 dilution = 0.5
mM in 2 .mu.M Store at -20 N2B27. Some cell lines >1 yr Add to
media at 1:250 for final may need to be concentration of 2 .mu.M
optimised, range = 1-5 .mu.M
[0124] Media
[0125] Preparation of DMEM/F12-N2 Medium
[0126] To 100 ml of DMEM/F12 (Gibco 42400-010) add 1 ml of N2
100.times. stock solution. The final concentration of each
component of N2 in the DMEM/F12 medium is:
[0127] Insulin 25 .mu.g/ml Putrescine 16 .mu.g/ml Transferrin 100
.mu.g/ml
[0128] Sodium Selenite 30 nM Progesterone 6 ng/ml BSA 50
.mu.g/ml
[0129] Preparation of Neurobasal/B27
[0130] To 100 ml Neurobasal medium (Gibco 21103-049) add 2 ml of
B27 (Gibco 17504-044) and 1-2M L-glutamine (TC stores 1:100)
[0131] Preparation of N2 B27 Medium
[0132] Mix DMEM/F12-N2 medium with Neurobasal/B27 medium at the
ratio of 1:1.
[0133] The media was used to dilute all compounds and grow the
cells.
[0134] The medium was used for maintenance of human ES cells and
for derivation and maintenance of ES cells from 129 strain mice,
and also for derivation of ES cells from the non-permissive mouse
strains CBA and C56/BL6.
Example 6
[0135] Mouse ES cells were cultured in the presence of an inhibitor
of the FGF receptor and a MEK inhibitor. Selective pharmacological
inhibitors SU5402 and PD184352 were used to inhibit FGF receptor
tyrosine kinase and activation of Erk1/2 via MEK1/2, respectively.
We found that addition of either inhibitor is sufficient for robust
ES cell propagation in N2B27 medium containing LIF without
provision of BMP4 (data not shown). Undifferentiated cultures can
be continuously passaged in these conditions while retaining
expression of the pluripotency markers Oct4, Nanog, and Rex1.
Neural commitment does not occur despite much lower expression of
Id genes than in cultures maintained with LIF plus BMP.
[0136] ES cells plated in N2B27 medium without added LIF,
conditions that normally elicit efficient neural commitment, remain
Oct4 positive and Sox1 negative for several days if either SU5402
or PD184352 are added (data not shown). However, these cells
invariably differentiate and/or die after passaging. To reduce
potential toxic side effects we used 2.5 fold lower doses and
combined the two inhibitors together. In N2B27 with 0.8 .mu.M
PD184352 plus 2 .mu.M SU5402, some differentiation is observed
initially, but ES cells persist and expand after passage (data not
shown). Viability is lower and population doubling time slower in
this two inhibitor (2i) condition than in the presence of LIF, but
differentiation is effectively restrained. This finding suggests
that the minimal requirements for ES cell self-renewal may be to
deflect differentiation signals emanating from FGF receptor and Erk
signaling while avoiding compromise to cell growth and
viability.
Example 7
[0137] We reasoned that reduced growth of ES cells in 2i media
could be due to increased activity of glycogen synthase kinase 3
(GSK-3) consequent to release of inhibitory phosphorylation by Rsk
downstream of pErk. CHIR99021 is a well-characterised highly
selective small molecule inhibitor of GSK-3 that does not cross
react with cyclin-dependent kinases (CDKs) at concentrations that
completely block GSK-3 activity. When added to cultures in the
presence of serum we found that CHIR99021 (3 .mu.M) actually
promotes differentiation, even in the presence of LIF. In
serum-free N2B27 medium the differentiation response is reduced and
some colonies appear morphologically undifferentiated for several
days. However, undifferentiated cells do not persist after
passaging (data not shown). Similar results were obtained with two
other widely used GSK-3 inhibitors, SB216763 and SB415286, although
both appeared somewhat toxic to ES cells.
[0138] However, when CHIR99021 is combined with 2i the
differentiation response is lost entirely. Furthermore, CHIR99021
modulated the response to 2i such that ES cells grew as compact
three dimensional colonies rather than flattened monolayers
typically seen in LIF plus serum/BMP or in 2i. Differentiation was
negligible in the three inhibitors (3i) and ES cells propagated
rapidly. Most importantly undifferentiated colonies grew up at high
efficiency after passaging (data not shown). Derivatives of two
independent parental ES cell lines, E14Tg2a and CGR8 showed robust
long term expansion in 3i with little or no overt differentiation.
They express Oct4, Nanog and Rex1 and do not exhibit appreciable
expression of lineage commitment markers, Gata4, Sox1, or brachyury
(data not shown). In bulk culture ES cells expand with a comparable
doubling rate in 3i as in LIF plus BMP, and the proportion of
Oct4-GFP positive undifferentiated cells remains over 90% (data not
shown).
[0139] Thus, 3i medium can be used to culture ES cells without
differentiation in the absence of serum or added cytokines.
Example 8
[0140] A rigorous test of the sufficiency of a culture formulation
to sustain ES cell self-renewal is formation of undifferentiated
colonies by individual cells. After single cell deposition, cloning
efficiency in N2B27 plus 3i is 25% (98/384), higher than with LIF
plus BMP (11%, 23/192)--data not shown. These colonies express
Oct4-GFP and are passageable as undifferentiated ES cells. Thus,
medium comprising a MEK inhibitor, an inhibitor of the FGF receptor
and a GSK3 inhibitor is able to sustain the formation of
undifferentiated ES cell colonies derived from single cells.
Example 9
[0141] We examined whether 3i was adequate for the derivation of
new ES cells directly from embryos or reflected an adaptation of
established lines. Blastocysts from the permissive 129 strain were
plated directly in N2B27 plus 3i on gelatin-coated plastic and
cultured for 5 days. After subsequent dissociation and replating of
the inner cell mass, ES cell colonies were obtained from 7 of 12
embryos. Three of these were expanded and injected into
blastocysts. All gave high rates of chimaerism and germline
transmission (Table 1). Subsequently we have derived multiple ES
cells from C57BL/6 and non-permissive CBA and MF1 strains
indicating that 3i facilitates the transition from epiblast to ES
cell. We conclude that 3i liberates ES cells from requirements for
exogenous LIF and BMP/serum without selection or compromise to
developmental potency.
TABLE-US-00002 TABLE 1 Contribution of ES cells derived in 3i to
chimaeras and production of germline offspring No. Cell embryos No.
liveborn No. No. No. line injected pups chimaeras* test-mated
transmitting* CPS1 64 16 12 8 (5m, 3f) 3f.sup.# CPS2 21 5 4 3 (1m,
2f) 2f.sup.# CPS3 20 15 11 4 (3m, 1f) 2m *Chimaerism and
transmission of the 129/Ola ES cell genome detected by coat colour
.sup.#ES cells assumed to be XX.
[0142] Thus, ES cells are maintained in a combination of a GSK3
inhibitor and a MEK inhibitor, a MEK inhibitor and an antagonist of
an FGF receptor or, optionally, a GSK3 inhibitor, a MEK inhibitor
and an antagonist of an FGF receptor and the invention also
provides culture methods and media therefor.
* * * * *